copy Koninklijke Brill NV Leiden 2008 DOI 101163157075608X383674
Animal Biology 58 (2008) 353ndash370 wwwbrillnlab
Quantitative description of collagen structure in the articular cartilage of the young and adult
equine distal metacarpus
Mark C van Turnhout Monique B Haazelager Merel AL Gijsen Henk Schipper Sander Kranenbarg Johan L van Leeuwen
Experimental Zoology Group Department of Animal Sciences Wageningen UniversityPO Box 338 6700 AH Wageningen Th e Netherlands
Abstract Th e orientation and organisation of collagen fi brils play an important role in the mechanical functioning of the articular cartilage (AC) that covers the surfaces in the diarthrodial joints In the adult animal typically an arcade like lsquoBenninghoff structurersquo is found Because the remodelling capacity of the collagen network in the adult animal is limited this Benninghoff structure needs to develop before the animal reaches maturity and it needs to develop correctly
Th e aim of this study is to use quantitative polarised light microscopy (qPLM) and scanning electron microscopy (SEM) techniques to investigate if this Benninghoff structure is already present in the young animal and to quantitatively investigate possible diff erences in collagen structure in the equine distal metacarpus of the young and adult animal
In total 21 forelimbs of 13 horses are used In animals of age 10 months and older we fi nd an arcade like Benninghoff structure for the collagen fi bril network in both the qPLM and SEM study Th e qPLM study shows that the collagenrsquos predominant orientation is parallel to the articular surface throughout the entire cartilage depth in two animals directly after birth Th ese fi ndings are supported by SEM results on a foal
We conclude that structural remodelling of the collagen network in AC occurs in the fi rst months after birth Because animals start with collagen parallel to the articular surface and need to remodel this structure to a Benninghoff architecture and because collagen structure is an important parameter for AC mechanics and mechanobiology these results suggest implications for AC epigenetics copy Koninklijke Brill NV Leiden 2008
Key words qPLM SEM equine articular cartilage collagen structure mechanobiology
354 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Introduction
Articular cartilage (AC) is the thin layer of tissue that covers the surfaces of bones in diarthrodial joints Its function is to provide a low friction environment for joint movement and to transmit loads To fulfi l this function AC needs a certain composition and structure which it develops during early life (Brama et al 2000b 2002 Helminen et al 2000 Hunziker et al 2007b ) At maturity AC composition may to some degree still be adapted to fi t changing functional demands (Brama et al 2000b Saadat et al 2006 ) but the remodelling capacity of the collagen network in the mature animal is limited (Brama et al 2000a Hyttinen et al 2001 Murray et al 2001 Saadat et al 2006 ) In fact most diff erences we observe in the collagen network in AC at this point are associated with wear trauma and pathologies
Th e orientation and organisation of the collagen fi brils play an important role in the mechanical function of AC (Bi et al 2005 Han et al 2002 Hughes et al 2005 Julkunen et al 2007 Kiviranta et al 2006 Rieppo et al 2003a ) Th e classical model of AC collagen architecture is that of Benninghoff ( 1925 ) From the articular surface to the bone this model describes fi rst a thin superfi cial zone with collagen fi brils arranged parallel to the articular surface next a thicker transitional zone where the col-lagen fi brils seem to lack a predominant orientation and fi nally the thickest deep zone where the collagen fi brils are oriented in the radial direction more or less perpendicular to the subchondral bone
Th is model has been confi rmed in a variety of species and anatomical sites (Hughes et al 2005 Kaumlaumlb et al 1998 ) but notably in specimens past the juvenile age Th e collagen network in the young developing animal is known to be subject to composi-tional remodelling For instance Bland and Ashhurst ( 1996a b ) looked at the temporal distribution of diff erent collagen types in fetal and young rabbit AC
Th ey were unable to show type II collagen the major collagen component in adult AC (over 90 ) in rabbit AC before 3 weeks post natal in the menisci and before 6 weeks post natal in the tibial plateau Brama et al ( 2000b ) showed an increase in collagen content in the equine metacarpophalangeal joint up to an age of 5 months and no changes afterwards Structural remodelling too has been shown in eg mouse (Hughes et al 2005 ) and rabbit AC (Hunziker et al 2007b ) Th e focus in these studies is on the diff erentiation in superfi cialtransitionaldeep zones and to the best of our knowledge a quantitative description and comparison of collagen structure in developing and full-grown AC has not yet been reported
Quantitative polarised light microscopy (qPLM) is a popular technique to evaluate collagen structure in AC (Massoumian et al 2003 Rieppo et al 2003b Ross et al 1997 Ugryumova et al 2005 ) and is sometimes called lsquothe gold standard of histologyrsquo (Alhadlaq et al 2004 Xia et al 2007 ) What is measured with qPLM are properties of birefringent structures cq collagen fi brils Two parameters are measured retardance and azimuth of the birefringent structure Th e retardance is a combined measure for structural anisotropy and collagen amount (Arokoski et al 1996 Bennett 1950 Kiraacutely et al 1997 ) low retardance indicates either a low amount of collagen or a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 355
degree of structural anisotropy or both In the transitional zone where according to the Benninghoff model the fi bril architecture shows low structural anisotropy compared to the other zones we therefore expect the retardance to show a minimum Th is fact is often used to determine zone thickness with PLM (Hughes et al 2005 Hyttinen et al 2001 Kiraacutely et al 1998 Kiviranta et al 2006 Li et al 2006 Saadat et al 2006 Xia et al 2002 2003 ) Th e second parameter is the azimuth which is the predominant orientation of the birefringent structures (Julkunen et al 2007 Rieppo et al 2003b Ross et al 1997 )
Scanning electron microscopy (SEM) has the advantage over qPLM that it is able to visualise individual fi brils It is particularly SEM studies that have stressed that the azimuth that we fi nd with qPLM is a predominant orientation only and not the orientation of every single fi bril (Clark 1985 1991 Hughes et al 2005 Speer and Dahners 1979 ) Th e objective of this study is to use qPLM and SEM techniques to quantitatively investigate diff erences in collagen structure in the equine distal metacarpus of the young and adult animal
Methods
Scanning Electron Microscopy (SEM)
Nine forelimbs of fi ve horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) and processed on the day of slaughter Th e approximate age in years of 4 horses was obtained from the owner Th e age of the fi fth horse (a foal) is only known to be below one year See table 1 for details
Horse age left right origin breed
sem1 1 year x x abattoir Frysiansem2 1 year x x abattoir Dutch warmbloodsem3 15 years x abattoir Dutch warmbloodsem4 2 years x x abattoir Dutch warmbloodsem5 lt 1 year x x abattoir Dutch warmbloodplm1 0 months x x Utrecht Shetlandplm2 0 months x Utrecht unknownplm3 45 months x Utrecht unknownplm4 10 months x x Utrecht unknownplm5 12 months x Utrecht unknownplm6 120 months x Utrecht unknownplm7 adult x x abattoir unknownplm8 adult x x abattoir unknown
Table 1 Th e age and the origin of the limbs used in this study Th e animals labelled 0 months of age were stillborn Horses labeled lsquosemrsquo were used for the scanning electron microscope study horses labeled lsquoplmrsquo were used for the polarised microscopy study
356 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Full depth cartilage plugs were taken from the distal metacarpus using a hollow drill and a chisel at the medial and lateral distal parts of the joint (MDi and LDi fi g 1 )
Th e cartilage plugs were then fi xed (25 glutaraldehyde in 02 M sodium cacodylate buff er) for 4 days washed and infi ltrated with sucrose (25 on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing Frozen samples were split in two with a scalpel and hammer placed in water and dehydrated with a series of alcohol solutions (70 80 90 and 96 for 15 min each 100 once for 5 min and twice for 10 min) and fi nally dried with a critical point dryer using CO 2 (CPD 020 Balzers Liechtenstein) to avoid surface tension and surface damage Th ese samples were glued on a sample holder with conductive carbon cement (Leit- C Neubauer Chemicalien Germany) and stored overnight for the glue to dry Th e surface of the sam-ple was sputter coated with 8 nm platinum in a dedicated preparation chamber (Oxford Instruments CT 1500 HF Eynsham England) for a better reflection of electrons
SEM imaging was performed with a fi eld emission scanning electron microscope (JEOL 6300 F Tokyo Japan) in vacuum at room temperature using a focussed elec-tron beam of 35 kV with a work distance of 16 mm Digital images were recorded at a scan rate of 100 secondsfull frame (Orion 6 ELI sprl Belgium) and stored in 8 bit TIFF format
We fi rst collected an overview of the full depth cartilage layer at a magnifi cation of 70x Next approximately ten to twenty high magnifi cation images (10000x) were collected at diff erent heights in the cartilage layer for analysis with a Fast Fourier Transformation (FFT) Th e resulting 2D power spectrum showed an ellipse like bright spot centred at the image with its long axis in the direction of the predominant fi bril orientation When we scanned the intensity on a line with a certain azimuth starting in the centre we therefore found the highest values when the azimuth of the line corresponds
Figure 1 Th e distal metacarpus (right limb) and the fi ve sample sites used in this study mediodorsal (MDo) laterodorsal (LDo) sagittal ridge (SR) mediodistal (MDi) and laterodistal (LDi)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 357
with the ellipsersquos long axis (Kim et al 2006 Petroll et al 1993 ) Figure 2a shows an example of a SEM image in the 2 year old animal with a magnifi cation of 10000x Th ese images measure 2528 pixels by 2030 pixels and were cropped to a circle posi-tioned in the centre with a radius of 927 pixels to minimise FFT artifacts Th e cropped image was subjected to 2D FFT and this resulted in a power spectrum (fi g 2b) Note that FFT introduced a 90deg phase shift in the power spectrum We corrected for this phase shift in this example From the spectrum we could detect the predominant orientation from the shape of the ellipse in the centre Th is is illustrated in fi g 2c a thresholded binary image of the power spectrum that shows the core of the ellipse
Th e analysis was implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005) Results were interpolated to 20 equidistant points and will be presented as a function of dimensionless height ie from the tidemark h = 0 to the articular surface h = 1 We expressed the azimuth with respect to the articular surface ie 0deg and 180deg are parallel to the articular surface 90deg is perpendicular to the articular
Figure 2 Example illustrating predominant fi bril orientation detection through 2D Fast Fourier Transform (FFT) analysis (a) Original SEM image at a magnifi cation of 10000x (b) Th e power spectrum after 2D FFT and correction for the phase shift introduced by the FFT Th e predominant orientation corresponds to the long axis of the ellipse in the centre (c) A binary version of the power spectrum illustrating the core of the ellipse
(a) (b)
(c)
358 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
surface Because of the small sample size we treated all measurements independently for the presentation of height dependent results
Quantitative polarised light microscopy (qPLM)
Eight forelimbs of six horses with known age were collected from the Faculty of Veteri-nary Medicine of the University of Utrecht (Th e Netherlands) and an additional four forelimbs from two adult horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) see table 1 for details Forelimbs provided by Utrecht University were obtained frozen and were thawed at 5 degC overnight before processing Limbs collected from the abattoir were fresh and processed on the day of slaughter
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Split line patterns were created on the articular surface of the distal metacarpus with a sharp round needle charged with Indian ink Th e needle was inserted perpendicular to the articular surface at 2 mm intervals and excess ink was removed by rinsing Th e resulting split line pattern was recorded with a Nikon D-100 digital camera with a Micro-Nikon 55 mm objective
Samples were taken from the sagittal ridge (SR) the distal part of the medial side (MDi) and lateral side (LDi) and dorsal parts of the medial side (MDo) and lateral side (LDo) see fi gure 1 A dentist drill was used to introduce rectangular carvings and from these full depth cartilage plugs (ie including a piece of the subchondral bone) were extracted using a chisel Th ese samples were fi xed with formalin decalcifi ed with EDTA (10 EDTA pH 74) for two weeks washed and infi ltrated with sucrose (25 sucrose on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing and fi nally cut parallel to the superfi cial split lines to 5 μm thick histological slices with a cryostat (Reichert 2800N)
Two macroscopically normal histological samples for each sample site were mounted with water and analysed with the LC-PolScope system for qPLM (Oldenbourg and Mei 1995 Oldenbourg 2004) Images were obtained with a Zeiss Axiovert 200M microscope at a 5x16 magnifi cation equipped with a Q-imaging monochrome HR Retiga EX 1350 camera Recorded intensity images had a resolution of 159 μm 2 pixel and were stored in 8 bit TIFF format We used the fi ve frame setting with background correction as described by Shribak and Oldenbourg ( 2003 ) with a swing of 003 [-] Th e recorded images were analysed for predominant collagen fi bril orientation and tis-sue retardance with custom written scripts implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005)
A representative section with a width of 170 pixels that reaches from the tidemark to the articular surface was extracted from the images Th e results were averaged over this width and are presented as a function of dimensionless height after interpolation to 400 equidistant points Th e azimuth was expressed with respect to the articular surface Average retardance was obtained by taking the arithmetic mean of the 170 pixels and used to determine the position of the birefringence minimum in the tran-sitional zone To obtain an average orientation over these 170 pixels with predomi-nant fi bril azimuth the arithmetic means did not suffi ce (Upton and Fingleton 1989 )
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 359
We therefore introduced a retardance weighted average azimuth (φ) that is obtained by maximising the function
( ) ( )[ ] ( )( )uacuteucirc
ugraveecirceuml
eacutetimesD=aring
= nn
nIN
n ϕϕ
ϕϕsincos
sincos1
ϕ
for φ Th e summation over the absolute values of the inner dot product found the line
with smallest diff erence in angles compared to all lines N described by the azimuth values φ Th e retardance φ ( n ) is a measure for the amount of collagen that is associated with azimuth φ ( n ) Th rough multiplication of the inner dot product with the retardance we assigned a lower importance to azimuth values with a lower retardance which may indicate that 1) there is fewer collagen in this pixel or that 2) the predomi-nant orientation belongs to a collagen structure with a low level of anisotropy or 3) a combination of these two Maximisation of equation (1) was done with Matlabrsquos built in function lsquofminbndrsquo on a interval of 0 lt φ le π Final curves for each site were averages from two separate qPLM slices taken from each sample
Results
SEM study
In the older animals SEM images show that the collagen fi brils exhibit structural aniso-tropy in varying degrees depending on the height in the cartilage Structural anisotropy is most clearly visible at the articular surface and at the tidemark In the foal however the cartilage appears more chaotic throughout the entire cartilage layer Figure 3 shows an example of the collagen structure close to the tidemark in the foal and in the adult animals Th e diff erence in structural anisotropy is also apparent from the power spectra the adult animal shows a more pronounced ellipse than the young animal When the spectrum resembled a circle more than an ellipse as in this example for the foal we rotated the image and repeated the analysis Th e observed predominant orientation then rotated with the image confi rming that we found an objective measure for orientation
Th e height dependent orientation patterns we fi nd in the foal diff er from those found in the other animals again particularly in the deep zone Figures 4 and 5 show these patterns for the foal and for the older animals In the foal the orientation of the fi brils appears to vary around 0deg (or 180deg) throughout the entire cartilage depth Visual inspection of the SEM images shows that the orientation we fi nd in the foal belongs to a near isotropic structure for h lt 08 see for instance fi g 3a Near the articular sur-face SEM images in the foal show an anisotropic structure as in the older animals
In the other animals an arcade like Benninghoff structure appears collagen is aligned perpendicularly to the subchondral bone in the deep zone (approximately 80 of the tissue height) then curves away to form an arcade and is fi nally aligned with the articular surface
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
354 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Introduction
Articular cartilage (AC) is the thin layer of tissue that covers the surfaces of bones in diarthrodial joints Its function is to provide a low friction environment for joint movement and to transmit loads To fulfi l this function AC needs a certain composition and structure which it develops during early life (Brama et al 2000b 2002 Helminen et al 2000 Hunziker et al 2007b ) At maturity AC composition may to some degree still be adapted to fi t changing functional demands (Brama et al 2000b Saadat et al 2006 ) but the remodelling capacity of the collagen network in the mature animal is limited (Brama et al 2000a Hyttinen et al 2001 Murray et al 2001 Saadat et al 2006 ) In fact most diff erences we observe in the collagen network in AC at this point are associated with wear trauma and pathologies
Th e orientation and organisation of the collagen fi brils play an important role in the mechanical function of AC (Bi et al 2005 Han et al 2002 Hughes et al 2005 Julkunen et al 2007 Kiviranta et al 2006 Rieppo et al 2003a ) Th e classical model of AC collagen architecture is that of Benninghoff ( 1925 ) From the articular surface to the bone this model describes fi rst a thin superfi cial zone with collagen fi brils arranged parallel to the articular surface next a thicker transitional zone where the col-lagen fi brils seem to lack a predominant orientation and fi nally the thickest deep zone where the collagen fi brils are oriented in the radial direction more or less perpendicular to the subchondral bone
Th is model has been confi rmed in a variety of species and anatomical sites (Hughes et al 2005 Kaumlaumlb et al 1998 ) but notably in specimens past the juvenile age Th e collagen network in the young developing animal is known to be subject to composi-tional remodelling For instance Bland and Ashhurst ( 1996a b ) looked at the temporal distribution of diff erent collagen types in fetal and young rabbit AC
Th ey were unable to show type II collagen the major collagen component in adult AC (over 90 ) in rabbit AC before 3 weeks post natal in the menisci and before 6 weeks post natal in the tibial plateau Brama et al ( 2000b ) showed an increase in collagen content in the equine metacarpophalangeal joint up to an age of 5 months and no changes afterwards Structural remodelling too has been shown in eg mouse (Hughes et al 2005 ) and rabbit AC (Hunziker et al 2007b ) Th e focus in these studies is on the diff erentiation in superfi cialtransitionaldeep zones and to the best of our knowledge a quantitative description and comparison of collagen structure in developing and full-grown AC has not yet been reported
Quantitative polarised light microscopy (qPLM) is a popular technique to evaluate collagen structure in AC (Massoumian et al 2003 Rieppo et al 2003b Ross et al 1997 Ugryumova et al 2005 ) and is sometimes called lsquothe gold standard of histologyrsquo (Alhadlaq et al 2004 Xia et al 2007 ) What is measured with qPLM are properties of birefringent structures cq collagen fi brils Two parameters are measured retardance and azimuth of the birefringent structure Th e retardance is a combined measure for structural anisotropy and collagen amount (Arokoski et al 1996 Bennett 1950 Kiraacutely et al 1997 ) low retardance indicates either a low amount of collagen or a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 355
degree of structural anisotropy or both In the transitional zone where according to the Benninghoff model the fi bril architecture shows low structural anisotropy compared to the other zones we therefore expect the retardance to show a minimum Th is fact is often used to determine zone thickness with PLM (Hughes et al 2005 Hyttinen et al 2001 Kiraacutely et al 1998 Kiviranta et al 2006 Li et al 2006 Saadat et al 2006 Xia et al 2002 2003 ) Th e second parameter is the azimuth which is the predominant orientation of the birefringent structures (Julkunen et al 2007 Rieppo et al 2003b Ross et al 1997 )
Scanning electron microscopy (SEM) has the advantage over qPLM that it is able to visualise individual fi brils It is particularly SEM studies that have stressed that the azimuth that we fi nd with qPLM is a predominant orientation only and not the orientation of every single fi bril (Clark 1985 1991 Hughes et al 2005 Speer and Dahners 1979 ) Th e objective of this study is to use qPLM and SEM techniques to quantitatively investigate diff erences in collagen structure in the equine distal metacarpus of the young and adult animal
Methods
Scanning Electron Microscopy (SEM)
Nine forelimbs of fi ve horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) and processed on the day of slaughter Th e approximate age in years of 4 horses was obtained from the owner Th e age of the fi fth horse (a foal) is only known to be below one year See table 1 for details
Horse age left right origin breed
sem1 1 year x x abattoir Frysiansem2 1 year x x abattoir Dutch warmbloodsem3 15 years x abattoir Dutch warmbloodsem4 2 years x x abattoir Dutch warmbloodsem5 lt 1 year x x abattoir Dutch warmbloodplm1 0 months x x Utrecht Shetlandplm2 0 months x Utrecht unknownplm3 45 months x Utrecht unknownplm4 10 months x x Utrecht unknownplm5 12 months x Utrecht unknownplm6 120 months x Utrecht unknownplm7 adult x x abattoir unknownplm8 adult x x abattoir unknown
Table 1 Th e age and the origin of the limbs used in this study Th e animals labelled 0 months of age were stillborn Horses labeled lsquosemrsquo were used for the scanning electron microscope study horses labeled lsquoplmrsquo were used for the polarised microscopy study
356 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Full depth cartilage plugs were taken from the distal metacarpus using a hollow drill and a chisel at the medial and lateral distal parts of the joint (MDi and LDi fi g 1 )
Th e cartilage plugs were then fi xed (25 glutaraldehyde in 02 M sodium cacodylate buff er) for 4 days washed and infi ltrated with sucrose (25 on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing Frozen samples were split in two with a scalpel and hammer placed in water and dehydrated with a series of alcohol solutions (70 80 90 and 96 for 15 min each 100 once for 5 min and twice for 10 min) and fi nally dried with a critical point dryer using CO 2 (CPD 020 Balzers Liechtenstein) to avoid surface tension and surface damage Th ese samples were glued on a sample holder with conductive carbon cement (Leit- C Neubauer Chemicalien Germany) and stored overnight for the glue to dry Th e surface of the sam-ple was sputter coated with 8 nm platinum in a dedicated preparation chamber (Oxford Instruments CT 1500 HF Eynsham England) for a better reflection of electrons
SEM imaging was performed with a fi eld emission scanning electron microscope (JEOL 6300 F Tokyo Japan) in vacuum at room temperature using a focussed elec-tron beam of 35 kV with a work distance of 16 mm Digital images were recorded at a scan rate of 100 secondsfull frame (Orion 6 ELI sprl Belgium) and stored in 8 bit TIFF format
We fi rst collected an overview of the full depth cartilage layer at a magnifi cation of 70x Next approximately ten to twenty high magnifi cation images (10000x) were collected at diff erent heights in the cartilage layer for analysis with a Fast Fourier Transformation (FFT) Th e resulting 2D power spectrum showed an ellipse like bright spot centred at the image with its long axis in the direction of the predominant fi bril orientation When we scanned the intensity on a line with a certain azimuth starting in the centre we therefore found the highest values when the azimuth of the line corresponds
Figure 1 Th e distal metacarpus (right limb) and the fi ve sample sites used in this study mediodorsal (MDo) laterodorsal (LDo) sagittal ridge (SR) mediodistal (MDi) and laterodistal (LDi)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 357
with the ellipsersquos long axis (Kim et al 2006 Petroll et al 1993 ) Figure 2a shows an example of a SEM image in the 2 year old animal with a magnifi cation of 10000x Th ese images measure 2528 pixels by 2030 pixels and were cropped to a circle posi-tioned in the centre with a radius of 927 pixels to minimise FFT artifacts Th e cropped image was subjected to 2D FFT and this resulted in a power spectrum (fi g 2b) Note that FFT introduced a 90deg phase shift in the power spectrum We corrected for this phase shift in this example From the spectrum we could detect the predominant orientation from the shape of the ellipse in the centre Th is is illustrated in fi g 2c a thresholded binary image of the power spectrum that shows the core of the ellipse
Th e analysis was implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005) Results were interpolated to 20 equidistant points and will be presented as a function of dimensionless height ie from the tidemark h = 0 to the articular surface h = 1 We expressed the azimuth with respect to the articular surface ie 0deg and 180deg are parallel to the articular surface 90deg is perpendicular to the articular
Figure 2 Example illustrating predominant fi bril orientation detection through 2D Fast Fourier Transform (FFT) analysis (a) Original SEM image at a magnifi cation of 10000x (b) Th e power spectrum after 2D FFT and correction for the phase shift introduced by the FFT Th e predominant orientation corresponds to the long axis of the ellipse in the centre (c) A binary version of the power spectrum illustrating the core of the ellipse
(a) (b)
(c)
358 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
surface Because of the small sample size we treated all measurements independently for the presentation of height dependent results
Quantitative polarised light microscopy (qPLM)
Eight forelimbs of six horses with known age were collected from the Faculty of Veteri-nary Medicine of the University of Utrecht (Th e Netherlands) and an additional four forelimbs from two adult horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) see table 1 for details Forelimbs provided by Utrecht University were obtained frozen and were thawed at 5 degC overnight before processing Limbs collected from the abattoir were fresh and processed on the day of slaughter
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Split line patterns were created on the articular surface of the distal metacarpus with a sharp round needle charged with Indian ink Th e needle was inserted perpendicular to the articular surface at 2 mm intervals and excess ink was removed by rinsing Th e resulting split line pattern was recorded with a Nikon D-100 digital camera with a Micro-Nikon 55 mm objective
Samples were taken from the sagittal ridge (SR) the distal part of the medial side (MDi) and lateral side (LDi) and dorsal parts of the medial side (MDo) and lateral side (LDo) see fi gure 1 A dentist drill was used to introduce rectangular carvings and from these full depth cartilage plugs (ie including a piece of the subchondral bone) were extracted using a chisel Th ese samples were fi xed with formalin decalcifi ed with EDTA (10 EDTA pH 74) for two weeks washed and infi ltrated with sucrose (25 sucrose on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing and fi nally cut parallel to the superfi cial split lines to 5 μm thick histological slices with a cryostat (Reichert 2800N)
Two macroscopically normal histological samples for each sample site were mounted with water and analysed with the LC-PolScope system for qPLM (Oldenbourg and Mei 1995 Oldenbourg 2004) Images were obtained with a Zeiss Axiovert 200M microscope at a 5x16 magnifi cation equipped with a Q-imaging monochrome HR Retiga EX 1350 camera Recorded intensity images had a resolution of 159 μm 2 pixel and were stored in 8 bit TIFF format We used the fi ve frame setting with background correction as described by Shribak and Oldenbourg ( 2003 ) with a swing of 003 [-] Th e recorded images were analysed for predominant collagen fi bril orientation and tis-sue retardance with custom written scripts implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005)
A representative section with a width of 170 pixels that reaches from the tidemark to the articular surface was extracted from the images Th e results were averaged over this width and are presented as a function of dimensionless height after interpolation to 400 equidistant points Th e azimuth was expressed with respect to the articular surface Average retardance was obtained by taking the arithmetic mean of the 170 pixels and used to determine the position of the birefringence minimum in the tran-sitional zone To obtain an average orientation over these 170 pixels with predomi-nant fi bril azimuth the arithmetic means did not suffi ce (Upton and Fingleton 1989 )
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 359
We therefore introduced a retardance weighted average azimuth (φ) that is obtained by maximising the function
( ) ( )[ ] ( )( )uacuteucirc
ugraveecirceuml
eacutetimesD=aring
= nn
nIN
n ϕϕ
ϕϕsincos
sincos1
ϕ
for φ Th e summation over the absolute values of the inner dot product found the line
with smallest diff erence in angles compared to all lines N described by the azimuth values φ Th e retardance φ ( n ) is a measure for the amount of collagen that is associated with azimuth φ ( n ) Th rough multiplication of the inner dot product with the retardance we assigned a lower importance to azimuth values with a lower retardance which may indicate that 1) there is fewer collagen in this pixel or that 2) the predomi-nant orientation belongs to a collagen structure with a low level of anisotropy or 3) a combination of these two Maximisation of equation (1) was done with Matlabrsquos built in function lsquofminbndrsquo on a interval of 0 lt φ le π Final curves for each site were averages from two separate qPLM slices taken from each sample
Results
SEM study
In the older animals SEM images show that the collagen fi brils exhibit structural aniso-tropy in varying degrees depending on the height in the cartilage Structural anisotropy is most clearly visible at the articular surface and at the tidemark In the foal however the cartilage appears more chaotic throughout the entire cartilage layer Figure 3 shows an example of the collagen structure close to the tidemark in the foal and in the adult animals Th e diff erence in structural anisotropy is also apparent from the power spectra the adult animal shows a more pronounced ellipse than the young animal When the spectrum resembled a circle more than an ellipse as in this example for the foal we rotated the image and repeated the analysis Th e observed predominant orientation then rotated with the image confi rming that we found an objective measure for orientation
Th e height dependent orientation patterns we fi nd in the foal diff er from those found in the other animals again particularly in the deep zone Figures 4 and 5 show these patterns for the foal and for the older animals In the foal the orientation of the fi brils appears to vary around 0deg (or 180deg) throughout the entire cartilage depth Visual inspection of the SEM images shows that the orientation we fi nd in the foal belongs to a near isotropic structure for h lt 08 see for instance fi g 3a Near the articular sur-face SEM images in the foal show an anisotropic structure as in the older animals
In the other animals an arcade like Benninghoff structure appears collagen is aligned perpendicularly to the subchondral bone in the deep zone (approximately 80 of the tissue height) then curves away to form an arcade and is fi nally aligned with the articular surface
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 355
degree of structural anisotropy or both In the transitional zone where according to the Benninghoff model the fi bril architecture shows low structural anisotropy compared to the other zones we therefore expect the retardance to show a minimum Th is fact is often used to determine zone thickness with PLM (Hughes et al 2005 Hyttinen et al 2001 Kiraacutely et al 1998 Kiviranta et al 2006 Li et al 2006 Saadat et al 2006 Xia et al 2002 2003 ) Th e second parameter is the azimuth which is the predominant orientation of the birefringent structures (Julkunen et al 2007 Rieppo et al 2003b Ross et al 1997 )
Scanning electron microscopy (SEM) has the advantage over qPLM that it is able to visualise individual fi brils It is particularly SEM studies that have stressed that the azimuth that we fi nd with qPLM is a predominant orientation only and not the orientation of every single fi bril (Clark 1985 1991 Hughes et al 2005 Speer and Dahners 1979 ) Th e objective of this study is to use qPLM and SEM techniques to quantitatively investigate diff erences in collagen structure in the equine distal metacarpus of the young and adult animal
Methods
Scanning Electron Microscopy (SEM)
Nine forelimbs of fi ve horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) and processed on the day of slaughter Th e approximate age in years of 4 horses was obtained from the owner Th e age of the fi fth horse (a foal) is only known to be below one year See table 1 for details
Horse age left right origin breed
sem1 1 year x x abattoir Frysiansem2 1 year x x abattoir Dutch warmbloodsem3 15 years x abattoir Dutch warmbloodsem4 2 years x x abattoir Dutch warmbloodsem5 lt 1 year x x abattoir Dutch warmbloodplm1 0 months x x Utrecht Shetlandplm2 0 months x Utrecht unknownplm3 45 months x Utrecht unknownplm4 10 months x x Utrecht unknownplm5 12 months x Utrecht unknownplm6 120 months x Utrecht unknownplm7 adult x x abattoir unknownplm8 adult x x abattoir unknown
Table 1 Th e age and the origin of the limbs used in this study Th e animals labelled 0 months of age were stillborn Horses labeled lsquosemrsquo were used for the scanning electron microscope study horses labeled lsquoplmrsquo were used for the polarised microscopy study
356 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Full depth cartilage plugs were taken from the distal metacarpus using a hollow drill and a chisel at the medial and lateral distal parts of the joint (MDi and LDi fi g 1 )
Th e cartilage plugs were then fi xed (25 glutaraldehyde in 02 M sodium cacodylate buff er) for 4 days washed and infi ltrated with sucrose (25 on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing Frozen samples were split in two with a scalpel and hammer placed in water and dehydrated with a series of alcohol solutions (70 80 90 and 96 for 15 min each 100 once for 5 min and twice for 10 min) and fi nally dried with a critical point dryer using CO 2 (CPD 020 Balzers Liechtenstein) to avoid surface tension and surface damage Th ese samples were glued on a sample holder with conductive carbon cement (Leit- C Neubauer Chemicalien Germany) and stored overnight for the glue to dry Th e surface of the sam-ple was sputter coated with 8 nm platinum in a dedicated preparation chamber (Oxford Instruments CT 1500 HF Eynsham England) for a better reflection of electrons
SEM imaging was performed with a fi eld emission scanning electron microscope (JEOL 6300 F Tokyo Japan) in vacuum at room temperature using a focussed elec-tron beam of 35 kV with a work distance of 16 mm Digital images were recorded at a scan rate of 100 secondsfull frame (Orion 6 ELI sprl Belgium) and stored in 8 bit TIFF format
We fi rst collected an overview of the full depth cartilage layer at a magnifi cation of 70x Next approximately ten to twenty high magnifi cation images (10000x) were collected at diff erent heights in the cartilage layer for analysis with a Fast Fourier Transformation (FFT) Th e resulting 2D power spectrum showed an ellipse like bright spot centred at the image with its long axis in the direction of the predominant fi bril orientation When we scanned the intensity on a line with a certain azimuth starting in the centre we therefore found the highest values when the azimuth of the line corresponds
Figure 1 Th e distal metacarpus (right limb) and the fi ve sample sites used in this study mediodorsal (MDo) laterodorsal (LDo) sagittal ridge (SR) mediodistal (MDi) and laterodistal (LDi)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 357
with the ellipsersquos long axis (Kim et al 2006 Petroll et al 1993 ) Figure 2a shows an example of a SEM image in the 2 year old animal with a magnifi cation of 10000x Th ese images measure 2528 pixels by 2030 pixels and were cropped to a circle posi-tioned in the centre with a radius of 927 pixels to minimise FFT artifacts Th e cropped image was subjected to 2D FFT and this resulted in a power spectrum (fi g 2b) Note that FFT introduced a 90deg phase shift in the power spectrum We corrected for this phase shift in this example From the spectrum we could detect the predominant orientation from the shape of the ellipse in the centre Th is is illustrated in fi g 2c a thresholded binary image of the power spectrum that shows the core of the ellipse
Th e analysis was implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005) Results were interpolated to 20 equidistant points and will be presented as a function of dimensionless height ie from the tidemark h = 0 to the articular surface h = 1 We expressed the azimuth with respect to the articular surface ie 0deg and 180deg are parallel to the articular surface 90deg is perpendicular to the articular
Figure 2 Example illustrating predominant fi bril orientation detection through 2D Fast Fourier Transform (FFT) analysis (a) Original SEM image at a magnifi cation of 10000x (b) Th e power spectrum after 2D FFT and correction for the phase shift introduced by the FFT Th e predominant orientation corresponds to the long axis of the ellipse in the centre (c) A binary version of the power spectrum illustrating the core of the ellipse
(a) (b)
(c)
358 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
surface Because of the small sample size we treated all measurements independently for the presentation of height dependent results
Quantitative polarised light microscopy (qPLM)
Eight forelimbs of six horses with known age were collected from the Faculty of Veteri-nary Medicine of the University of Utrecht (Th e Netherlands) and an additional four forelimbs from two adult horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) see table 1 for details Forelimbs provided by Utrecht University were obtained frozen and were thawed at 5 degC overnight before processing Limbs collected from the abattoir were fresh and processed on the day of slaughter
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Split line patterns were created on the articular surface of the distal metacarpus with a sharp round needle charged with Indian ink Th e needle was inserted perpendicular to the articular surface at 2 mm intervals and excess ink was removed by rinsing Th e resulting split line pattern was recorded with a Nikon D-100 digital camera with a Micro-Nikon 55 mm objective
Samples were taken from the sagittal ridge (SR) the distal part of the medial side (MDi) and lateral side (LDi) and dorsal parts of the medial side (MDo) and lateral side (LDo) see fi gure 1 A dentist drill was used to introduce rectangular carvings and from these full depth cartilage plugs (ie including a piece of the subchondral bone) were extracted using a chisel Th ese samples were fi xed with formalin decalcifi ed with EDTA (10 EDTA pH 74) for two weeks washed and infi ltrated with sucrose (25 sucrose on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing and fi nally cut parallel to the superfi cial split lines to 5 μm thick histological slices with a cryostat (Reichert 2800N)
Two macroscopically normal histological samples for each sample site were mounted with water and analysed with the LC-PolScope system for qPLM (Oldenbourg and Mei 1995 Oldenbourg 2004) Images were obtained with a Zeiss Axiovert 200M microscope at a 5x16 magnifi cation equipped with a Q-imaging monochrome HR Retiga EX 1350 camera Recorded intensity images had a resolution of 159 μm 2 pixel and were stored in 8 bit TIFF format We used the fi ve frame setting with background correction as described by Shribak and Oldenbourg ( 2003 ) with a swing of 003 [-] Th e recorded images were analysed for predominant collagen fi bril orientation and tis-sue retardance with custom written scripts implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005)
A representative section with a width of 170 pixels that reaches from the tidemark to the articular surface was extracted from the images Th e results were averaged over this width and are presented as a function of dimensionless height after interpolation to 400 equidistant points Th e azimuth was expressed with respect to the articular surface Average retardance was obtained by taking the arithmetic mean of the 170 pixels and used to determine the position of the birefringence minimum in the tran-sitional zone To obtain an average orientation over these 170 pixels with predomi-nant fi bril azimuth the arithmetic means did not suffi ce (Upton and Fingleton 1989 )
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 359
We therefore introduced a retardance weighted average azimuth (φ) that is obtained by maximising the function
( ) ( )[ ] ( )( )uacuteucirc
ugraveecirceuml
eacutetimesD=aring
= nn
nIN
n ϕϕ
ϕϕsincos
sincos1
ϕ
for φ Th e summation over the absolute values of the inner dot product found the line
with smallest diff erence in angles compared to all lines N described by the azimuth values φ Th e retardance φ ( n ) is a measure for the amount of collagen that is associated with azimuth φ ( n ) Th rough multiplication of the inner dot product with the retardance we assigned a lower importance to azimuth values with a lower retardance which may indicate that 1) there is fewer collagen in this pixel or that 2) the predomi-nant orientation belongs to a collagen structure with a low level of anisotropy or 3) a combination of these two Maximisation of equation (1) was done with Matlabrsquos built in function lsquofminbndrsquo on a interval of 0 lt φ le π Final curves for each site were averages from two separate qPLM slices taken from each sample
Results
SEM study
In the older animals SEM images show that the collagen fi brils exhibit structural aniso-tropy in varying degrees depending on the height in the cartilage Structural anisotropy is most clearly visible at the articular surface and at the tidemark In the foal however the cartilage appears more chaotic throughout the entire cartilage layer Figure 3 shows an example of the collagen structure close to the tidemark in the foal and in the adult animals Th e diff erence in structural anisotropy is also apparent from the power spectra the adult animal shows a more pronounced ellipse than the young animal When the spectrum resembled a circle more than an ellipse as in this example for the foal we rotated the image and repeated the analysis Th e observed predominant orientation then rotated with the image confi rming that we found an objective measure for orientation
Th e height dependent orientation patterns we fi nd in the foal diff er from those found in the other animals again particularly in the deep zone Figures 4 and 5 show these patterns for the foal and for the older animals In the foal the orientation of the fi brils appears to vary around 0deg (or 180deg) throughout the entire cartilage depth Visual inspection of the SEM images shows that the orientation we fi nd in the foal belongs to a near isotropic structure for h lt 08 see for instance fi g 3a Near the articular sur-face SEM images in the foal show an anisotropic structure as in the older animals
In the other animals an arcade like Benninghoff structure appears collagen is aligned perpendicularly to the subchondral bone in the deep zone (approximately 80 of the tissue height) then curves away to form an arcade and is fi nally aligned with the articular surface
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
356 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Full depth cartilage plugs were taken from the distal metacarpus using a hollow drill and a chisel at the medial and lateral distal parts of the joint (MDi and LDi fi g 1 )
Th e cartilage plugs were then fi xed (25 glutaraldehyde in 02 M sodium cacodylate buff er) for 4 days washed and infi ltrated with sucrose (25 on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing Frozen samples were split in two with a scalpel and hammer placed in water and dehydrated with a series of alcohol solutions (70 80 90 and 96 for 15 min each 100 once for 5 min and twice for 10 min) and fi nally dried with a critical point dryer using CO 2 (CPD 020 Balzers Liechtenstein) to avoid surface tension and surface damage Th ese samples were glued on a sample holder with conductive carbon cement (Leit- C Neubauer Chemicalien Germany) and stored overnight for the glue to dry Th e surface of the sam-ple was sputter coated with 8 nm platinum in a dedicated preparation chamber (Oxford Instruments CT 1500 HF Eynsham England) for a better reflection of electrons
SEM imaging was performed with a fi eld emission scanning electron microscope (JEOL 6300 F Tokyo Japan) in vacuum at room temperature using a focussed elec-tron beam of 35 kV with a work distance of 16 mm Digital images were recorded at a scan rate of 100 secondsfull frame (Orion 6 ELI sprl Belgium) and stored in 8 bit TIFF format
We fi rst collected an overview of the full depth cartilage layer at a magnifi cation of 70x Next approximately ten to twenty high magnifi cation images (10000x) were collected at diff erent heights in the cartilage layer for analysis with a Fast Fourier Transformation (FFT) Th e resulting 2D power spectrum showed an ellipse like bright spot centred at the image with its long axis in the direction of the predominant fi bril orientation When we scanned the intensity on a line with a certain azimuth starting in the centre we therefore found the highest values when the azimuth of the line corresponds
Figure 1 Th e distal metacarpus (right limb) and the fi ve sample sites used in this study mediodorsal (MDo) laterodorsal (LDo) sagittal ridge (SR) mediodistal (MDi) and laterodistal (LDi)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 357
with the ellipsersquos long axis (Kim et al 2006 Petroll et al 1993 ) Figure 2a shows an example of a SEM image in the 2 year old animal with a magnifi cation of 10000x Th ese images measure 2528 pixels by 2030 pixels and were cropped to a circle posi-tioned in the centre with a radius of 927 pixels to minimise FFT artifacts Th e cropped image was subjected to 2D FFT and this resulted in a power spectrum (fi g 2b) Note that FFT introduced a 90deg phase shift in the power spectrum We corrected for this phase shift in this example From the spectrum we could detect the predominant orientation from the shape of the ellipse in the centre Th is is illustrated in fi g 2c a thresholded binary image of the power spectrum that shows the core of the ellipse
Th e analysis was implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005) Results were interpolated to 20 equidistant points and will be presented as a function of dimensionless height ie from the tidemark h = 0 to the articular surface h = 1 We expressed the azimuth with respect to the articular surface ie 0deg and 180deg are parallel to the articular surface 90deg is perpendicular to the articular
Figure 2 Example illustrating predominant fi bril orientation detection through 2D Fast Fourier Transform (FFT) analysis (a) Original SEM image at a magnifi cation of 10000x (b) Th e power spectrum after 2D FFT and correction for the phase shift introduced by the FFT Th e predominant orientation corresponds to the long axis of the ellipse in the centre (c) A binary version of the power spectrum illustrating the core of the ellipse
(a) (b)
(c)
358 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
surface Because of the small sample size we treated all measurements independently for the presentation of height dependent results
Quantitative polarised light microscopy (qPLM)
Eight forelimbs of six horses with known age were collected from the Faculty of Veteri-nary Medicine of the University of Utrecht (Th e Netherlands) and an additional four forelimbs from two adult horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) see table 1 for details Forelimbs provided by Utrecht University were obtained frozen and were thawed at 5 degC overnight before processing Limbs collected from the abattoir were fresh and processed on the day of slaughter
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Split line patterns were created on the articular surface of the distal metacarpus with a sharp round needle charged with Indian ink Th e needle was inserted perpendicular to the articular surface at 2 mm intervals and excess ink was removed by rinsing Th e resulting split line pattern was recorded with a Nikon D-100 digital camera with a Micro-Nikon 55 mm objective
Samples were taken from the sagittal ridge (SR) the distal part of the medial side (MDi) and lateral side (LDi) and dorsal parts of the medial side (MDo) and lateral side (LDo) see fi gure 1 A dentist drill was used to introduce rectangular carvings and from these full depth cartilage plugs (ie including a piece of the subchondral bone) were extracted using a chisel Th ese samples were fi xed with formalin decalcifi ed with EDTA (10 EDTA pH 74) for two weeks washed and infi ltrated with sucrose (25 sucrose on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing and fi nally cut parallel to the superfi cial split lines to 5 μm thick histological slices with a cryostat (Reichert 2800N)
Two macroscopically normal histological samples for each sample site were mounted with water and analysed with the LC-PolScope system for qPLM (Oldenbourg and Mei 1995 Oldenbourg 2004) Images were obtained with a Zeiss Axiovert 200M microscope at a 5x16 magnifi cation equipped with a Q-imaging monochrome HR Retiga EX 1350 camera Recorded intensity images had a resolution of 159 μm 2 pixel and were stored in 8 bit TIFF format We used the fi ve frame setting with background correction as described by Shribak and Oldenbourg ( 2003 ) with a swing of 003 [-] Th e recorded images were analysed for predominant collagen fi bril orientation and tis-sue retardance with custom written scripts implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005)
A representative section with a width of 170 pixels that reaches from the tidemark to the articular surface was extracted from the images Th e results were averaged over this width and are presented as a function of dimensionless height after interpolation to 400 equidistant points Th e azimuth was expressed with respect to the articular surface Average retardance was obtained by taking the arithmetic mean of the 170 pixels and used to determine the position of the birefringence minimum in the tran-sitional zone To obtain an average orientation over these 170 pixels with predomi-nant fi bril azimuth the arithmetic means did not suffi ce (Upton and Fingleton 1989 )
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 359
We therefore introduced a retardance weighted average azimuth (φ) that is obtained by maximising the function
( ) ( )[ ] ( )( )uacuteucirc
ugraveecirceuml
eacutetimesD=aring
= nn
nIN
n ϕϕ
ϕϕsincos
sincos1
ϕ
for φ Th e summation over the absolute values of the inner dot product found the line
with smallest diff erence in angles compared to all lines N described by the azimuth values φ Th e retardance φ ( n ) is a measure for the amount of collagen that is associated with azimuth φ ( n ) Th rough multiplication of the inner dot product with the retardance we assigned a lower importance to azimuth values with a lower retardance which may indicate that 1) there is fewer collagen in this pixel or that 2) the predomi-nant orientation belongs to a collagen structure with a low level of anisotropy or 3) a combination of these two Maximisation of equation (1) was done with Matlabrsquos built in function lsquofminbndrsquo on a interval of 0 lt φ le π Final curves for each site were averages from two separate qPLM slices taken from each sample
Results
SEM study
In the older animals SEM images show that the collagen fi brils exhibit structural aniso-tropy in varying degrees depending on the height in the cartilage Structural anisotropy is most clearly visible at the articular surface and at the tidemark In the foal however the cartilage appears more chaotic throughout the entire cartilage layer Figure 3 shows an example of the collagen structure close to the tidemark in the foal and in the adult animals Th e diff erence in structural anisotropy is also apparent from the power spectra the adult animal shows a more pronounced ellipse than the young animal When the spectrum resembled a circle more than an ellipse as in this example for the foal we rotated the image and repeated the analysis Th e observed predominant orientation then rotated with the image confi rming that we found an objective measure for orientation
Th e height dependent orientation patterns we fi nd in the foal diff er from those found in the other animals again particularly in the deep zone Figures 4 and 5 show these patterns for the foal and for the older animals In the foal the orientation of the fi brils appears to vary around 0deg (or 180deg) throughout the entire cartilage depth Visual inspection of the SEM images shows that the orientation we fi nd in the foal belongs to a near isotropic structure for h lt 08 see for instance fi g 3a Near the articular sur-face SEM images in the foal show an anisotropic structure as in the older animals
In the other animals an arcade like Benninghoff structure appears collagen is aligned perpendicularly to the subchondral bone in the deep zone (approximately 80 of the tissue height) then curves away to form an arcade and is fi nally aligned with the articular surface
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 357
with the ellipsersquos long axis (Kim et al 2006 Petroll et al 1993 ) Figure 2a shows an example of a SEM image in the 2 year old animal with a magnifi cation of 10000x Th ese images measure 2528 pixels by 2030 pixels and were cropped to a circle posi-tioned in the centre with a radius of 927 pixels to minimise FFT artifacts Th e cropped image was subjected to 2D FFT and this resulted in a power spectrum (fi g 2b) Note that FFT introduced a 90deg phase shift in the power spectrum We corrected for this phase shift in this example From the spectrum we could detect the predominant orientation from the shape of the ellipse in the centre Th is is illustrated in fi g 2c a thresholded binary image of the power spectrum that shows the core of the ellipse
Th e analysis was implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005) Results were interpolated to 20 equidistant points and will be presented as a function of dimensionless height ie from the tidemark h = 0 to the articular surface h = 1 We expressed the azimuth with respect to the articular surface ie 0deg and 180deg are parallel to the articular surface 90deg is perpendicular to the articular
Figure 2 Example illustrating predominant fi bril orientation detection through 2D Fast Fourier Transform (FFT) analysis (a) Original SEM image at a magnifi cation of 10000x (b) Th e power spectrum after 2D FFT and correction for the phase shift introduced by the FFT Th e predominant orientation corresponds to the long axis of the ellipse in the centre (c) A binary version of the power spectrum illustrating the core of the ellipse
(a) (b)
(c)
358 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
surface Because of the small sample size we treated all measurements independently for the presentation of height dependent results
Quantitative polarised light microscopy (qPLM)
Eight forelimbs of six horses with known age were collected from the Faculty of Veteri-nary Medicine of the University of Utrecht (Th e Netherlands) and an additional four forelimbs from two adult horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) see table 1 for details Forelimbs provided by Utrecht University were obtained frozen and were thawed at 5 degC overnight before processing Limbs collected from the abattoir were fresh and processed on the day of slaughter
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Split line patterns were created on the articular surface of the distal metacarpus with a sharp round needle charged with Indian ink Th e needle was inserted perpendicular to the articular surface at 2 mm intervals and excess ink was removed by rinsing Th e resulting split line pattern was recorded with a Nikon D-100 digital camera with a Micro-Nikon 55 mm objective
Samples were taken from the sagittal ridge (SR) the distal part of the medial side (MDi) and lateral side (LDi) and dorsal parts of the medial side (MDo) and lateral side (LDo) see fi gure 1 A dentist drill was used to introduce rectangular carvings and from these full depth cartilage plugs (ie including a piece of the subchondral bone) were extracted using a chisel Th ese samples were fi xed with formalin decalcifi ed with EDTA (10 EDTA pH 74) for two weeks washed and infi ltrated with sucrose (25 sucrose on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing and fi nally cut parallel to the superfi cial split lines to 5 μm thick histological slices with a cryostat (Reichert 2800N)
Two macroscopically normal histological samples for each sample site were mounted with water and analysed with the LC-PolScope system for qPLM (Oldenbourg and Mei 1995 Oldenbourg 2004) Images were obtained with a Zeiss Axiovert 200M microscope at a 5x16 magnifi cation equipped with a Q-imaging monochrome HR Retiga EX 1350 camera Recorded intensity images had a resolution of 159 μm 2 pixel and were stored in 8 bit TIFF format We used the fi ve frame setting with background correction as described by Shribak and Oldenbourg ( 2003 ) with a swing of 003 [-] Th e recorded images were analysed for predominant collagen fi bril orientation and tis-sue retardance with custom written scripts implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005)
A representative section with a width of 170 pixels that reaches from the tidemark to the articular surface was extracted from the images Th e results were averaged over this width and are presented as a function of dimensionless height after interpolation to 400 equidistant points Th e azimuth was expressed with respect to the articular surface Average retardance was obtained by taking the arithmetic mean of the 170 pixels and used to determine the position of the birefringence minimum in the tran-sitional zone To obtain an average orientation over these 170 pixels with predomi-nant fi bril azimuth the arithmetic means did not suffi ce (Upton and Fingleton 1989 )
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 359
We therefore introduced a retardance weighted average azimuth (φ) that is obtained by maximising the function
( ) ( )[ ] ( )( )uacuteucirc
ugraveecirceuml
eacutetimesD=aring
= nn
nIN
n ϕϕ
ϕϕsincos
sincos1
ϕ
for φ Th e summation over the absolute values of the inner dot product found the line
with smallest diff erence in angles compared to all lines N described by the azimuth values φ Th e retardance φ ( n ) is a measure for the amount of collagen that is associated with azimuth φ ( n ) Th rough multiplication of the inner dot product with the retardance we assigned a lower importance to azimuth values with a lower retardance which may indicate that 1) there is fewer collagen in this pixel or that 2) the predomi-nant orientation belongs to a collagen structure with a low level of anisotropy or 3) a combination of these two Maximisation of equation (1) was done with Matlabrsquos built in function lsquofminbndrsquo on a interval of 0 lt φ le π Final curves for each site were averages from two separate qPLM slices taken from each sample
Results
SEM study
In the older animals SEM images show that the collagen fi brils exhibit structural aniso-tropy in varying degrees depending on the height in the cartilage Structural anisotropy is most clearly visible at the articular surface and at the tidemark In the foal however the cartilage appears more chaotic throughout the entire cartilage layer Figure 3 shows an example of the collagen structure close to the tidemark in the foal and in the adult animals Th e diff erence in structural anisotropy is also apparent from the power spectra the adult animal shows a more pronounced ellipse than the young animal When the spectrum resembled a circle more than an ellipse as in this example for the foal we rotated the image and repeated the analysis Th e observed predominant orientation then rotated with the image confi rming that we found an objective measure for orientation
Th e height dependent orientation patterns we fi nd in the foal diff er from those found in the other animals again particularly in the deep zone Figures 4 and 5 show these patterns for the foal and for the older animals In the foal the orientation of the fi brils appears to vary around 0deg (or 180deg) throughout the entire cartilage depth Visual inspection of the SEM images shows that the orientation we fi nd in the foal belongs to a near isotropic structure for h lt 08 see for instance fi g 3a Near the articular sur-face SEM images in the foal show an anisotropic structure as in the older animals
In the other animals an arcade like Benninghoff structure appears collagen is aligned perpendicularly to the subchondral bone in the deep zone (approximately 80 of the tissue height) then curves away to form an arcade and is fi nally aligned with the articular surface
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
358 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
surface Because of the small sample size we treated all measurements independently for the presentation of height dependent results
Quantitative polarised light microscopy (qPLM)
Eight forelimbs of six horses with known age were collected from the Faculty of Veteri-nary Medicine of the University of Utrecht (Th e Netherlands) and an additional four forelimbs from two adult horses were collected from a local abattoir (VOF Paardenslachterij Nijkerk Th e Netherlands) see table 1 for details Forelimbs provided by Utrecht University were obtained frozen and were thawed at 5 degC overnight before processing Limbs collected from the abattoir were fresh and processed on the day of slaughter
Skin and subcutaneous tissue were removed and the metacarpophalangeal joint was carefully opened Split line patterns were created on the articular surface of the distal metacarpus with a sharp round needle charged with Indian ink Th e needle was inserted perpendicular to the articular surface at 2 mm intervals and excess ink was removed by rinsing Th e resulting split line pattern was recorded with a Nikon D-100 digital camera with a Micro-Nikon 55 mm objective
Samples were taken from the sagittal ridge (SR) the distal part of the medial side (MDi) and lateral side (LDi) and dorsal parts of the medial side (MDo) and lateral side (LDo) see fi gure 1 A dentist drill was used to introduce rectangular carvings and from these full depth cartilage plugs (ie including a piece of the subchondral bone) were extracted using a chisel Th ese samples were fi xed with formalin decalcifi ed with EDTA (10 EDTA pH 74) for two weeks washed and infi ltrated with sucrose (25 sucrose on PBS) overnight snap frozen in liquid nitrogen and stored at -80 degC until further processing and fi nally cut parallel to the superfi cial split lines to 5 μm thick histological slices with a cryostat (Reichert 2800N)
Two macroscopically normal histological samples for each sample site were mounted with water and analysed with the LC-PolScope system for qPLM (Oldenbourg and Mei 1995 Oldenbourg 2004) Images were obtained with a Zeiss Axiovert 200M microscope at a 5x16 magnifi cation equipped with a Q-imaging monochrome HR Retiga EX 1350 camera Recorded intensity images had a resolution of 159 μm 2 pixel and were stored in 8 bit TIFF format We used the fi ve frame setting with background correction as described by Shribak and Oldenbourg ( 2003 ) with a swing of 003 [-] Th e recorded images were analysed for predominant collagen fi bril orientation and tis-sue retardance with custom written scripts implemented in Matlab (version 72R14 Th e MathWorks Inc 1984-2005)
A representative section with a width of 170 pixels that reaches from the tidemark to the articular surface was extracted from the images Th e results were averaged over this width and are presented as a function of dimensionless height after interpolation to 400 equidistant points Th e azimuth was expressed with respect to the articular surface Average retardance was obtained by taking the arithmetic mean of the 170 pixels and used to determine the position of the birefringence minimum in the tran-sitional zone To obtain an average orientation over these 170 pixels with predomi-nant fi bril azimuth the arithmetic means did not suffi ce (Upton and Fingleton 1989 )
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 359
We therefore introduced a retardance weighted average azimuth (φ) that is obtained by maximising the function
( ) ( )[ ] ( )( )uacuteucirc
ugraveecirceuml
eacutetimesD=aring
= nn
nIN
n ϕϕ
ϕϕsincos
sincos1
ϕ
for φ Th e summation over the absolute values of the inner dot product found the line
with smallest diff erence in angles compared to all lines N described by the azimuth values φ Th e retardance φ ( n ) is a measure for the amount of collagen that is associated with azimuth φ ( n ) Th rough multiplication of the inner dot product with the retardance we assigned a lower importance to azimuth values with a lower retardance which may indicate that 1) there is fewer collagen in this pixel or that 2) the predomi-nant orientation belongs to a collagen structure with a low level of anisotropy or 3) a combination of these two Maximisation of equation (1) was done with Matlabrsquos built in function lsquofminbndrsquo on a interval of 0 lt φ le π Final curves for each site were averages from two separate qPLM slices taken from each sample
Results
SEM study
In the older animals SEM images show that the collagen fi brils exhibit structural aniso-tropy in varying degrees depending on the height in the cartilage Structural anisotropy is most clearly visible at the articular surface and at the tidemark In the foal however the cartilage appears more chaotic throughout the entire cartilage layer Figure 3 shows an example of the collagen structure close to the tidemark in the foal and in the adult animals Th e diff erence in structural anisotropy is also apparent from the power spectra the adult animal shows a more pronounced ellipse than the young animal When the spectrum resembled a circle more than an ellipse as in this example for the foal we rotated the image and repeated the analysis Th e observed predominant orientation then rotated with the image confi rming that we found an objective measure for orientation
Th e height dependent orientation patterns we fi nd in the foal diff er from those found in the other animals again particularly in the deep zone Figures 4 and 5 show these patterns for the foal and for the older animals In the foal the orientation of the fi brils appears to vary around 0deg (or 180deg) throughout the entire cartilage depth Visual inspection of the SEM images shows that the orientation we fi nd in the foal belongs to a near isotropic structure for h lt 08 see for instance fi g 3a Near the articular sur-face SEM images in the foal show an anisotropic structure as in the older animals
In the other animals an arcade like Benninghoff structure appears collagen is aligned perpendicularly to the subchondral bone in the deep zone (approximately 80 of the tissue height) then curves away to form an arcade and is fi nally aligned with the articular surface
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 359
We therefore introduced a retardance weighted average azimuth (φ) that is obtained by maximising the function
( ) ( )[ ] ( )( )uacuteucirc
ugraveecirceuml
eacutetimesD=aring
= nn
nIN
n ϕϕ
ϕϕsincos
sincos1
ϕ
for φ Th e summation over the absolute values of the inner dot product found the line
with smallest diff erence in angles compared to all lines N described by the azimuth values φ Th e retardance φ ( n ) is a measure for the amount of collagen that is associated with azimuth φ ( n ) Th rough multiplication of the inner dot product with the retardance we assigned a lower importance to azimuth values with a lower retardance which may indicate that 1) there is fewer collagen in this pixel or that 2) the predomi-nant orientation belongs to a collagen structure with a low level of anisotropy or 3) a combination of these two Maximisation of equation (1) was done with Matlabrsquos built in function lsquofminbndrsquo on a interval of 0 lt φ le π Final curves for each site were averages from two separate qPLM slices taken from each sample
Results
SEM study
In the older animals SEM images show that the collagen fi brils exhibit structural aniso-tropy in varying degrees depending on the height in the cartilage Structural anisotropy is most clearly visible at the articular surface and at the tidemark In the foal however the cartilage appears more chaotic throughout the entire cartilage layer Figure 3 shows an example of the collagen structure close to the tidemark in the foal and in the adult animals Th e diff erence in structural anisotropy is also apparent from the power spectra the adult animal shows a more pronounced ellipse than the young animal When the spectrum resembled a circle more than an ellipse as in this example for the foal we rotated the image and repeated the analysis Th e observed predominant orientation then rotated with the image confi rming that we found an objective measure for orientation
Th e height dependent orientation patterns we fi nd in the foal diff er from those found in the other animals again particularly in the deep zone Figures 4 and 5 show these patterns for the foal and for the older animals In the foal the orientation of the fi brils appears to vary around 0deg (or 180deg) throughout the entire cartilage depth Visual inspection of the SEM images shows that the orientation we fi nd in the foal belongs to a near isotropic structure for h lt 08 see for instance fi g 3a Near the articular sur-face SEM images in the foal show an anisotropic structure as in the older animals
In the other animals an arcade like Benninghoff structure appears collagen is aligned perpendicularly to the subchondral bone in the deep zone (approximately 80 of the tissue height) then curves away to form an arcade and is fi nally aligned with the articular surface
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
360 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
qPLM study
Figure 6 shows the azimuth results for the 5 anatomical sites for three animals of ages 0 months 45 months and 10 years We can see that these three animals show diff erent patterns from each other and that within the stillborn (0 months) and adult animal there seems to be little variation with anatomical site Th e animal of age 45 months does show some variation between the anatomical sites
When we collect all orientation pattern results again two distinct collagen orienta-tion profi les are found in the qPLM study We cannot fi nd diff erences between left or right limbs or between the anatomical sites and therefore pool the data as independent measurements (due to the sample size as in the SEM study) Th e fi rst pattern belongs to the stillborn animals and is shown in fi gure 7 Here we fi nd that the predominant fi bril orientation is aligned with the articular surface over the complete cartilage height
(a) (b)
Figure 3 Example of SEM results close to the tidemark in the young and adult animal Top (panel a and b) SEM images at a magnifi cation of 10000x bottom panel c and d) binary representation of the centre of the power spectrum (white on a gray background) with detected predominant orientation (black line) left (panel a and c) results for the foal right (panel b and d) results for an adult horse
(c) (d)
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 361
Figure 4 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for three samples from the foal ( n = 3)
Figure 5 Average predominant fi bril orientation plusmn standard deviation from the SEM study as a function of dimensionless height for all samples in the animals 1 year and older ( n = 14)
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
362 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
in these animals Th e second pattern is found in the animals of age 10 months and older and is shown in fi gure 8 Th ese animals show an arcade like Benninghoff struc-ture with fi brils perpendicular to the subchondral bone in the deep zone that bend in
Figure 6 Predominant collagen fi bril orientation in the stillborn animals (dotted) the animal of 45 months old (dashed) and the animal of 10 years old (solid) for the fi ve diff erent sample sites (a) Mediodorsal (b) Laterodorsal (c) Mediodistal (d) Laterodistal and (e) Sagittal ridge
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 363
Figure 7 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the stillborn animals ( n = 15)
Figure 8 Average predominant fi bril orientation plusmn standard deviation (upper half of the graph left axis) and corresponding average retardance plusmn standard deviation (bottom half of the graph right axis) from the qPLM study as a function of dimensionless height for all sites in the animals age 10 months and older ( n = 40)
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
364 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
the transitional zone until they are aligned parallel with the articular surface Th e pat-terns we observe in the animal of age 45 months show a mixture of these two primary patterns ( fi g 6 )
Figures 7 and 8 show the average retardance patterns that correspond to these orien-tation patterns Retardance appears to be larger in the older than in the stillborn ani-mals Th is apparent rise in retardance is accompanied by an increase in variation that is just as high Th e minimum in the retardance pattern that is associated with the transitional zone can be found in all samples and the height of this minimum d m is collected in table 2
Finally we present the overall cartilage thickness as a function of age in table 3 It shows signifi cantly higher values for the stillborn animals than in the other animals From age 45 months onwards no signifi cant diff erences in cartilage thickness are detected
Discussion
In both parts of the study we were able to confi rm the Benninghoff model in our sam-ple of animals of age 10 months and older Strikingly we fi nd in the qPLM study that collagen is predominantly arranged parallel to the articular surface throughout the
Table 2 Dimensionless height of the retardance minimum in the transitional zone d m
per animal site and leftright limb and the mean d
mdash m per age (in months) Th e star for the horses of age 0 indicates that this is the mean
for both horses of age 0 taken together not for the individual animals
Table 3 Cartilage thickness as a function of age in the qPLM study Th e last column collects the data for the two adult animals and the animal with age 120 months
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 365
entire cartilage depth in the stillborn animal At 45 months we seem to fi nd interme-diate results between this parallel structure and the adult Benninghoff structure Th is marked result is confi rmed in the SEM study were we observe such a parallel arrange-ment in the foal Even if the exact age of this foal is not known and the variation we fi nd is large the results suggest that the Benninghoff structure is not found in this animal whereas it is found in the SEM experiments with older animals Th e SEM experiments further show that the predominant orientation in the young animals belongs to collagen fi brils with a nearly isotropic architecture
Another interesting fi nding is that the minimum in retardance that is associated with the isotropic transitional zone is already present in the stillborn animals while no zonal diff erentiation is apparent from the orientation patterns Th is onset of a transi-tional zone without a change in collagen fi bril orientation has also been reported by Hughes et al ( 2005 ) in 7 day old mouse AC Th is indicates that some degree of struc-tural diff erentiation over the cartilage height may already be present at birth It also shows that the use of orientation patterns for determination of the diff erent zones as recently proposed by Julkunen et al ( 2007 ) will not suffi ce in young animals
Brama and coworkers performed biochemical studies on the developing AC in the same equine joint Th ey did not investigate depth dependent or structural parameters but concentrated on compositional diff erences as a function of site age and exercise Looking at the data they present for collagen content we fi nd that they report a large increase in collagen content up to an age of 5 months (Brama et al 2000b ) and little or no adaptation after this age (Brama et al 2000a 1999 ) Furthermore they report site related diff erences in a variety of parameters that do not exist at birth but are fully developed at an age of 5 months (Brama et al 2000b 2002 ) Th e current study con-centrates on height dependent collagen structure and the results indicate that in line with the work by Brama and coworkers structural changes in the collagen fi bre net-work also occur in the fi rst 5 months after birth
Brama et al ( 2000b c ) report compositional diff erences between the chosen sites from an age of 5 months onwards It is very likely that the structural changeover in developing AC is a function of biomechanical loading as is the compositional change-over Th erefore diff erences in collagen structure between sites that are subject to diff er-ent loading patterns would not be unexpected In this study however the number of samples probably is too small to show this in terms of collagen structure Because of the small sample size it is conceivable that diff erences in loading patterns between animals may have confounded diff erences in loading patterns between the sites within an animal We do fi nd that cartilage thickness is signifi cantly higher in the stillborn animals than in the older animals which is in line with the results of Brommer et al ( 2005 ) and Firth and Greydanus ( 1987 ) However we add that this is due to one of the two stillborn animals that had a very thick cartilage layer (gt 2 mm) Cartilage thick-ness in the second stillborn animal is in line with that found in the other animals
Caveats in this preliminary study are the small number of animals we were able to use the temporal distribution of the sample points and the limited knowledge on the ages and loading history of the animals Th e diff erence in cartilage thickness between the two stillborn animals for instance may be explained by diff erences in the
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
366 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
developmental stage of the fetuses since we do not know the gestational age of these animals Furthermore we had to resign ourselves to the use of diff erent breeds of horses which is likely to have increased variations we fi nd between animals and we had no control over the ages of the animals that we could use Still we are confi dent that the results we report on collagen orientation in stillborn and adult horses will hold since these follow unambiguously from both age categories even when little or no diff erences can be found in the other parameters
As such these fi ndings suggest implications for AC epigenetics AC remodels during growth and maturation from a homogenous tissue to a tissue with site specifi c compo-sition (Brama et al 2000b c Hunziker et al 2007b ) and mechanical properties (Brommer et al 2005 ) It does so under the influence of mechanical loads (Brama et al 1999 Helminen et al 2000 Murray et al 2001 ) that vary over the joint surfaces (Brama et al 2001 Hodge et al 1986 Palmer et al 1994 ) Th e collagen fi bre network plays an important role in the mechanical properties of AC in general (Arokoski et al 1999 Kiviranta et al 2006 Korhonen et al 2002 Wilson et al 2005a ) and in the diff erentiation in cartilage mechanics over the tissue height global cartilage loads will result in diff erent strains over the cartilage height due to its depth dependent structure and composition (Schinagl et al 1997 Wilson et al 2006b 2007 ) In fact the relationship between cartilage loads and depth dependent collagen structure is strong enough for Wilson et al ( 2006a ) to be able to predict depth and site dependent collagen structure as a function of global cartilage loads
Given that the collagen fi bre network in the newborn animal is nearly uniform over the cartilage height the question now arises to what degree the depth dependent mechanical properties manifest in the newborn animal Also one might ask to what degree this is the result of the diff erences in collagen fi bre network to what degree such depth dependent mechanical properties in the newborn animal are necessary to develop the distinct depth dependent collagen structure at maturity and what the role is of the other cartilage components in the development of cartilage mechanics and structure We intent to address these questions in future work
Conclusions
In the cartilage on the equine distal metacarpus we fi nd diff erent collagen structures in young (2 stillborn animals) and older animals (9 animals older than 10 months) Th e transition between the two structures takes place in the fi rst months after birth as we see a transitional structure in a 45 months old animal
In the two stillborn animals that we analysed the predominant collagen fi bril orien-tation is parallel to the articular surface throughout the entire cartilage depth Th e minimum in the retardance patterns suggest the onset of a transitional zone but this zone is not yet apparent from fi bril arrangement
In the animals older than 10 months that we analysed we fi nd the classical Benninghoff structure with the three zones In the deep zone collagen fi brils are pre-dominantly arranged perpendicular to the subchondral bone in the superfi cial zone they are aligned parallel to the articular surface In the transitional zone we fi nd a low
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 367
retardance which indicates a less anisotropic collagen structure and that the azimuth patterns describe an arcade like structure
Acknowledgements
We thank Prof R Oldenbourg for kindly making his code and algorithm for analysis of the raw PLM results available to us We thank VOF Paardenslagerij Nijkerk Th e Netherlands and Prof Dr PR van Weeren and Dr H Brommer at the Faculty of Veterinary Medicine of Utrecht University Th e Netherlands for the generous gift of the equine limbs
From Wageningen University we thank Mr A van Aelst at the Wageningen Electron Microscopy Centre for the help with the SEM experiments and Dr N de Ruijter at the Plant Cell Biology group for the help with the PLM experiments
References
Alhadlaq H Xia Y Moody J amp Matyas J ( 2004 ) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy Ann Rheum Dis 63 709 ndash 717
Arokoski JP Hyttinen MM Helminen HJ amp Jurvelin JS ( 1999 ) Biomechanical and structural characteristics of canine femoral and tibial cartilage J Biomed Mater Res 48 99 ndash 107
Arokoski JP Hyttinen MM Lapvetelaumlinen T Takaacutecs P Kosztaacuteczky B Moacutedis L Kovanen V amp Helminen HJ ( 1996 ) Decreased birefringence of the superfi cial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarised light microscopy Ann Rheum Dis 55 253 ndash 264
Bennett SH ( 1950 ) Methods applicable to the study of both fresh and fi xed materials - the microscopi-cal investigation of biological materials with polarized light in McClung Jones R (Ed) McClungrsquos handbook of microscopical technique Cassel and company limited chap IX 3rd ed 591 ndash 677
Benninghoff A ( 1925 ) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion Zweiter Teil Der Aufbau des Gelenkknorpels in seinen Beziehungen zur Funktion Z Zellforsch 2 783 ndash 862
Bi X Li G Doty S amp Camacho N ( 2005 ) A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS) Osteoarthritis Cartilage 13 1050 ndash 1058
Bland YS amp Ashhurst DE ( 1996a ) Changes in the content of the fi brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing Histochem J 28 265 ndash 274
Bland YS amp Ashhurst DE ( 1996b ) Development and ageing of the articular cartilage of the rabbit knee joint distribution of the fi brillar collagens Anat Embryol (Berl) 194 607 ndash 619
Brama P Karssenberg D Barneveld A amp van Weeren P ( 2001 ) Contact areas and pressure distribu-tion on the proximal articular surface of the proximal phalanx under sagittal plane loading Equine Vet J 33 26 ndash 32
Brama P Tekoppele J Bank R Barneveld A Firth E amp van Weeren P ( 2000a ) Th e influence of strenuous exercise on collagen characteristics of articular cartilage in Th oroughbreds age 2 years Equine Vet J 32 551 ndash 554
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2000b ) Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year Equine Vet J 32 217 ndash 221
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
368 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
Brama P TeKoppele J Bank R Barneveld A amp van Weeren P ( 2002 ) Development of biochemical heterogeneity of articular cartilage influences of age and exercise Equine Vet J 34 265 ndash 269
Brama P Tekoppele J Bank R Karssenberg D Barneveld A amp van Weeren P ( 2000c ) Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint Equine Vet J 32 19 ndash 26
Brama P TeKoppele J Bank R van Weeren P amp Barneveld A (1999) Influence of diff erent exercise levels and age on the biochemical characteristics of immature equine articular cartilage Equine Vet J Suppl 55 ndash 61
Brommer H Brama P Laasanen M Helminen H van Weeren P amp Jurvelin J ( 2005 ) Functional adaptation of articular cartilage from birth to maturity under the influence of loading a biomechani-cal analysis Equine Vet J 37 148 ndash 154
Clark JM ( 1985 ) Th e organization of collagen in cryofractured rabbit articular cartilage a scanning electron microscopic study J Orthop Res 3 17 ndash 29
Clark JM ( 1991 ) Variation of collagen fi ber alignment in a joint surface a scanning electron microscope study of the tibial plateau in dog rabbit and man J Orthop Res 9 246 ndash 257
Firth E amp Greydanus Y ( 1987 ) Cartilage thickness measurement in foals Res Vet Sci 42 35 ndash 46 Han B Cole A Shen Y Brodie T amp Williams J ( 2002 ) Early alterations in the collagen meshwork
and lesions in the ankles are associated with spontaneous osteoarthritis in guinea-pigs Osteoarthritis Cartilage 10 778 ndash 784
Helminen HJ Hyttinen MM Lammi MJ Arokoski JP Lapvetelaumlinen T Jurvelin J Kiviranta I amp Tammi MI ( 2000 ) Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life a hypothesis J Bone Miner Metab 18 245 ndash 257
Hodge W Fijan R Carlson K Burgess R Harris W amp Mann R ( 1986 ) Contact pressures in the human hip joint measured in vivo Proc Natl Acad Sci USA 83 2879 ndash 2883
Hughes L Archer C amp ap Gwynn I ( 2005 ) Th e ultrastructure of mouse artic-ular cartilage collagen orientation and implications for tissue functionality A polarised light and scanning electron micro-scope study and review Eur Cell Mater 9 68 ndash 84
Hunziker E Kapfi nger E amp Geiss J ( 2007a ) Corrigendum to ldquoTh e structural architecture of adult mam-malian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal developmentrdquo [Osteoarthritis Cartilage 15 (2007) 403ndash413] Osteoarthritis Cartilage
Hunziker E Kapfi nger E amp Geiss J ( 2007b ) Th e structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development Osteoarthritis Cartilage 15 403 ndash 413 corrigendum in Hunziker et al (2007a)
Hyttinen M Arokoski J Parkkinen J Lammi M Lapvetelaumlinen T Mauranen K Kiraacutely K Tammi M amp Helminen H ( 2001 ) Age matters collagen birefringence of superfi cial articular car-tilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading Osteoarthritis Cartilage 9 694 ndash 701
Julkunen P Kiviranta P Wilson W Jurvelin JS amp Korhonen RK ( 2007 ) Characterization of articu-lar cartilage by combining microscopic analysis with a fi bril-reinforced fi nite-element model J Biomech 40 1862 ndash 1870
Kaumlaumlb M ap Gwynn I amp Noumltzli H ( 1998 ) Collagen fi bre arrangement in the tibial plateau articular cartilage of man and other mammalian species J Anat 193 (Pt 1) 23 ndash 34
Kim A Lakshman N amp Petroll WM ( 2006 ) Quantitative assessment of local collagen matrix remod-eling in 3-D culture the role of Rho kinase Exp Cell Res 312 3683 ndash 3692
Kiraacutely K Hyttinen M Lapvetelaumlinen T Elo M Kiviranta I Dobai J Moacutedis L Helminen H amp Arokoski J ( 1997 ) Specimen preparation and quantifi cation of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy Histochem J 29 317 ndash 327
Kiraacutely K Hyttinen M Parkkinen J Arokoski J Lapvetelaumlinen T Toumlrroumlnen K Kiviranta I amp Helminen H ( 1998 ) Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading Anat Rec 251 28 ndash 36
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
M C van Turnhout et al Animal Biology 58 (2008) 353ndash370 369
Kiviranta P Rieppo J Korhonen RK Julkunen P Toumlyraumls J amp Jurvelin JS ( 2006 ) Collagen net-work primarily controls Poissonrsquos ratio of bovine articular cartilage in compression J Orthop Res 24 690 ndash 699
Korhonen R Wong M Arokoski J Lindgren R Helminen H Hunziker E amp Jurvelin J ( 2002 ) Importance of the superfi cial tissue layer for the indentation stiff ness of articular cartilage Med Eng Phys 24 99 ndash 108
Li C Pruitt LA amp King KB ( 2006 ) Nanoindentation diff erentiates tissue-scale functional properties of native articular cartilage J Biomed Mater Res A 78 729 ndash 738
Massoumian F Juškaitis R Neil M amp Wilson T ( 2003 ) Quantitative polarized light microscopy J Microsc 209 13 ndash 22
Murray R Birch H Lakhani K amp Goodship A ( 2001 ) Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specifi c manner Osteoarthritis Cartilage 9 625 ndash 632
Oldenbourg R amp Mei G ( 1995 ) New polarized light microscope with precision universal compensator J Microsc 180 140 ndash 147
Oldenbourg R ( 2004 ) Polarization microscopy with the LC-microscope in RD Goldman amp DL Spector (Eds) Live Cell Imaging A Laboratory Manual Cold Spring Harbor Laboratory Press chap 13 205 ndash 238
Palmer JL Bertone AL amp Litsky A ( 1994 ) Contact area and pressure distribution changes of the equine third carpal bone during loading Equine Vet J 26 197 ndash 202
Petroll WM Cavanagh HD Barry P Andrews P amp Jester JV ( 1993 ) Quantitative analysis of stress fi ber orientation during corneal wound contraction J Cell Sci 104 353 ndash 363
Rieppo J Toumlyraumls J Nieminen MT Kovanen V Hyttinen MM Korhonen RK Jurvelin JS amp Helminen HJ ( 2003a ) Structure-function relationships in enzymatically modifi ed articular carti-lage Cells Tissues Organs 175 121 ndash 132
Rieppo J Hallikainen J Jurvelin J Helminen H amp Hyttinen M ( 2003b ) Novel quantitative polari-zation microscopic assessment of cartilage and bone collagen birefringence orientation and aniso-tropy Transactions of the ORS 28 paper nr 0570 at the 49th Annual Meeting of the Orthopaedic Research Soceity New Orleans Louisiana
Ross S Newton R Zhou YM Haff egee J Ho MW Bolton J amp Knight D ( 1997 ) Quantitative image analysis of birefringent biological material J Microsc 187 62 ndash 67
Saadat E Lan H Majumdar S Rempel DM amp King KB ( 2006 ) Long-term cyclical in vivo load-ing increases cartilage proteoglycan content in a spatially specifi c manner an infrared microspectro-scopic imaging and polarized light microscopy study Arthritis Res Th er 8 R147
Schinagl RM Gurskis D Chen AC amp Sah RL ( 1997 ) Depth-dependent confi ned compression modulus of full-thickness bovine articular cartilage J Orthop Res 15 499 ndash 506
Shribak M amp Oldenbourg R ( 2003 ) Techniques for fast and sensitive measurements of two-dimen-sional birefringence distributions Appl Opt 42 3009ndash 3017
Speer DP amp Dahners L ( 1979 ) Th e collagenous architecture of articular cartilage Correlation of scan-ning electron microscopy and polarized light microscopy observations Clin Orthop Relat Res 267 ndash 275
Ugryumova N Attenburrow DP Winlove CP amp Matcher SJ ( 2005 ) Th e collagen structure of equine articular cartilage characterized using polarizationsensitive optical coherence tomography J Phys D Appl Phys 38 2612 ndash 2619
Upton GJ amp Fingleton B ( 1989 ) Spatial data analysis by example ndash Volume 2 Categorical and direc-tional data Wiley series in probability and mathematical statistics John Wiley and Sons
Wilson W van Donkelaar C van Rietbergen B amp Huiskes R ( 2005a ) A fi bril-reinforced poroviscoe-lastic swelling model for articular cartilage J Biomech 38 1195 ndash 1204 erratum in J Biomech 38(10)2138ndash2140 see Wilson et al (2005b)
Wilson W van Donkelaar C van Rietbergen B Ito K amp Huiskes R ( 2005b ) Erratum to ldquoStresses in the local collagen network of articular cartilage a poroviscoelastic fi bril-reinforced fi nite element studyrdquo [Journal of Biomechanics 37 (2004) 357ndash366] and ldquoA fi bril-reinforced poroviscoelastic
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
370 M C van Turnhout et al Animal Biology 58 (2008) 353ndash370
swelling model for articular cartilagerdquo [Journal of Biomechanics 38 (2005) 1195ndash1204] J Biomech 38 2138 ndash 2140
Wilson W Driessen N van Donkelaar C amp Ito K ( 2006a ) Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm Osteoarthritis Cartilage 14 1196 ndash 1202
Wilson W Huyghe J amp van Donkelaar C ( 2006b ) A composition-based cartilage model for the assess-ment of compositional changes during cartilage damage and adaptation Osteoarthritis Cartilage 14 554 ndash 560
Wilson W Huyghe J amp van Donkelaar C ( 2007 ) Depth-dependent compressive equilibrium proper-ties of articular cartilage explained by its composition Biomech Model Mechanobiol 6 43 ndash 53
Xia Y Ramakrishnan N amp Bidthanapally A ( 2007 ) Th e depth-dependent anisotropy of articular car-tilage by Fourier-transform infrared imaging (FTIRI) Osteoarthritis Cartilage 15 780 ndash 788
Xia Y Moody JB amp Alhadlaq H ( 2002 ) Orientational dependence of T 2 relaxation in articular carti-lage A microscopic MRI ( μ MRI) study Magn Reson Med 48 460 ndash 469
Xia Y Moody JB Alhadlaq H amp Hu J ( 2003 ) Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution J Magn Reson Imaging 17 365 ndash 374
