A novel murine model of orthopaedic wear-debris associatedosteolysis

W Ren*, S-Y Yang*, PH Wooley

Department of Orthopaedic Surgery, Wayne State University, Detroit, MI, USA

Objective: To develop a mouse model of bone resorption to quantitatively evaluate wear-debris induced osteolysis.

Methods: Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section

of femur or calvaria from a syngeneic mouse donor. One group of bone-implanted pouches was stimulated with ultra-

high molecular weight polyethylene (UHMWPE) debris, and the remaining bone-implanted pouches received saline

alone as controls. The tissues were harvested at 2, 7, and 14 days after bone implantation for molecular and

histological analyses.

Results: Marked inflammatory responses (thicker membrane and increased cellular infiltration) were observed in

UHMWPE-stimulated pouches, compared with the saline control. Intensive tartrate-resistant acid phosphatase(TRAP) staining was identified in the UHMWPE-stimulated pouches, especially at the attachment site of

inflammatory tissue with implanted bone, where active osteolysis occurred. Image analysis showed that the bone

collagen loss was closely related to the amount of UHMWPE within the tissue, and was most prevalent at the contact

site of bone with inflammatory tissue. UHMWPE stimulation also significantly increased the release of free calcium

into the pouch fluids.

Conclusion: This model demonstrates a sensitive, rapid, and reproducible method for studying wear-debris induced

osteolysis seen in patients with aseptic loosening.

Aseptic loosening has become the most commonlong-term complication of total joint arthroplasty(1, 2), and is associated with the generation ofwear debris particles, which accumulate at theinterface between implant component and sur-rounding bone. Macrophage phagocytosis of debrisinitiates the release of inflammatory mediators, suchas interleukin-1 (IL-1) and tumour necrosis factor-a(TNF-a) (3 – 6), which in turn promotes osteoclasto-genesis and bone resorption, and subsequently leadsto implant loosening (7, 8).

Due to the limitations of in vitro modelling, andlimited access to human joint tissues (particularlyjoints from patients in early stages of asepticloosening), an accurate animal model is requiredto study the pathogenesis of aseptic loosening and toseek novel pharmaceutical agents to retard theprocess. Although several large-animal prostheticimplant models have been reported (9 – 12), thesemodels are time-consuming and expensive to use,and are not generally applicable for pharmaceuticalscreening purposes. We and others have utilized the

rodent air-pouch model to investigate the inflamma-tory response to biomaterials, including orthopaedicwear debris, under controlled experimental condi-tions (13 – 18). However, this model cannot be usedto evaluate wear-debris induced osteoclastogenesisand bone resorption, due to the lack of bone tissue.

In this study, we report the development of amodified mouse pouch model, with the capacity tomodel osteoclastogenesis and bone resorptionresponses to debris. This model utilizes implantationof femoral or calvaria bone sections from geneticallyidentical donor mice into an established air pouch.We show evidence that implantation of these boneswithin the ultra-high molecular weight polyethylene(UHMWPE) particulate-stimulated air pouch pro-vides a quantitative model to evaluate wear debris-provoked bone changes. This model appears to beuseful for screening the effects of pharmaceuticalintervention, including gene therapy targeted againstinflammatory cytokines and receptor activator ofnuclear factor kappa beta (RANK/NFkB) signalingpathway, on bone resorption.

Materials and methods

UHMWPE particles

UHMWPE particles were generously provided by DrJohn Cuckler, University of Alabama, Birmingham,* W Ren and S-Y Yang contributed equally to this work.

Paul H Wooley, Department of Orthopaedic Surgery, Wayne

State University, 1 South, Hutzel Hospital, 4707 St Antoine Blvd,

Detroit, MI 48201, USA.

E-mail: ad8754@wayne.edu

Scand J Rheumatol 2004;33:349–357 349

www.scandjrheumatol.dk

# 2004 Taylor & Francis on license from Scandinavian Rheumatology Research Foundation

DOI: 10.1080/03009740410005944

AL, USA. Particle analysis (19) with a Coulter

channelizer (Coulter Electronics, Hialeah, CA, USA)

and scanning electron microscopy demonstrated that90% of the particles were v5.5 mm in diameter, with

a mean size of 2.6 mm [v0.6 – 21 mm, standard

deviation (SD) 2.4 mm], and a mean aspect ratio of

0.984. The particles were washed in 70% ethanol

solution and resuspended in sterile phosphate buf-

fered saline (PBS) containing 10% of foetal bovine

serum (FBS) at a concentration of 10 mg/mL

(1.5|107 particles/mL). The particle suspension wasdetermined to be endotoxin-free using the Limulus

assay (Endosafe; Charles Rivers, Charlestown, SC,

USA).

Establishment of pouch model of bone resorption

Female BALB/c mice (8 – 10 weeks old) were pur-

chased from The Jackson Laboratory (Bar Harbor,

ME, USA) and quarantined within our facility prior

to experimentation. Air pouches were generated by

injection of sterile air, as described previously (13).

Six days after air pouch formation, congeneric

littermates were sacrificed as bone donors (each

donor mouse provided bone implants for six airpouch recipients). Femurs and calvaria caps were

surgically harvested by dissection and adherent tissue

removed, taking care not to damage the bone

surfaces. Femurs were divided into proximal and

distal sections, and each parietal bone was trimmed

to approximately 4 mm in diameter. Donor bones

were placed in sterile PBS and immediately

implanted.Mice with established air pouches were anesthe-

tized with 50 mg/kg of pentobarbital (Fisher Scien-

tific, Pittsburgh, PA, USA) by intraperitoneal

injection. A 0.5 cm incision overlying the pouch

was made, and a 0.6 cm long femur section with

cartilage intact, or 0.4|0.25 cm fragment of calvar-

ium was inserted into the pouch using fine forceps.

Since preliminary studies showed that the weights ofproximal and distal sections of femurs were almost

identical with similar bone surface areas, we

combined data from the two femur parts for

analysis. The results for calvaria implantation were

analysed separately from femur implants. Sterile PBS

(0.3 mL) containing 1:100 penicillin/streptomycin

(GIBCO-BRL, Gaithersburg, MD, USA) was

injected into the pouch, and the pouch layers andskin incision were closed using 4-0 Prolene sutures.

The entire procedure was performed under sterile

conditions in a laminar flow hood.

The following day, 5 mg UHMWPE particles

suspended in 0.5 mL of 10% FBS/PBS were injected

into each pouch of the experimental group to

provoke inflammatory responses, and 0.5 mL of

10% FBS/PBS vehicle was injected into control bone-implanted pouches. Mice were sacrificed in a CO2

chamber at Days 2, 7, and 14 after particle

stimulation, and both the pouch membrane and

implanted bone tissue harvested. Pouch fluidscombined with 0.2 mL of saline pouch lavage were

collected for determination of free calcium and

cytokine production, and a small portion of the

pouch process was collected for molecular analyses.

The remainder of the pouch tissue, with the intact

bone implant, was either snap-frozen for crystallized

sectioning, or fixed in 10% buffered formalin (Fisher

Scientific) for paraffin embedding. All procedureswere approved by the University Animal Investiga-

tion Committee.

Assessment of osteoclastogenesis

To assess debris-induced osteoclastogenesis, real-time reverse transcriptase-polymerase chain reaction

(RT-PCR) methods (20) were performed to quantify

the gene expression of cathepsin K (CK) in pouch

membrane and implanted calvaria tissue (21, 22).

Total ribonucleic acid (RNA) was extracted follow-

ing the kit manufacturer’s instruction (Tel-Test Inc.,

Friendswood, TX, USA). Complementary deoxy-

ribonucleic acid (cDNA) was reverse transcribedfrom 0.5 mg of total RNA in 40 mL reaction mixture

containing 500 mM each of deoxynucleotide tripho-

sphates (dNTP), 0.4 U/mL of RNase inhibitor,

2.5 mM random hexamers, 50 mM tris-HCl (pH 7.5),

5.5 mM MgCl2, 75 mM KCl and 1.25 U/mL of reverse

transcriptase (GIBCO-BRL). The reaction mixture

was incubated in a DNA Thermal Cycler (Perkin

Elmer, CT, USA) at 25‡C for 10 min, 48‡C for5 min, followed by 95‡C for 5 min. Real-time PCR

was performed following manufacturer’s instruc-

tions. To standardize target gene level with respect

to variability in ribonucleic acid and cDNA quality,

the level of transcription of glyceraldehyde-3-phos-

phate dehydrogenase (GAPDH), a housekeeping

gene, was used as an internal control. Reaction

mixtures of 50 mL contained 25 mL of 2|SYBRGreen PCR Master Mix (containing 5 mM MgCl2,

200 mM dATP, dCTP, dGTP, 400 mM dUTP,

1.25 U AmpliTag Gold DNA polymerase, 0.5 U

AmpErase uracil N-glycosylase), 400 nM forward

and reverse primers, and 4 mL of cDNA (Perkin

Elmer/Applied Biosystems).

The PCR was set in a MicroAmp optical 96-well

reaction plate with MicroAmp optical caps for 40cycles (95‡C/15 s, 60‡C/1 min) in the ABI Prism 7700

Sequence Detector (PE-Applied Biosystems, Foster

City, CA, USA) and the fluorescent signals recorded

dynamically. Normalization and analysis of the

reporter signals (DRn) at the threshold cycle was

recorded by the machine, and target gene copies

(mRNA expression) were calculated against the

regression of the standard curve.Expression of tartrate-resistant acid phosphatase

350 W Ren, S-Y Yang, PH Wooley

www.scandjrheumatol.dk

(TRAP, EC3.1.3.2) was used to evaluate the

presence of osteoclast-like cells (23). Histochemical

TRAP staining using a commercial kit (Sigma) wasperformed on pouches implanted with calvarium, to

localize the osteoclast-like cells in the pouch tissue —

as described elsewhere (24). Briefly, cryosections

(6 mm) were prepared and fixed in buffered acetone

for 30 s. Sections were incubated at 37‡C for 1 h in

100 mM acetate buffer (pH 5.2), containing 0.5 mM

naphthol AS-BI phosphoric acid, 2.2 mM Fast

Garnet GBC and 8 mM sodium tartrate. Thesections were then washed in several changes of

distilled water, and mounted in Crystal mount

(Biomeda). The presence of dark purple staining

granules in the cytoplasm was determined as the

specific criterion for TRAP positive cells.

Determination of osteolysis

Modified Masson’s Trichrome and van Gieson histo-

logical stains (25) were performed on 5 mm paraffin-

embedded sections of pouch membrane, to quantify

bone collagen content as a parameter of bone

osteolysis, using methods described previously (26).

In brief, sections were deparaffinized and hydratedbefore equilibrating in Bouin’s solution (70% picric

acid, 5% glacial acetic acid, and 10% formaldehyde)

at 56‡C for 1 h. The sections were then incubated in

phosphomolybdic (0.21% w/v)-phosphotungstic acid

(0.21% w/v) for 10 min, followed by aniline blue

solution (2.5% aniline blue in 2% acetic acid) stain

for 5 min. The sections were then incubated in 1%

acetic acid for 4 min before dehydration in gradedalcohol. The slides were cleared in xylene, and

mounted with Permont.

Bone collagen stained dark blue, with colour

density proportional to the content of collagen

deposit. We have determined that this modified

Trichrome stain is a sensitive measure of bone

collagen content of femoral bone, whereas van

Gieson stain (another histological stain for collagen;0.1% aqueous acid fuchsin in saturated aqueous

picric acid) exhibited superior results using calvar-

ium implants. Haematoxylin and eosin (H&E)

stains were routinely performed to examine bone

erosion and particle-induced inflammatory cellular

invasion.

Calcium concentration in air pouch fluid was also

assessed as a measure of bone demineralisation,using an automatic fluorometric titration method

with a fluorescent spectrophotometer (Model 810

Photomultiplier Detection System, Photon Technol-

ogy International, NJ, USA) equipped with a

fluorescent Fura-2 probe (Precision Systems). The

size of implanted femur or calvarium bone and

volume of pouch lavage fluid were kept constant, to

standardize calcium concentration per pouch. Theconcentration of calcium released from implanted

femur or calvarium fragment was calculated respec-

tively according to Grynkiewicz et al (27) and

expressed as nanomoles per litre.

Image analysis and quantification

The images of histological stained tissue sections

were digitally photographed using an Axiophot light

microscope (Zeiss, Jena, Germany) equipped with a

Toshiba CCD colour camera (IK-TU40A). Air

pouch membrane thickness and cellular infiltration

were quantified by a computerized image analysis

system with the software ‘Image-Pro Plus’ (Media

Cybernetics, Silver Spring, MD, USA), as described

previously (18).

A modification of our established method was

adopted to determine the changes of bone collagen

content. Magnified images (100|) of modified

Trichrome Blue-stained sections containing femur

implants, or van Gelson-stained pouches with

calvaria, were analysed with the software package

Image-Pro Plus. Integrated optical densities (IODs)

of the areas at the bone surface contiguous with

particle-containing inflammatory pouch membranes

were recorded, and normalized with the IODs

measured at the same-sized inner part of the bone

distal from the inflammatory membrane. This

provided an accurate measurement, avoiding possi-

ble differences due to section thickness and staining

time variance. The obtained ratio was expressed as

percentage of collagen content preserved in response

to particle-stimulated inflammation. Six pairs of

IOD readings at different regions of each bone

section were determined in a minimum of eight mice

per group.

The number of osteoclasts in each calvarium

section was quantified from a TRAP-stained section

by two independent, blinded observers who counted

the large red stained cells. A minimum of four mice

were used for each datum point.

Statistical analysis

Mice were randomly assigned to experimental

groups with at least eight mice in each group.

All statistical analyses were conducted using

SPSS (version 7.5; SPSS Inc., Chicago, IL, USA)

software. Statistical analysis between two groups

was performed by the two-tailed paired-samples

T test. Correlation coefficient (r) analysis between

the various parameters was tested by regression

analysis, and the significance of the r-value deter-

mined by the Student’s t-test. A probability (p)

value of v0.05 was considered as significant. Data

was expressed as mean¡standard error of the

mean (SEM).

Murine osteolysis model 351

www.scandjrheumatol.dk

Results

UHMWPE-induced local inflammation in the model

Bone implants were tolerated well in BALB/c

mice up to 14 days, and a recovery rate of 100%

with no infection was achieved. UHMWPE debrisinduced an inflammatory response within the pouch

membrane, as demonstrated previously (13, 28).

Polyethylene-particle-stimulated pouches exhibited

pronounced erythematous and oedematous changes

compared with control (saline stimulated) pouches

(Figure 1). Image analysis of histological sections

revealed marked inflammatory cellular infiltration

and membrane hyperplasia. Pouch membranes con-taining UHMWPE debris averaged 154¡30 mm in

thickness, significantly thicker than non-particle-

containing control pouches (94¡24 mm, pv0.01)

(Figure 2A). Cellular infiltration in pouches with

polyethylene debris (7888¡855 per mm2) was also

significantly increased compared with the non-

particle control pouches (4800¡855/mm2, pv0.01)

(Figure 2B). There was no difference in theseparticle-induced inflammation parameters between

femur bone and calvaria implanted pouches.

Quantification of osteoclastogenesis

Two experimental approaches were performed to

evaluate orthopaedic wear debris-induced inflamma-

tory osteoclastogenesis. Investigation of the expres-sion and localization of TRAP stained cells

(osteoclasts) revealed few TRAP-positive osteoclasts

present in the control calvaria-implanted pouches

without particle stimulation (Figure 3A). Due to

technical difficulties, TRAP staining was only

performed on frozen sectioned calvaria-pouch tis-

sues. The TRAP (z) cells were mainly located along

the inner endosterum, at the interface between pouchmembranes and implanted within calvaria, and bone

morphology was well preserved with no obvious

bone erosion. In addition, no TRAP positive cells

could be demonstrated in the surrounding pouch

membrane tissue.

In contrast, UHMWPE particle stimulation

resulted in a pronounced increase in TRAP expres-

sion in both the inflammatory pouch membranes and

the implanted calvaria. Clusters of TRAP stained

cells were located in regions in contact with

Figure 1. Typical macroscopic appearance of air pouches dissected from mice 14 days after bone implantation. Panel (A) illustrates

a parcel-free control air pouch with a piece of calvarium implant: Panel (B) shows a bone-implanted air pouch with UHMWPE

stimulation.

Figure 2. Histological assessment of pouch membrane thickness

(panel A) and total cell counts (panel B) by computerized image

analysis system.

352 W Ren, S-Y Yang, PH Wooley

www.scandjrheumatol.dk

inflammatory tissue containing polyethylene particles

(Figure 3B). TRAP positive staining was intense in

regions of implanted calvaria at the outer edge of the

calvaria periosterun, and on the invasion front,

where active osteolysis occurred. These histological

changes were similar to previous findings using an ex

vivo calvaria organ culture system (29).

Enhanced osteoclastogenesis was also indicated by

the over-expression of CK, an osteoclast marker

gene, determined using the real time RT-PCR

technique. UHMWPE debris elevated gene expres-

sion of CK as soon as 2 days after particle

stimulation (Figure 4), and this result was signifi-

cantly increased in comparison with CK gene

expression in saline-stimulated control tissues

(pv0.01). Constitutive expression of a housekeeping

gene, GAPDH, was essentially constant, with a

relative standard error of ¡3% for all groups in the

presence or absence of UHMWPE debris stimulation

(data not shown).

Assessment of UHMWPE debris-induced osteolysis

Histological analysis of H&E stained bone-

implanted pouch sections revealed the frequent

occurrence of bone erosions at surfaces in contact

with particle-induced inflammatory pouch mem-

branes where inflammatory cells invaded the bone

cortex (Figure 5). In contrast, bone surfaces without

contact with the inflammatory membrane remained

histologically normal. Modified Masson’s Trichrome

and van Gilson histological stains were performed

on decalcified implanted bone sections, to quantify

relative bone matrix collagen changes using compu-

terized image analysis. Figure 6 shows representative

images of Modified Masson’s Trichrome stained sec-

tions. UHMWPE particle stimulation dramatically

increased the loss of bone collagen content at the bone

surface in close contact with the inflammatory pouch

membranes (Figure 6B), in com- parison with the

bone collagen changes in sections from control (saline-

stimulated) pouches (Figure 6A). Computerized image

analysis quantitatively summarized the collagen degra-

dation (Figure 7, pv0.01). This suggests that bone

collagen content represents an accurate parameter of

osteolysis in this model.

Free calcium concentration in pouch fluid was

determined as a measure of bone resorption.

Previous studies have confirmed that there was no

detectable calcium in pouch fluids without bone

implantation. In this study of the bone resorption

model, introduction of UHMWPE particles induced

a significant increase in calcium release from

implanted femurs into the pouch fluid as soon as

2 days after particle introduction. Figure 8 sum-

marizes a time-course of free calcium ion release in

femoral bone-implanted pouches. It is apparent that

the significance of calcium level difference between

the groups was abolished after 14 days implantation,

Figure 3. TRAP staining in mouse pouch with calvaria implantation at 14 days (|200). Panel (A) shows a calvarium-containing pouch

with PBS injection (control): panel (B) demonstrates a bone-pouch with UHMWPE treatment. An extensive brown stain along the

bone surface on panel B indicates the deposits of osteoclast-like cells.

Figure 4. Gene expression of CK evaluated in bone-implanted

pouches determined by real time RT-PCR. Data is converted as

target gene copies reverse-transcribed from 1 ng of total RNA

(*pv0.05).

Murine osteolysis model 353

www.scandjrheumatol.dk

probably due to disruption of the normal blood

supply to the implanted bone.

Discussion

Aseptic loosening induced by particulate wear-debris

from implant materials has been recognized as the

major cause of long-term failure in total joint

replacements, and currently no treatment has been

proved to prevent or inhibit this condition. An

accurate animal model would be useful in thedevelopment of pharmaceutical intervention for this

complication. Large animal prosthetic implant

models (9 – 12) are time-consuming and expensive,

and are not generally appropriate for broad

applications in screening therapeutics for activity

in aseptic loosening. We and others have described

orthopaedic wear-debris related inflammation in

mouse air pouch model, and demonstrated advan-

tages of this model, including the close resemblance

to inflammatory periprosthetic tissue, the ability to

reproduce experimental conditions, the capacity to

quantify cell influx and mediators of inflammation

responding to debris stimulation, and its cost-

effective nature (13 – 16, 18, 30). The obvious

limitation of the conventional murine air pouch is

that it cannot be used to evaluate osteolysis.

We report here on the development of a mouse

pouch model adapted to assess bone resorption

and quantitatively evaluate wear-debris induced

osteolysis. This novel model preserves the sensitivity

of pouch tissue membranes in the response to

particulate debris stimulation, and provides the

addition of bone tissue to resemble the environment

of debris-associated loosening prosthetic joint.

Further, this model behaves as an ‘in vivo culture’

system. In comparison with ‘in vitro’ bone resorp-

tion assays described by Glant and ourselves (29),

the bone-pouch model has advantages over in vitro

cell/organ culture systems.

The model represents in vivo wear-debris inter-

acting inflammatory cellular infiltration, including

the expression of inflammation mediators, and

resembles the biological activity in peri-prosthetic

membranes associated with aseptic loosening. This

has advantages over a single population of cells

responding to stimuli in an in vitro environment. It

allows the quantitative evaluation of the histological

morphology of wear-debris associated inflammation,

Figure 5. Representative histological sections of bone-implanted

pouch stained with H&E. (A) Particle-free saline control pouch

showing the smooth and clean-cut surface of implanted bone,

surrounded by thin and less inflammatory pouch tissue. (B)

Pouch containing polyethylene debris showing proliferated

pouch membrane, dense inflammatory cell aggregation, and

pitted bone erosion (|100).

Figure 6. Representative micrographs of modified Masson’s Trichrome stained sections for bone collagen content. (A) Particle-free sal-

ine control pouch with femur implantation for 7 days; (B) femur-containing pouch treated with UHMWPE particles for 7 days (|50).

354 W Ren, S-Y Yang, PH Wooley

www.scandjrheumatol.dk

osteoclastogenesis, and osteolysis under controlled

experimental condition, and provides a useful tool to

investigate the interaction between inflammatory

tissue and implanted bones.

Using this model, we were able to assess wear-

debris associated inflammation, and monitor osteo-

clastogenesis changes and bone resorption. The

results suggest that this system models the relation-

ship between orthopaedic debris particles, local

tissue inflammation, and the consequent bone

resorption. Our findings show that UHMWPE

debris-induced osteolysis was reproducible, with

linear implanted-bone resorption associated with

local erosion at the contact sites of inflammatory

pouch membrane. This observation suggests that

polyethylene debris-stimulated inflammatory tissue is

directly responsible for the implanted-bone resorption.

The proliferation, adhesion, and invasion of

inflammatory cells (mainly macrophages) at the

contact zone of implanted bone and pouch mem-

brane were similar to those observed in the tissue

morphology of aseptic loosening patients at the

interface of implant components and surrounding

bone (31, 32). Our findings also provide supportive

evidence that wear-debris induced osteolysis is a

direct consequence of the response to the inflamma-

tory tissue residing on the interface of bone. We are

currently using this model to investigate the size,

shape, and composition of the particulate debris

accumulated in the pathology of bone resorption,

showing the model to be a sensitive tool to

accomplish the task (unpublished observation).

The data demonstrate that femur or calvaria

implanted in inflammatory pouches under stringent

sterile conditions can be maintained for up to

14 days without obvious host reactions. The

implanted bones were obtained from genetically

identical donor mice; hence there were no obvious signs

of immunological rejection. The implanted bone can

undergo cell differentiation and proliferation within

UHMWPE debris-stimulated pouches for up to

14 days, as assessed by TRAP staining and CK gene

expression. This indicates that local inflammatory

tissue, either through direct cell-contact, or by releasing

inflammatory mediators, can stimulate implanted bone

to undergo osteoclastogenesis changes. Enhanced

osteoclastogenesis, in turn, accelerates osteolysis in

the model. Our findings confirmed previous observa-

tions (29) that the biological properties of implanted

calvaria tissue are well preserved.

Schwarz et al recently reported a quantitative

small-animal surrogate of wear-debris induced

osteolysis (33, 34) using implanted titanium particles

on surfaces of murine calvaria, with the assessment

of sagittal suture area as a parameter of osteolysis.

This model was utilized to study drug efficacy in

preventing debris-induced osteolysis, and appears to

be the only other small animal model to have the

advantages of being sensitive, rapid, and low-cost in

screening therapeutic agents with potential to pre-

vent wear-debris associated bone loss. Both models

may be applicable to study osteolytic response to

wear debris at different concentrations, shapes and

sizes in vivo, and are suitable to screen the effect of

pharmaceutical intervention on wear-debris induced

osteolysis. The air-pouch model may have certain

advantages, since the air pouch is the only blind

connective tissue cavity apart from the synovium

that lacks a mesothelial basement membrane. With

bone implantation and injection of wear-debris

particles, this model has many features of failed

total joint arthroplasty. Pouch fluid is available to

examine the release of proinflammatory mediators

(cytokines), and measure free calcium to indicate

bone resorption. In addition, it provides sufficient

tissue for the analysis of histological, molecular and

biochemical aspects of the response, including

Figure 7. Collagen loss of implanted bones was illustrated by

van Gelson staining (for calvaria) or modified Trichrome stain

(for femur implants), and quantified by Image-Pro software to

compare IOD of bone area closely contacting inflammatory

pouch membrane and density measured on the inner part of

bone tissue. The ratio was plotted as percentage of bone col-

lagen loss (*pv0.05).

Figure 8. Free calcium ion concentrations in the pouch fluid

were determined as a measure of bone demineralization (see

above, *pv0.05).

Murine osteolysis model 355

www.scandjrheumatol.dk

sensitive and reliable techniques such as real-time

RT-PCR for the evaluation of cytokine and

osteoclast markers.There are several limitations to this mouse model.

One of the most noticeable is the lack of blood

supply to the implanted bone. Although the air

pouch has been referred as an in vivo culture system

(15), implanted bone will deteriorate after extended

periods of implantation without a vascular supply.

We observed that beyond 14 days of implantation,

the overall elevated calcium release and degradationof bone collagen due to avascularity of implanted

bone masked particle-associated bone resorption.

This observation restricts the model to the study of

acute osteolysis, rather than the chronic osteolysis

seen in aseptic loosening. Different types of bone

implants may be employed in this model, and while

femoral implants provide assessment of a typical

articulating bone, they cannot be sectioned withoutdecalcification, which affects some immunological or

histological determination, such as TRAP staining

for the study of osteoclastogenesis. While calvaria

implantation can be adopted for the latter purposes,

this bone is not anatomically typical of bony

surfaces involved in aseptic loosening. Separate

analyses may therefore be required in the evaluation

of bone collagen and calcium changes. Despite theselimitations, this model appears to be useful in the

basic in vivo investigation of cellular responses to

various types of wear-debris and their osteolytic

effects under strict control experimental condition,

and in the screening of therapeutic agents/therapy

for debris-associated bone resorption.

In summary, this mouse air pouch model of bone

resorption provides a sensitive, rapid, economicaland reproductive way to obtain insights into the

cellular and molecular basis of wear-debris induced

osteolysis in aseptic loosening.

Acknowledgements

This work has been supported in part by research grants from the

Veterans Administration (Rehabilitation Section) and the Arthritis

Foundation. The authors acknowledge the excellent technical

assistance of Bin Wu and Lois Mayton.

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