Modifications to Nano‐ and Microstructural Qualityand the Effects on Mechanical Integrity in Paget’sDisease of BoneElizabeth A Zimmermann,1,2 Till Köhne,1 Hrishikesh A Bale,3 Brian Panganiban,3 Bernd Gludovatz,2
Joszef Zustin,4 Michael Hahn,1 Michael Amling,1 Robert O Ritchie,2,3 and Björn Busse1,2
1Department of Osteology and Biomechanics, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany2Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA3Department of Materials Science and Engineering, University of California, Berkeley, CA, USA4Institute of Pathology, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany
KEY WORDS: PAGET’S DISEASE OF BONE; PATHOMECHANISM; FRACTURE RISK; BONE QUALITY; MECHANICAL PROPERTIES; COLLAGENCHARACTERISTICS
Introduction
Paget’s disease of bone (PDB) was first described by Sir JamesPaget in 1876 after observing distinct proportion changes
and deformities in patients’ bones.(1) Today, PDB is the secondmost common bone disease behind osteoporosis. The disease isusually triggered after the age of 50 years possibly by geneticand/or environmental factors.(2,3) PDB has a high prevalence inwestern European countries as well as regions around the worldformerly colonized by people fromwestern European descent.(4,5)
PDB localizes at one or more skeletal sites, most commonly thepelvis, spine, femur, and tibia,(2,3,5–7) leading to outwardlyobservable abnormalities in the bone’s size and shape. Althoughapproximately 90% of patients do not have any symptoms, 10%of patients with PDB suffer from pain in bones, joints, andmuscles; headaches; hearing loss; gait disturbances; compression
of nerves; local temperature increases; and secondary osteoar-thritis.(5,8–10) However, the hallmark diagnostic feature of PDBunder X‐ray examination is the reorganization of the boneemphasized through a combination of osteolytic, sclerotic, anddeformed bone regions indicating hypervascularity, trabeculardensification, and cortical thickening (Fig. 1A).(8,11,12) Thispronounced disease pattern is accompanied by blood serummarkers of bone remodeling showing abnormally high alkalinephosphatase activity and bone‐specific alkaline phosphataseactivity,(10,13) which are indicators of excessive bone remodeling.
At the bone cellular level, where previously a delicate balanceof bone resorption by osteoclast cells and bone deposition byosteoblast cells produced healthy bone, changes in the osseouscell activity after the onset of PDB reflect a defective boneremodeling pattern. The appearance of abnormally shapedosteoclasts, so called “giant osteoclasts” characteristic of PDB, are
Received in original form June 11, 2014; revised form August 4, 2014; accepted August 7, 2014. Accepted manuscript online August 12, 2014.Address correspondence to: Björn Busse, PhD, Department of Osteology and Biomechanics, University Medical Center, Lottestrasse 59, 22529 Hamburg,Germany. E‐mail address: [email protected]
related to enhanced bone resorption followed by osteoblasticoverstimulation causing increased bone volume,(3,14) whichcontributes to the typical enlargement of the affected bones.Essentially, both increased osteoclast and osteoblast activitycause the striking high bone turnover in PDB.(3,15) As a result,increased proportions of rapidly synthesized and non‐organizedcollagen matrix are deposited followed by a brief mineralizationperiod,(15) producing a bonematrix with a structure resembling amosaic of woven bone.(15–17)
The changes in bone remodeling as well as the resultingoutwardly observable changes in whole‐bone geometry atdiseased skeletal sites indicate a shift in bone quality. Bonequality describes the integrity of bone’s hierarchical structuralfeatures (Fig. 1B), which span collagen molecules (�300nm) andmineral nanoparticles (�10nm) at small‐length scales to cylindri-cal features called osteons at the size‐scale of 100s of microns incortical bone to the interconnecting architecture in trabecularbone. Bone’s mechanical integrity arises from the quality of thebone structure and how it resists deformation and fracture.(18–21)
The hierarchical structure contributes to the mechanical integrityin terms of intrinsic and extrinsic mechanisms that resistdeformation and fracture. Specifically, the intrinsic materialresistance results in bone’s inherent stiffness, strength, andresistance to crack initiation. The intrinsic resistance originatesfrom the composition and assembly of bone’s constituents atsmall‐length scales and how these features promote or restrictplasticity (whereas elastic deformation refers to the stretching ofbonds, plastic or inelastic deformation implies permanent,irreversible deformation). In bone, the primary intrinsic mecha-nisms are thought to befibrillar sliding and sacrificial bonding, andmodifications, eg, in the cross‐linking or mineralization profiles,are thought to impact the generation of plasticity at this length‐scale.(22) In contrast, the extrinsic material resistance results inbone’s resistance to the growth of a crack. The extrinsic resistanceoriginates from larger‐length scales on the microstructural scalethat are large enough to stop/interfere with crack growth. Ineffect, extrinsic mechanisms shield the growth of cracks throughcrack deflection or bridging mechanisms.(22)
Epidemiological studies have quantified fracture risk in cohortsof patients with PDB.(10,23–26) Various studies have found a slightto no increase in overall fracture risk (because PDB localizes at oneor more skeletal sites, it is important to differentiate betweenfracture risk at pathological bone sites and overall fracture risk) inpatients with PDB.(23,24) However, higher rates of fracture havebeen reported through pathological bone, even after bisphosph-onate treatment.(23,26) Even though fracture events at pathologi-cal skeletal sites are uncommon (occurring in �2% of patients),fracture does represent a concern in patients with PDB and maybe accompanied by further fracture‐related complications, suchas subsequent fracture, nonunion of fractured site, andpseudofracture or fissure fractures.(23,24,26–28) Fractures at patho-logical skeletal sites are commonly transverse (ie, “chalk‐stick”fractures) and preceded by the presence of incomplete fractures,termed pseudofractures or fissure fractures.(12,29) Regions withsevere bowing and deformity commonly contain the fissurefractures, which occur on the convex side of the bone undertensile stress and contribute to the sensation of bone pain.(28,30,31)
Because PDB clearly disrupts the mechanical integrity of bonetissue, leading to bowing, deformity, and fissure fractures inclinical cases, our aimherewas to use a cohort of human iliac crestbone biopsies from control and PDB cases to experimentallycharacterize the structure, composition, and mechanical proper-ties. Thus, modifications to the multiscale bone structure in PDB
Fig. 1. Hierarchical structure of bone. (A) Radiologic signs of PDB in theareamarkedby the yellow arrow,where the bone has a larger density andsize. (B) In this study, biopsies from the iliac crest were used to analyze thestructural and mechanical properties in control and PDB cases. At thisskeletal site, the bone architecture consists of a dense cortical shellsurrounding a porous trabecular core. At themicrostructural length‐scale,the cortical bone consists of osteons, which have a hypermineralizedcement line delineating their outer boundary and lamellae concentricallysurrounding a central vascular cavity termed the Haversian canal. Thelamellae are composed of arrays of fibers, which are composed of fibrilarrays. The fibril is a composite of collagen molecules and mineralplatelets. PDB¼Paget’s disease of bone.
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were related to the mechanical properties to investigate thefracture risk and the origins of reduced mechanical integrity (ie,bowing, deformities, cracking) commonly found in clinical cases.
Materials and Methods
Study design
The objective of this study was to characterize the alterations tothe structure and composition as well as the mechanicalproperties of bone from healthy patients and those with PDB.Here, 49 control and 49 PDB methylmethacrylate (MMA)‐embedded iliac crest biopsies were obtained from the HamburgBone Registry at the University Medical Center, Hamburg‐Eppendorf, Germany. The control samples stem from a previousbone histomorphometry study and did not show any sign ofmineralization defects or pathologic tissue.(32,33) All individualssuffering from cancer, renal diseases, primary hyperparathyroid-ism, and/or showing any other circumstances, such as immobili-zation or hospitalization, potentially leading to secondary bonediseases, were excluded from the study. The PDB biopsies weretaken to diagnose the source of abnormal X‐rays and/orscintigraphy in patients with bone pain and/or suspicion ofbreast and prostate cancer. Therefore, the PDB cases exhibitedpathological tissue at this skeletal region and had previously notbeen treated for PDB. The study was approved by the LawrenceBerkeley National Laboratory (BUA‐120). The PDB cohortconsisted of 19 females and 30 males with an average age of72.2� 7.3 years. The control cohort consisted of 16 females and33 males with an average age of 59.3� 7.3 years.
Histomorphometry
Prior to embedding, the sampleswere first fixed in 4%phosphate‐buffered formaldehyde and then dehydrated in an ascendingethanol series (80%, 90%, 94%, 96%, 100%ethanol). Undecalcifiedspecimens were infiltrated in two steps with MMA solutions(Merck, Darmstadt, Germany). Afterward, the polymerization ofdestabilized MMA augmented with N,N dimethyl‐p‐toluidine(DMPT) as an initiator/catalyst took place under a N2 saturatedatmosphere. The polymerization of resin in all of the samples’voids took place at a temperature of 4 °C. Static histomorphom-etry was performed on toluidine blue or Giemsa‐stainedundecalcified sections. The following parameters were measuredaccording to ASBMR standards(34) with an Osteo‐Measurehistomorphometry system (Osteometrics, Atlanta, GA, USA) anda Zeiss microscope (Carl Zeiss, Jena, Germany): bone volume(BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N),trabecular separation (Tb.Sp), osteoid volume (OV/BV), osteoidsurface (OS/BS), osteoclast number (N.Oc/B.Pm), osteoclastsurface (Oc.S/BS), osteoblast number (N.Ob/B.Pm), and osteoblastsurface (Ob.S/BS).
Quantitative backscattered electron imaging
Although undecalcified histology is able to capture soft tissueand bone cells, backscattered electron imaging has the capabilityto focus on the different degrees of mineralization within thebone tissue.(35–38) Here, the bone mineral density distribution(BMDD) was measured via quantitative backscattered electronimaging (qBEI) on 46 controls and 49 PDB cases. The measure-ments were performed at 20 kV and 580 pA (LEO 435 VP; LeoElectron Microscopy Ltd., Cambridge, UK) with a constantworking distance of 20mm using a solid state backscattered
electron detector (BSE Detector, Type 202; K.E. DevelopmentsLtd., UK). The electron beamwas kept constant at 580 pA using aFaraday cup (MAC Consultants Ltd., UK). The signal amplification(brightness and contrast) was calibrated during the entireprocedure by keeping measurements of carbon and aluminumstandards (MAC Consultants Ltd., UK).(39) The gray‐level histo-grams of bone were standardized using a threshold routine(Image J 1.42; National Institutes of Health, Bethesda, MD, USA).The obtained gray values were transformed into calcium weightpercentages as described.(33,39) We evaluated the value (Camean), standard deviation (SD) (Ca width), and peak (Ca peak) ofthe calcium distribution, which refer to the mean calciumcontent, the heterogeneity of the calcium content, and the mostfrequent calcium content, respectively.(38,40) Additionally, wecalculated the mean value of the distributions’ 5th and 95thpercentiles, which were 16.54 and 27.15wt % Ca, respectively.For every distribution curve, we also evaluated the portion left ofthe mean 5th percentile (Ca low) and right of the mean 95thpercentile (Ca high). These BMDD parameters represent the areaof low and highly mineralized bone, respectively.
Fourier transform infrared spectroscopy
To assess the quality of the bone matrix, Fourier transforminfrared (FTIR) spectroscopy was performed on 5 control and 5PDB cases. From the embedded bone sections, 5‐mm‐thicksections were cut with a microtome to acquire FTIR spectra intransmission with a FTIR imaging system (Spotlight 400; PerkinElmer, Waltham, MA, USA). Over a specified bone area, spectrawere acquired at 6.25‐mm intervals over the spectral range of 570to 4000 cm�1 at a spectral resolution of 4 cm�1 and 128 scans. Intotal, at least 8000 pixels of bone (roughly 560� 560mm2) wereanalyzed per sample in both the trabecular and corticalcompartments. Spectra were analyzed using a custom programin Matlab (MathWorks, Natick, MA, USA). Each spectrum wasbaseline corrected and the contribution from the embeddingmaterial was subtracted from the measured spectrum.
At each pixel, area ratios were calculated from the spectra toquantify the mineral‐to‐matrix ratio, carbonate‐to‐phosphate ratio,and 1660/1690 cm�1 collagen crosslink ratio.(41–43) The mineral‐to‐matrix ratio was measured as the area ratio of the phosphate y1(915 to 1180 cm�1) to amide I peaks (1590 to 1725 cm�1). Thecarbonate‐to‐phosphate ratio was measured as the area ratio ofthe carbonate (850 to 900 cm�1) to phosphate y1 peaks (915 to1180 cm�1). The collagen crosslink ratio was determined by peak‐fitting the amide I and II bands between 1490 and 1725 cm�1.Specifically, the amide I and II bands were smoothed with aSavitzky‐Golay filter using 21 points and a 2nd degree polynomial.For the 1660/1690 cm�1 collagen crosslink ratio, the secondderivative of the bandswas used to determine the locations of ninesubbands and the collagen crosslink ratio was then correlated tothe area ratio of the 1660 cm�1 to 1690 cm�1 subbands.(42)
The average mineral‐to‐matrix, carbonate‐to‐phosphate, andcollagen crosslink ratios were obtained for each case bycalculating the average and SD of the parameters from eachpixel over the area of interest.
Polarized light microscopy
Histological sections were toluidine blue–stained and observedunder linearly polarized light. Collagen fibrils or bundles of fibrils,which are cut longitudinally and run parallel to the polarizer oranalyzer plane, appear bright on the dark background, whereascross‐sectioned fibrils or fibers appear dark. The application of
266 ZIMMERMANN ET AL. Journal of Bone and Mineral Research
linearly polarized light on histological sections qualitativelydistinguished between woven and lamellar bone within thespecimen taken from control and PDB cases.(44)
Nanoindentation
The mechanical properties of 14 control and 14 PDB cases wereassessed via nanoindentation measurements. The embeddedand polished biopsy specimens were indented with a Berkovichtip in a Triboindenter (Hysitron, Minneapolis, MN, USA)perpendicular to the cross‐section. The indent was loaded at arate of 100mN/s. When a peak load of 600mN was reached, theload was held for 10 s and then the sample was unloaded at thesame rate. Three sets of 10 indent points were performed in afield with at least a 5‐mm separation. The Young’s modulus andthe hardness of the bone samples were acquired from thenanoindentation measurements.
Reference point indentation
The mechanical properties of 10 controls and 10 PDB cases wereanalyzed with reference point indentation (RPI) measurements.Microindentations perpendicular to the cross‐section weremadeon polished embedded bone biopsies with a Biodent ReferencePoint Indenter (ActiveLife Tech, Inc., Santa Barbara, CA, USA). ABP2 probe was used to apply an indentation force of 6 N at anindentation rate of 2 Hz with 10 indentations per measurementcycle. Three indents were made in the cortical compartment ofeach iliac crest biopsy, and the first‐cycle indentation distance,indentation distance increase, first‐cycle creep indentationdistance, and average energy dissipated were reported.
In situ fracture toughness tests
Four control and 3 PDB cases fulfilled the criteria for a validfracturemechanics experiment according to ASTM standard 1820with an external cortex of roughly 12mm in length and 1.4mm inwidth.(45) The samples were polished into beams, notched with awater‐irrigated low‐speed saw, and then the saw‐cut notch wassharpened to a crack tip radius of roughly 10mmby polishing theroot of the notch with a razor blade irrigated with 0.5mmdiamond solution. The features of the bone structure (ie, osteons,cement lines, mineralized collagen fibrils, etc.) are predominantlyaligned in a certain orientation. Because of this anisotropy, bonefracture toughness can be measured either parallel (ie,longitudinal orientation) or perpendicular (ie, transverse orienta-tion) to the structure’s orientation; here, the bone fracturetoughness was measured in the transverse orientation. Thesurface of the sample was polished to a 0.5‐mm finish and thesamples were hydrated in Hanks’ Balanced Salt Solution (HBSS)for at least 12 hours prior to testing. The toughness of the notchedsamples was tested with a Gatan Microtest 2kN bending stage(Gatan, Abington, UK) in a S‐4300SE/N variable pressure scanningelectron microscope (Hitachi America, Pleasanton, CA, USA),allowing continuous observation of the crack length on thesample’s surface throughout mechanical testing.The linear elastic stress‐intensity factor was measured as a
function of crack growth following standard ASTM 1820.(45)
Corrections were made to the load to account for the porosity inthe control and pagetic samples. A change in porosity will reducethe load bearing area and increase the load in the material asfollows: Pcorr¼ P/(1–p), where P is the experimentally measuredload, Pcorr is the porosity‐corrected load, and p is the porosity,which was measured on the bulk sample via synchrotron micro–computed tomography (mCT).
Corrections were also made to the stress intensity to accountfor crack deflection. The average deflection angle, u, wasmeasured through the thickness of each sample via X‐ray mCT.The globally applied mode‐I stress intensity, KI, was converted tothe local mode I, k1, and mode II, k2, stress intensities at the cracktip by the following relationship for in‐plane tilted cracks:k1¼ a11(u)KIþ a12(u)KII and k2¼ a21(u)KIþ a22(u)KII, where aij(u)are mathematical functions dependent on the angle of crackdeflection, u.(46) The local stress intensities can then be convertedto an effective stress intensity using the following relationshipbased on the strain energy release rate: Keff¼ (k1
2þ k22)1/2.
Assuming a yield strength of 100MPa and the initiationtoughness of K¼ 1.15MPam1/2, the minimum sample thicknessfor plane‐strain conditions of 0.33mm and minimum in‐planedimensions of 0.007mm to satisfy the criterion for small‐scaleyielding were both met to ensure validity of the test.
3D synchrotron mCT
The crack paths from the fracture tests were assessed in thecortical regions of control and PDB samples by microtomog-raphy. The microtomography was performed after mechanicaltesting to avoid changes in mechanical properties associatedwith high doses of irradiation.(20) Briefly, at beamline 8.3.2 at theAdvanced Light Source (Lawrence Berkeley National Laboratory,Berkeley, CA, USA), scans were conducted at 17 keV withmonochromatic X‐rays at a minimum sample‐to‐detectordistance of 50mm and a 600‐ms exposure at a 1.8‐mm/pixelspatial resolution around the crack path. Tomography slices werereconstructed with Octopus (Octopus v8, IIC UGent) from 1440exposures acquired over 180‐degree sample rotation in 0.125‐degree angular increments and visualized in Avizo 6.1 (Visuali-zation Sciences Group, Inc.).
Statistics
Results are presented as means� SD. Statistical analysis wasperformedwithOriginPro 8 (OriginLab Inc.). To test for differencesbetween the study groups, we used the unpaired two‐sided t teston normally distributed data. The normal distribution of the datawas tested using the Kolmogorov‐Smirnov test. Values of p� 0.05were considered statistically significant. For data that was notnormally distributed, a nonparametric two‐sided Mann‐Whitneytest was used.
Results
Characterization of mineral and collagen quality
Here, we find significant changes in the composition and qualityof the Paget’s bone structure at small‐length scales. qBEI of theBMDD indicates a distinctly lower mineral content in PDB cases(Fig. 2A–C). From the distribution of the mineral content, thehistogram showing the frequency of eachmineral density can beused to quantify the Ca mean, Ca peak, Ca low, Ca high, and Cawidth (heterogeneity). Here, in the PDB cases, the Ca mean andCa peak values are both �19% lower (Fig. 2A–C, Table 1) andcontained six times more bone with a low mineral densitydistribution as well as 86% less bone with a high BMDD (Fig. 2A–C, Table 1). The PDB cases also had a 17% greater degree ofheterogeneity in bone mineralization, as measured through thewidth of the histograms (Fig. 2A–C, Table 1). All of these BMDDparameters indicate a prominent lower degree of mineralizationin the PDB cases.
Journal of Bone and Mineral Research NANO/MICROSTRUCTURE EFFECTS ON MECHANICAL INTEGRITY IN PDB 267
FTIR was also used to characterize the collagen and mineralquality (Fig. 3A–D), where the peak area ratios correlate to specificbone quality parameters: mineral‐to‐matrix ratio (MMR), carbon-ate‐to‐phosphate ratio (CPR), and 1660/1690 cm�1 collagencrosslink ratio. FTIR measurements confirm a 12% lower MMR inPDB (control 2.96� 0.20, PDB 2.59� 0.13, p¼ 0.009) (Fig. 3B) andalso indicate a 15% lower CPR (control 0.0104� 0.0006, PDB0.0088� 0.0005, p¼ 0.003) (Fig. 3C). The CPR corresponds tocarbonate substitution for phosphate in the mineral lattice andgenerally increases with tissue age (ie, the relative age of theosteons). For the organic component, FTIR showed a significantlyhigher collagen crosslink ratio in PDB (control 3.40� 0.41, PDB3.94� 0.18, p¼ 0.040) (Fig. 3D), which corresponds to changes inthe collagen’s secondary structure and/or an increased presenceof noncollagenous proteins (eg, osteonectin, osteocalcin, andosteopontin) in PDB.(47,48) Thus, the qBEI and FTIR results indicatechanges to the composition and quality of the bone tissue in PDBresulting in a lower, heterogeneous bone mineralization and ayounger tissue age.
Characterization of trabecular and cortical morphology
In the trabecular region of the iliac crest, static histomorphom-etry reveals elevated bone turnover and a denser bone volume inthe PDB cases (Table 2). Indeed, the PDB cases have a significantincrease in bone volume (Table 2) measured through a 2.5‐foldincrease in trabecular bone volume (BV/TV), threefold increase intrabecular number (Tb.N), and nearly 4.5‐fold decrease intrabecular spacing (Tb.Sp). However, the trabecular thicknessdid not significantly change. Thus, the bone volume increases
through the creation of new trabeculae and not throughapposition or growth of preexisting trabeculae.(3) Additionally,the PDB cases had a significant increase in bone formationmeasured through increases in osteoid as well as osteoclast andosteoblast numbers (Table 2).
In the cortical structure, synchrotron X‐ray CT images revealthat the parallel Haversian canals characteristic of healthy humanbone are replaced by disorganized clusters of porosity (ie,regions of hypervascularity) without a certain directional pattern(Fig. 4A, B). Polarized light microscopy (Fig. 4C, D) shows thatthese clusters are a patchwork of lamellar and woven bone,which is characteristic of PDB,(15–17) whereas the control caseshave a normal lamellar structure.(49,50) Thus, on the microstruc-tural level, the sandwich structure of the iliac crest, consisting of atrabecular core surrounded by a cortical frame in control cases, isreplaced by a dense clumsy bone structure that lacks a well‐defined directional orientation of collagen fibers and osteons.
Mechanical properties
Classical nanoindentation and RPI were used to assess thedeformation resistance of the control and PDB cases. Nano-indentation reveals a 14% lower Young’s modulus (p¼ 0.002)and a 19% lower hardness (p¼ 0.003) in PDB samples (Fig. 5A, B).RPI was also used to investigate the bone’s mechanicalresistance.(51,52) RPI is a microindentation technique thatcyclically loads the bone with an indenter in relation to areference point. The RPI parameters showed significantly higherindentation depths in the PDB samples (Fig. 5C–E) with nochange in the average energy dissipated (Fig. 5F). A previousstudy using RPI in this orientation found that bone with a lowermodulus also had higher indentation depth values.(52) Thus, theindentation techniques reveal that the PDB cases have a lowermodulus and less resistance to plastic deformation.
Fracture mechanics tests were performed on the hydratedcortices of control and PDB samples. The fracture toughness interms of the linear‐elastic stress intensity, K, was measured as afunction of crack extension, Da, to determine the crack growthresistance curve (ie, R‐curve) (see Fig. 6A). The toughness ofhealthy bone is highly dependent on orientation, mainly due todifferent extrinsicmechanisms that are active in either orientation.
Fig. 2. Small length‐scales: Quantitative backscattered electron imaging. The BMDDwas assessed in the control and PDB cases with qBEI, where the grayvalues reflect the calcium content. The stark differences in the BMDD are clearly visible in the pseudo‐colored backscattered electron images of (A) controland (B) PDB samples, as well as the (C) histogram of the density distribution. BMDD¼bone mineral density distribution; PDB¼ Paget’s disease of bone;qBEI¼quantitative backscattered electron imaging; B.Ar¼bone area.
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Therefore, fracture toughness is generally higher in the transverseorientation where crack deflection along the microstructuralfeatures is most active, in comparison to the longitudinalorientation, where this deflection mechanism is not favoredbecause the osteons are parallel to the crack.(53,54) Because bonewith PDB loses its parallel aligned Haversian systems, thebone could be expected to have a fracture toughness similarto the longitudinal orientation. However, our results indicate thatthe fracture toughness of the transversely‐oriented control andPDB samples was not significantly different as measured throughthe intercept of the R‐curve (p¼ 0.34), the slope of the R‐curve inFig. 6A (p¼ 0.76), and through the energy dissipated during RPI(Fig. 5F, p¼ 0.06).To further investigate the fracture toughness measurements,
we imaged the path of the crack via scanning electronmicroscopy during testing and synchrotron X‐ray mCT after
testing (Fig. 6B–E). Although there was no change in fracturetoughness, we did observe the effect of the extreme changes inmicrostructural morphology on the crack path. Because of thenormalmicrostructural orientation of the osteons, the crack takesa deflected path in control cases, which can account for theincrease in bone toughness with crack extension (Fig. 6B, D).(53)
However, the crack path in the PDB samples is straighter than thecontrol cases and still contains crack bridges, which occur atinterfaces within the microstructure such as the interfacebetween bone packets and lamellae (Fig. 6C, E).
Discussion
Through the bowing, deformities, and fissure fractures observedin clinical cases, PDB has a clear effect on the bone’s mechanical
Table 2. Static Histomorphometry
Histomorphometric indices Control PDB Percent change (%) p
The static histomorphometry of the control and PDB cases was evaluated according to standards set by the ASBMR.(34) The values are reported asmean� SD.
Fig. 3. Small length‐scales: FTIR. The quality of the collagen and mineral components was assessed via FTIR mapping. (A) Spectra were collected at 6.25‐mm intervals across a defined region of interest. (B) From the data, the mineral‐to‐matrix ratio was significantly 12% lower in the PDB cases (p¼ 0.009). (C)The carbonate‐to‐phosphate ratio was 15% lower in the PDB cases (p¼ 0.003) and (D) the collagen crosslink ratio was 15% higher in the PDB cases(p¼ 0.040). Scale bars¼ 100mm. FTIR¼ Fourier transform infrared spectroscopy; PDB¼ Paget’s disease of bone.
Journal of Bone and Mineral Research NANO/MICROSTRUCTURE EFFECTS ON MECHANICAL INTEGRITY IN PDB 269
integrity, which results from a combination of intrinsic mecha-nisms at small‐length scales that generate/restrict plasticity andof extrinsic mechanisms at larger‐length scales that interferewith the crack growth. Here, through amultiscale investigation ofbone quality and mechanical properties in control and PDBcases, we investigate how the extreme changes to the multiscalebone structure (Fig. 1) lead to the pathological changes observedin the clinic.
In PDB cases, the bone quality was significantly altered at small‐length scales. Specifically, the mineral content and distributionmeasured through qBEI (Fig. 2) and FTIR (Fig. 3) show that the PDBcases have a significantly lower degree of mineralization. Thiscomposition change directly relates to the significantly lowerstiffness of the PDB tissue measured via nanoindentation andpossibly also the higher indentation distance valuesmeasured viaRPI(52) (Fig. 5), because in most biological materials, the Young’smodulus (ie, stiffness) scales with mineral content.(55)
In addition to affecting the bone stiffness, the deviations inbone quality at small‐length scales (Figs. 2, 3) influence how thediseased bone generates plastic deformation.(56,57) Indeed, thelower hardness and the deeper indentation values (Fig. 5)indicate that the pathological bone tissue will generate moreplasticity than the control cases and suggests that themodifications to the quality of the tissue alter the intrinsicmechanismswithin the structure (ie, fibrillar sliding and sacrificial
bonding). Thus, the structural and compositional changes atsmall‐length scales in PDB affect both the elastic (ie, stretching ofbonds generating stiffness) and plastic (ie, permanent deforma-tion promoting ductility and energy absorption) mechanicalproperties resulting in a lower stiffness and more plasticity.
In PDB cases, the bone quality was also significantly altered atlarger‐length scales. In the trabecular region of the iliac crest, theelevated bone turnover results in more trabeculae as reflected bythe higher BV/TV and trabecular number(3) (Table 2). In the corticalcompartment, the parallel aligned Haversian canals characteristicof healthy human bone are replaced by a patchwork of lamellarand woven bone in PDB cases, with less organized collagen fiberorientation(15–17) (Fig. 4). Thus, on the microstructural level, thesandwich structure of the iliac crest consisting of a trabecular coresurrounded by dense cortical frame in control cases is replaced by
Fig. 4. Large length‐scales: cortical microstructure. SynchrotronmCT andpolarized light microscopy were used to observe changes at the osteonallength‐scale in the cortical bone. (A) The 3D tomography reconstructionsshow that in control cases, the osteons have a predominant orientationwith parallel‐aligned Haversian canals, which is absent in (B) the PDBcases. Additionally, polarized light microscopy indicates that (C) theosteons in control cases have alternating light and dark lamellaereflecting normal collagen fiber orientation, whereas the (D) PDB casesare a mosaic of immature woven and lamellar bone. mCT¼micro–computed tomography; PDB¼ Paget’s disease of bone.
Fig. 5. Mechanical properties: Nanoindentation and RPI. Nanoindenta-tion of the control and PDB cases reveals (A) a 14% lower modulus(p¼ 0.002) and (B) a 19% lower hardness in PDB (p¼ 0.003). RPIcharacterizes the bone’s mechanical resistance by cyclically loading thebone with a microindenter in relation to a reference point. (C–E) The RPIparameters indicate significantly higher indentation depths in PDB (allp< 0.001), which supports the nanoindentation trends of a lowermodulus and hardness. However, (F) the average energy dissipated wasnot significantly different (p¼ 0.06). Values reported as mean� SD.RPI¼ reference point indentation; PDB¼ Paget’s disease of bone.
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a dense clumsybone structure that lacks awell‐defineddirectionalorientation of collagen fibers and osteons.Even though PDB resulted in significant changes to the
structure at large‐length scales, the fracture toughness of thediseased bone measured through the energy dissipated duringRPI (Fig. 5) and the crack‐growth toughness (Fig. 6) during
fracture mechanics experiments was not significantly different incomparison to the transversely‐oriented controls. This is in linewith some of the limitations of this study, which are (1) thelimited number of fracture toughness samples, which restrictsthe statistical comparisons, and (2) that the embedding andinfiltration procedures may limit the effects of sample rehydra-tion, which affects the mechanical property measurements.Although future studies with larger sample sets are required toprecisely distinguish a difference in fracture toughness betweenthe control and PDB samples, there was still a clearly higherfracture toughness in the transversely‐oriented controls and thePDB samples in comparison to the longitudinally‐oriented bone,which has a comparatively weak resistance to crack growth.(53)
Therefore, in both the transversely‐oriented controls and thePDB samples, there appears to be a form of extrinsic resistance tocrack growth. In the controls, the mechanical resistance to crackpropagation is primarily derived through crack deflection (seeFig. 6), which has been shown to increase fracture toughness.(53) InPDB, the crack deflectionmechanism is lost, resulting in straightercrack paths (see Fig. 6) due to the microstructural alterations.However, one possible route to generate further mechanicalresistance would be through increased plastic deformation. Thus,the fracture toughness measurements may indicate that thebone’s intrinsic resistance (ie, lower mineralization leading tolower hardness, more plasticity) compensates for the loss in theextrinsic crack deflection mechanism (ie, due to the loss in theparallel‐aligned Haversian systems). This increase in plasticitywould act to absorb energy during crack propagation, leading toan increased fracture toughness and is supported by studies onother low‐mineralized tissues that have also found significantplastic deformation.(22,56) Thus, even though PDB samples losetheir microstructural orientation, which is critical to the fracturetoughness of healthy bone, the altered, heterogeneous structurecharacteristic of the pathological tissue may compensate bygenerating more intrinsic plasticity to resist crack growth.
In terms of clinical relevance, bone disorders associated withan underlying imbalance in the remodeling process can lead toincreased fracture risk, particularly when the disorder createsstructural and compositional changes. Clearly, the modificationsto the bone tissue caused by the high bone turnover in PDBuniquely affect the bone structure, leading to a higher bonevolume, lower, heterogeneous mineral content/distribution,significantly younger tissue age, and loss in lamellar osteonalbone structure. These characteristics of the bone structure andcomposition of PDB are not associated with known bone fragilityin other diseases. However, even though the characteristics ofthe Paget bone structure are contrary to other bone disorderswith fracture risk, it is necessary to recognize the impact of PDBon the mechanical integrity.
The specific effects on the pathological bone tissue, in particularbonedeformities and fissure fractures, can nowbe further clarifiedfrom the present multiscale characterization of the bone structureand mechanical properties. Indeed, the excess amount of osteoidand mineralized bone produced in PDB leads to deformities andbowing in clinical PDB cases, where incomplete or “fissurefractures” occur in deformed load‐bearing tissue.(28,30) Here, ourexperimental data, revealing a lower spatially‐resolved mineralcontent and tissue age of the bone with a corresponding lowerstiffness and lower resistance to deformation, could directlyaccount for the occurrence of harmful bone deformities inpatients suffering from PDB. The deformities can in turn lead toosteoarthritis due to the gait problems encountered whendeformities occur in load‐bearing limbs.(58)
Fig. 6. Mechanical properties: fracture toughness and crack path. (A) Thefracture toughness in terms of the linear‐elastic stress intensity, K, ofcontrol and PDB cases wasmeasured as a function of crack extension,Da,which is called a crack growth resistance curve or R‐curve. The fracturetoughness of control (ie, transversely oriented) and PDB cases was notsignificantly different, as measured through the intercept (p¼ 0.34) andslope of the R‐curve (p¼ 0.76). Because the PDB cases do not have adefined orientation for crack deflection due to their mosaic structure, thefact that the toughness is comparable to the transverse orientation andhigher than the longitudinal orientation (which is also not optimized forcrack deflection) is surprising.(53) Based on our observations (B, C) of thecrack path after testing (via synchrotron X‐ray computed microtomog-raphy) and (D, E) during testing (via scanning electron microscopy), (B, D)the control cases toughen extrinsically by deflecting along the interfacesof the osteons, whereas (C, E) the PDB cases take a straighter crack paththrough the disordered structure with large crack bridges. PDB¼ Paget’sdisease of bone.
Journal of Bone and Mineral Research NANO/MICROSTRUCTURE EFFECTS ON MECHANICAL INTEGRITY IN PDB 271
The other interesting phenomenon is the presence ofsubcritical (ie, stable) cracks, so‐called fissure fractures, in PDB.The fissure fractures most likely occur because of the bonedeformities/bowing, but the fact that these fractures remain inthe tissue and do not completely cause bone failure is in line withthe same propensity for the altered structure to resist crackgrowth through plastic deformation. In this way, the alteredcomposition (ie, the reduced mineral content) has a negativeimpact on the bone stiffness (ie, causes bowing/deformity), butcompensates for the reorganized bone microstructure bygenerating plastic deformation to resist the growth of cracksallowing stable fissure cracks. In this connection, although bonefracture is an important issue in patients with PDB and stablecracks do occur, our fracture toughness data suggests that thematerial properties of the bone may compensate to a certaindegree to prevent complete bone fracture.
In conclusion, on a set of humanbonebiopsies fromcontrol andPDB cases, we found that the high bone turnover associated withPDB causes a significantly lowermineral content and tissue age. Atlarger structural‐length scales, the trabecular region is known tobecomedensified, whereas the cortex loses the lamellar Haversianosteon structure with its regular arrangement, which is replacedby a mosaic of immature woven and lamellar bone. Through theindentation measurements presented here, the structuralchanges at small‐length scales clearly reduce the stiffness andpromote plastic deformation in PDB cases. In turn, the loss of theosteonal structures should deteriorate the fracture toughness, butthe larger degree of plastic deformation at small‐length scalescompensates for the lack of structure and may be the reason forthe maintained fracture toughness presented here. Therefore, thealterations to the structure in PDB produce bowing/deformities,namely from the low mineral content, but may also improve themechanical integrity of the tissue by promoting plastic deforma-tion to stop the growth of cracks, leading to the presence of stablefissure fractures characteristic of the disease.
Disclosures
All authors state that they have no conflicts of interest.
Acknowledgements
This study was supported by the German Research Foundation(DFG) under grants BU 2562/2‐1 and BU 2562/1‐1. Weacknowledge use of the mCT beam line 8.3.2 at the AdvancedLight Source (ALS) synchrotron at Lawrence Berkeley NationalLaboratory (LBNL), Berkeley, CA, USA. The Advanced Light Sourceis supported by the Director, Office of Science, Office of BasicEnergy Sciences, of the U.S. Department of Energy underContract No. DE‐AC02‐05CH11231. We thank Dr. Björn Jobke(DKFZ, Heidelberg) for plain film images of PDB.
Authors’ roles: EAZ, ROR, and BB designed the study. EAZ, TK,HAB, BP, BG, and BB performed the experiments. EAZ, TK, HAB,BP, BG, MA, ROR, and BB analyzed the data. EAZ, HAB, JZ, MH, MA,ROR, and BB contributed reagents or analytic tools. MH and JZgave technical support and conceptual advice. EAZ and BBwrotethe manuscript. EAZ, TK, HAB, BP, BG, JZ, MH, MA, ROR, and BBapproved the final version of the manuscript.
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