Defects in articular cartilage metabolism and early arthritis in fibroblast growth factor receptor 3...

Defects in articular cartilage metabolism and early arthritis in

fibroblast growth factor receptor 3 deficient mice

G. Valverde-Franco 1,3, J.S. Binette 3, W. Li 1,3, H. Wang 1,3, S. Chai 1,3, F. Laflamme 4, N. Tran-

Khanh 5, E. Quenneville 5, T. Meijers 6, A.R. Poole 2 , J.S. Mort 2, M. D. Buschmann 5 and

J.E. Henderson 1,3.

1 J.T.N. Wong Laboratories for Mineralized Tissue Research, 2 Joint Diseases Laboratory,

Shriner’s Hospital for Children and 3 Centre for Bone and Periodontal Research, McGill

University, Montréal, Québec, Canada. Departments of 4 Engineering Physics and 5 Chemical

Engineering, École Polytechnique, Montréal, Québec, Canada. 6 TNO Pharma, Utrechtseweg 48,

The Netherlands

Corresponding Author

Janet E. Henderson PhD

Associate Professor, Associate Dean Research

Faculty of Medicine, McGill University

J.T.N. Wong Laboratories for Mineralized Tissue Research

Centre for Bone and Periodontal Research

740 Ave Dr Penfield, Room 2200

Montreal H3A 1A4

Tel: 514-398-3523

[email protected]

© The Author 2006. Published by Oxford University Press. All rights reserved

HMG Advance Access published April 19, 2006 by guest on M

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ABSTRACT

Fibroblast growth factor (FGF) receptor 3 has been identified as a key regulator of endochondral

bone development and of post natal bone metabolism through its action on growth plate

chondrocytes and osteoblasts respectively. It has also been shown to promote chondrogenesis

and cartilage production by cultured pre-chondrogenic cells in response to FGF18. In the current

studies we show that the absence of signalling through Fgfr3 in the joints of Fgfr3-/- mice leads to

premature cartilage degeneration and early arthritis. Degenerative changes in cartilage matrix

included excessive proteolysis of aggrecan core protein and type II collagen, as measured by

neoepitope immunoreactivity. These changes were accompanied by increased expression of

MMP13, type X collagen, cellular hypertrophy and loss of proteoglycan at the articular surface.

Using a novel micro-mechanical indentation protocol, it was shown that articular cartilage in the

humeral head of four month old Fgfr3-/- mice was less resistant to compressive force and less

stiff than that of littermate controls. These results identify Fgfr3 signaling as a potential target for

intervention in degenerative disorders of cartilage metabolism. by guest on May 3, 2014


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INTRODUCTION

Hyaline cartilage is found in the developing growth plates and covering the articulating

surfaces of long bones in the adult skeleton (1,2). Growth plate cartilage becomes mineralised

and is partially resorbed to form a template on which trabecular bone of the primary spongiosa is

laid down during endochondral bone development (3). Articular cartilage remains un-

mineralised to form a resilient, low friction surface that acts to absorb the shock caused by high

impact mechanical loading. It is composed primarily of an avascular type II collagen and

proteoglycan (aggrecan) matrix that is secreted and maintained by relatively few chondrocytes in

the adult. Articular cartilage therefore has a very limited capacity for repair. Focal lesions caused

by trauma, infection or biochemical imbalance do not heal adequately and tend to progress

rapidly to osteoarthritis (4,5). This is accompanied by increased proteolysis of proteoglycan and

collagen leading to cartilage matrix degradation. Many lines of investigation are thus focused on

the development of improved technology for cartilage tissue engineering and assisted repair.

Assisted repair of focal defects in articular cartilage may be accomplished by ex-vivo

expansion of chondroprogenitor cells and subsequent transplantation in a scaffold with a source

of growth factors to promote differentiation and cartilage production (6-8). Growth factors that

have been used with some degree of success include bone morphogenic proteins (BMPs) and

transforming growth factor β (TGF β) (9-12), platelet-derived growth factor (PDGF) (13) and

fibroblast growth factors (FGFs) (14-17). In chondrocytes, FGFs signal through three of four

related receptors that undergo alternative splicing and are linked to the MEKK, phospholipase C

and STAT signaling pathways (18). Targeted mutations of the gene encoding FGF receptor

three (fgfr3) in mice resulted in developmental defects in endochondral bone that mimicked

those seen in humans carrying the equivalent activating point mutations (19-22). Mice deficient

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in Fgfr3 signaling are phenotypically normal at birth but develop severe kyphosis, osteomalacia

and osteopenia by four months of age (23,24). Additional in vivo and in vitro work has shown

that these effects on growth plate cartilage and endochondral bone are mediated primarily

through FGF2 and FGF18 interactions with Fgfr3 (25-27). Furthermore, mRNA encoding

FGF18, FGFR2 and FGFR3 have been identified in articular cartilage explants removed from the

talus joints of adult human donors (28)

Previous work has therefore identified Fgfr3 signaling as a key regulator of chondrocyte

function in the developing growth plates and of osteoblast function in post natal bone. FGF2 has

also been identified as an important mitogen for articular chondrocytes ex vivo. The current

work was focused on defining the role of Fgfr3 signaling in post natal articular cartilage. The

shoulder and knee joints of Fgfr3+/+ and Fgfr3-/- mice were examined at four and nine months of

age for radiologic, histologic and molecular evidence of cartilage degradation. Compared with

their wild type littermates, Fgfr3-/- mice demonstrated narrowing of the humeral joint space and a

reduction in subchondral bone volume as early as four months of age. These changes were

accompanied by increased expression of type X collagen and increased cleavage of aggregan and

type II collagen, as evidenced by immunochemical staining with neo-epitope antibodies. These

results demonstrate an important role for FGFR3 in the maintenance of articular cartilage

integrity and identify the FGF-FGFR3 signaling pathway as a potential target for early

intervention in degenerative cartilage disease.



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RESULTS

In previous work, signaling through Fgfr3 in chondrocytes was shown to play an important role

in the development and maintenance of growth plate cartilage. An extensive literature documents

show similarities in the regulation of growth plate and articular chondrocyte biology. We

therefore hypothesized that the congenital absence of Fgfr3 signaling would lead to altered

cartilage development and metabolism of cartilage in the joints of Fgfr3-/- mice.

Radiologic evidence of joint degeneration in FGFR3-/- mice

X-ray analysis of the knee (A, B, E, F left panel), shoulder (A, B, E, F right panel) and paws (C,

D, G, H) of Fgfr3+/+ and Fgfr3-/- mice at 4 months (A-D) and 12 months (E-H) showed

progressive joint deterioration (arrows), narrowing of the joint space (arrowheads) and soft tissue

swelling (asterisk) in Fgfr3-/- compared with Fgfr3+/+ mice (Fig 1). The micro-architecture of the

shoulder joints was assessed and quantified using micro CT. Three-dimensional rendering and

reconstruction of the scans from 4mo (Fig 2A-B) and 9mo (Fig 2C-D) mice showed progressive

collapse of the humeral neck, roughened bone surfaces, numerous osteophytes protruding into

the joint cavity and abnormal joint articulation in Fgfr3-/- mice compared with FGFR3+/+ mice.

These changes were accompanied by a narrowing of the joint space (Fig 2E) and a reduction in

sub-chondral bone volume in the Fgfr3-/- mice (Fig 2F).

Quantifiable, destructive changes in the joints of mice lacking FGFR3 signaling were

therefore evident as early as 4mo of age. To characterize these changes at the molecular level we

performed histological analyses using molecular markers of cartilage matrix degradation.

Premature cartilage degradation and type X collagen in the knees of FGFR3-/- mice



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The knee joints of 4mo old mice (Fig 3A-D) stained with Safranin O (Fig 3A-B) appeared

similar except for an apparent increase in cell number and large lacunae and faint staining for

proteoglycan in the underlying cartilage layer in Fgfr3-/- compared with FGFR3+/+ mice. Faint

type X collagen immunoreactivity (Fig 3C-D), which is associated with chondrocyte

hypertrophy, was also apparent at the articular surface in Fgfr3-/- but not in the Fgfr3+/+ mice. By

9mo of age (Fig E-H), weak proteoglycan staining was also seen in the underlying mineralized

cartilage in both Fgfr3+/+ and Fgfr3-/- mice (Fig 3E-F). Fgfr3-/- mice also demonstrated a

significant reduction in proteoglycan at the articular surface, fibrillation and meniscal

hypertrophy, as well as an increase in type X collagen immunoreactivity (Fig 3G-H), Taken

together these changes suggested progression of the articular chondrocytes toward a terminally

differentiated phenotype, as seen in the transition zone of growth plate cartilage. It has been

proposed that these hypertrophic changes require a concomitant increase in the cleavage of

proteoglycan and type II collagen by locally active matrix metalloproteinases, particularly

MMP13 (29).

Increased cleavage of proteoglycan and type II collagen in the joints of Fgfr3-/- mice

The DIPEN antibody was used to distinguish fragments of aggregan generated by specific

cleavage of DIPEN341F342FGVG by MMP. The COL2-3/4Cs antibody was used to recognizes a

unique amino acid sequence neoepitope in the COOH-terminus of the large 3/4 fragment of the

a1(II) chain of type (II) collagen generated by cleavage by collagenase. Fig 4 shows a modest

increase in COL2-3/4Cs staining at 4mo in Fgfr3-/- joints (Fig 4D) and a significant increase in

both DIPEN (Fig 4F) and COL2-3/4Cs (Fig 4H) staining at 9mo compared with littermate

controls (Fig 4C, E, G). The increase in immunostaining in situ for type II collagen cleavage



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products was corroborated by an increase in the circulating level of type II collagen metabolites

in Fgfr3-/- mice compared with age-matched controls (Table 1). In keeping with the reduction in

subchondral bone there was also an increase in circulating C-telopeptide, a breakdown product of

type I collagen, the major collagen present in bone

Increased MMP13 in the joints of Fgfr3-/- mice

Metalloproteinases (MMPs) 13 and 9 are the principal enzymes responsible for degradation of

hyaline cartilage in the growth plates and the articular joints. Using antisera raised against

MMP13 and MMP9 it was shown that MMP13 (Fig 5A-B, E-F) was increased in articular

cartilage of the humeral head at 4 and 9 months in Fgfr3-/- mice (Fig 5B, F) and at 9 months in

Fgfr3+/+ mice (Fig 5E). MMP9 immunoreactivity was not apparent in articular cartilage of either

genetic strain at any age tested (Fig 5C-D, G-H). In contrast, MMP was detected in bone marrow

cells, in trabecular bone and in growth plate chondrocytes (data not shown) in all mice at all

ages.

Altered biomechanical properties of articular cartilage in Fgfr3-/- mice

Cartilage stiffness and integrity are largely dependent on the presence of an intact cross-linked

network of collagen entrapping the highly charged proteoglycan, aggrecan, which in turn

effectively retains interstitial water. Biomechanical properties of cartilage are most accurately

assessed using isolated cylindrical disks (30). However, the small size of the mouse joint

precludes such an approach. We therefore developed a micro-indentation test that can be applied

to intact small joints, similar to a previously published method (31). The mean thickness of

articular cartilage at the head of the humerus, as shown in Fig 6, was 24.75 ± 6.93 in Fgfr3+/+



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mice and 38.61 ± 7.65 microns in Fgfr3-/- mice (p <0.02). We then tested the resilience and

stiffness of articular cartilage on freshly dissected left humeral heads from 4-month old Fgfr3-/-

and Fgfr3+/+ mice. Although there was no detectable increase in aggregan cleavage at 4mo in

Fgfr3-/- mice, the loss of intact type II collagen resulted in a significant decrease in both cartilage

resilience (Fig 7A) and stiffness (Fig 7B). Similar, but more variable, changes were seen when

the right humerus was substituted for the left (data not shown).



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DISCUSSION

Generalized destruction of articular cartilage, such as that seen in osteoarthritis, is a common

occurrence in people over the age of 60. In many instances it is believed to be a consequence of

prior joint injury that does not heal due to the limited capacity of cartilage for self repair. While

the signaling pathways linked to receptors for FGFs have been identified as potential targets for

intervention in assisted cartilage repair, little is known about their role in chondrogenesis and

cartilage metabolism. In previous work we demonstrated a critical role for Fgfr3 signaling in

cartilage production in vitro. In this study we have found that the absence of signaling through

Fgfr3 in chondrocytes in vivo leads to a similar degeneration of articular cartilage to that seen in

human OA. Significant differences in proteoglycan content, expression of MMP13 and type X

collagen and cleavage of aggrecan and type II collagen were seen as early as 4mo in Fgfr3-/-

mice compared with their littermate controls. This work identifies FGFR3 signaling as a critical

regulator of articular cartilage metabolism and a potential pathway for early intervention in

degenerative cartilage disease.

Reduction in joint space in Fgfr-/- mice

Under physiological circumstances the head of the humerus fits into a socket formed by the outer

edge of the scapula, where it is held in place by the rotator cuff. Micro CT analyses showed

misalignment of the humerus with the scapula, collapse of the humeral neck and an apparent

reduction in the joint space by 4mo of age in FGFR3-/- mice. The reduced space could not be

accounted for by a reduction in the thickness of articular cartilage in the Fgfr3-deficient mice, in

which the layer of uncalcified tissue was significantly thicker at 4mo of age. It did however



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contain more hypertrophic chondrocytes and less matrix than that of the wild type mice, which

would have resulted in less resistance to compression when the joint was straightened for

imaging (see discussion of biomechanical properties). These defects were seen in association

with reduced sub-chondral bone volume and with the appearance of marginal osteophytes, a

typical feature of late OA (32-34). The fact that C-telopeptide was elevated in the serum of

Fgfr3-/- mice argues against focal bone loss due to inflammation, and for generalized bone loss as

a consequence of Fgfr3 deficiency (35-37). The later conjecture is supported by previous work

demonstrating increased MMP13, cortical thinning and osteopenia in 4mo old Fgfr3-/- mice

resulting from an intrinsic osteoblast defect (24).

Expression of type X collagen at the articular surface in Fgfr3-/- mice

The growth plates of long bones contain gradients of chondrocytes from committed chondro-

progenitor cells to fully differentiated hypertrophic cells that undergo apoptosis at the chondro-

osseous junction (3). The transition from immature matrix-producing cells to mature cells that

mineralize their surrounding matrix, is accompanied by a switch from primarily type II to type X

collagen. This transition is tightly regulated by coordinated signaling through growth factor

receptors, including PTH1R and FGFR3 (38-40). Expression of type X collagen in articular

cartilage is usually restricted to the layer of hypertrophic cells below the tidemark (41). Type X

collagen immunoreactivity was apparent in a few cells at the articular surface in 4mo old Fgfr3-/-

mice and greatly increased in 9mo mice deficient in Fgfr3 signaling. These results were

consistent with the decrease in safranin O-positive matrix and the increase in large lacunae at the

articular surface. These results support the hypothesis that Fgfr3 signaling is required to stabilize

the phenotype of articular chondrocytes and prevent progression to terminal differentiation.



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Corroborating evidence comes from previous studies demonstrating Fgfr3 deficiency promoted

type X collagen expression and hypertrophic differentiation of growth plate chondrocytes

(23,40,42).

Increase in aggrecan and type II collagen cleavage in Fgfr3-/- mice

The tensile strength of articular cartilage is conferred by a healthy fibrillar network of type II

collagen that traps aggrecan complexes, which in turn confer stiffness. The initial cleavage of

type II collagen by endogenous collagenase/s generates a ¾ fragment that is recognized by the

site-specific antibody Col2-3/4Cshort (43). Similarly, cleavage of aggrecan by MMP is site-

specific between the G1 and G2 domains to generate a neo-epitope recognised by the VDIPEN

antibody (44). The increase in type II collagen and aggrecan cleavage products in the articular

cartilage of Fgfr3-/- mice resembled that seen in mice over-expressing MMP-13 (45). This

predicted increase in protease activity in the articular cartilage of Fgfr3-/- mice is consistent with

that shown previously in diaphyseal osteoblasts in 4mo old mice (24). FGF2 is a universal ligand

for all FGF receptors (46,47) and accumulates in the synovial fluid of arthritic joints, where it is

believed to stimulate MMP-13 (collagenase 3) production by articular chondrocytes (48,49).

Compensatory signaling by FGF2 interacting with FGFR1 or FGFR2, which are both up-

regulated in the absence of Fgfr3 (24,27), could have resulted in the observed increase in MMP-

13, which would result in increased type II collagen cleavage in the joints of Fgfr3-/- mice. The

fact that circulating levels of the cleavage products of both type II (3/4Cshort) and type I (CTX-1)

collagen were elevated in the circulation of Fgfr3-/- mice argues strongly for a generalized defect

in MMP-13 mediated collagen metabolism. This conjecture is supported by the observed

increase in MMP13 immunoreactivity in cartilage and bone over time. Consistent with the



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published literature, MMP9 expression was apparent in growth plate but not in articular cartilage

(29, 45, 50). The increase in VDIPEN epitope is also consistent with upregulation of MMP-13

activity, although proteoglycan cleavage could also occur as a remodeling and repair response to

the loss of type II collagen integrity.

Impaired biomechanical properties of articular cartilage in Fgfr3-/- mice

The current literature indicates that the biomechanical properties and functional integrity of

articular cartilage deteriorates prior to the appearance of visible signs of surface erosion. Using

an arthroscopic indentation probe it was shown in a cohort of 50 patients with chronic anterior

cruciate ligament deficiency that cartilage stiffness decreased over time after injury and often

preceded the appearance of visible cartilage defects at the time of reconstructive surgery (51). In

other studies using focal indentation probes cartilage stiffness correlated with proteoglycan

content, as measured by gadolinium-enhanced magnetic resonance imaging but not with cartilage

thickness (52,53). Using a micro-mechanical indentation tester we showed a similar reduction in

cartilage stiffness in the shoulders of 4 month old Fgfr3-/- mice, in which the depth of articular

cartilage was in fact greater than that seen in Fgfr3+/+ mice. The loss of stiffness could be

attributed to increased degradation of type II collagen, demonstrated by in situ immunostaining

and ex vivo by Elisa assay of circulating levels of metabolites. Increased degradation of collagen

type II is in turn related to the increase in type X collagen hypertrophic cells seen at the articular

surface, which produce less proteoglycan than non-hypertrophic cells and occupy a larger

volume of the cartilage matrix. Further credence is given to this hypothesis by the observed

increase in type II collagen metabolism, demonstrated by in situ immunostaining and ex vivo by

Elisa assay of circulating levels of metabolites. This decrease in cartilage stiffness in association



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with impaired type II collagen metabolism is reminiscent of that seen in 15 month old Col2a1+/-

mice (31), which synthesize approximately 50% less type II collagen than their wild type

littermates. Interestingly, these mice also exhibited a reduction in sub-chondral bone volume, as

seen in our own Fgfr3-/- mice at 4 months of age. The reduction in sub-chondral bone volume no

doubt reflects the defects in FGFR3-/- osteoblast function described previously (24) and most

probably led to the obvious deformation of the articulating surfaces seen at 9 months of age.

Another potential explanation for the loss of cartilage stiffness is joint mis-alignment, as shown

by X-ray and micro CT imaging, which could lead to reduced mobility. In fact it was shown that

11 weeks of joint immobilization in dogs resulted in a 42% decrease in cartilage stiffness (54).

However, the morphological defects in the skeletons of 4 month old Fgfr3-/- mice do not appear

to inhibit their activity or their access to food. The combination of joint misalignment and

reduced sub-chondral bone volume could however compromise the overall biomechanical

properties of diarthrodial joints and combined with premature cartilage degradation, could lead

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MATERIALS and METHODS

Mice

All mouse procedures were performed in accordance with McGill University guidelines, which

are set by the Canadian Council on Animal Care. Mice homozygous for targeted inactivation of

the gene encoding Fgfr3, and wild type littermates, were obtained from intercross of FGFR+/-

maintained on a C3H background as described (24). Cohorts of mice were anesthetized and

euthanised by exsanguination at two, four, six, nine and 12 months for analyses. Representative

data is shown for 4mo and 9mo old Fgfr3+/+ and Fgfr3-/- mice

Radiologic imaging

High resolution X-rays of the shoulder joints, knees, forepaws and hindpaws were obtained on

euthanised mice or the dissociated limbs using a Faxitron MX20, equipped with an FPX-2

Imaging system (Dalsa Medoptics, Waterloo, Ontario, Canada). Immediately after X-ray the left

shoulder was dissected free of soft tissue, fixed overnight in 4% paraformaldehyde, rinsed three

times with PBS and scanned on a Skyscan1072 microCT instrument (Skyscan, Antwerp,

Belgium). Image acquisition was performed at 100 kV and 98 µA with a 0.9° rotation between

frames to obtain two-dimensional images, which were used to generate three-dimensional

reconstructions. The joint space and subchondral bone volume were calculated with the three-

dimensional Creator software supplied with the instrument.

Histology, histochemistry and immunochemistry



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All histology, histochemistry and immunohistochemistry procedures were performed essentially

as described previously (24,55). The right knee and shoulder joints were dissected free of soft

tissue, fixed for 16h in 4% paraformaldehyde at 4°C and rinsed three times in PBS. Bones were

decalcified in 4.13% EDTA for 14 days at 4°C, embedded in paraffin and 4 m sections cut on a

rotary microtome for staining with safranin-O and fast green to identify proteoglycan. Left

shoulder joints were embedded in LR white acrylic resin (London Resin Co. Ltd., London, UK)

and sections cut on an ultra microtome for staining with 0.2% toluidine blue for 1 minute to

identify mineralized and un-mineralized cartilage. Immunohistochemical analyses were

performed on 4µm sections of paraffin embedded tissue using the following primary antisera:

rabbit anti- C- terminal peptide Val-Asp-Ile-Pro-Glu-Asn of aggrecan G1 domain, (DIPEN

1: 800) (44); rabbit anti-peptide Gly-Pro-Hyp-Gly-Pro-Gln-Gly, which represents a type II

collagen primary cleavage site (COL 2 3/4C short peptide 1:200) (43); mouse anti-type X

collagen peptide Tyr-Asp-Pro-Arg-Thr-Gly-Ile-Phe-Thr (1:300) (56); rabbit anti-MMP9 peptide

(Sigma Saint Louis, MO, USA 1:200) and rabbit anti-MMP13 peptide ovalbumin conjugates

kindly provide by Dr. J.S. Mort (1:300).

Biomechanical testing

The left humeral heads of 4mo old Fgfr3+/+ and Fgfr3-/- mice were used for testing the

biomechanical properties of articular cartilage using a novel indentation test developed on a

Mach-1® micro-mechanical tester (BioSyntech Canada Inc., Laval, Canada). Samples were

dissected free of soft tissue and the distal metaphysis fixed with dental stone in a small plastic

container before dissecting the joint capsule to expose the articular surface. The humeral head

was immersed in PBS for 30 minutes at room temperature to equilibrate before controlled



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compression was applied by an actuator with 0.1 µm displacement precision, which moved a

cylindrical flat tip indentor of 60 µm diameter positioned perpendicular to the articular surface.

Ramp compressions at 5µm/s were applied until a 2g load was reached at two different positions

on each humeral head and the force response captured by a load cell with 7 mg precision. The

point of contact was determined by visual inspection of the loading curve and cartilage stiffness

was assessed by calculating load-displacement slopes in the displacement intervals of 2.5 to 5.5

m (S1), 5.5 to 8.5 m (S2) and 8.5 to 11.5 μm (S3). The results were analyzed by MANOVA,

using the general linear model with Fgfr3+/+ vs Fgfr3-/- as a categorical predictor and

displacement as a continuous predictor.

Biochemical analyses

A commercial ELISA assay was used to determine circulating levels of the type I collagen

degradation product CTX-I (Mouse C-telopeptide, Nordic Bioscience Diagnostics, USA).

Circulating type II collagen cleavage products were measured with the DELFIA® ELISA assay (

Perkin Elmer, Wellesley, Ma. USA) that uses a monoclonal C2C antibody raised against the

carboxyl-terminal 3/4 fragment of the degraded 1 (II) chain. This assay uses a time-resolved

immunofluorometric technique that greatly reduces background fluorescence (57).



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ACKNOWLEDGEMENTS

This work was supported by grants to JEH from the Canadian Institutes of Health Research

(CIHR), the Arthritis Society of Canada (TAS) and the Canadian Arthritis Network Centres of

Excellence, and to MDB from CIHR. GVF was supported by a studentship from the Fonds de la

recherche en santé du Québec (FRSQ) and SC, FL and NT-K by training awards from the CIHR

Strategic Training Program in Skeletal Health Research. JEH is a Chercheur Boursier Senior of

the FRSQ and MDB holds a Canada Research Chair in Cartilage Tissue Engineering. The

authors acknowledge the expert technical assistance of Ailian Li (J.T.N. Wong Laboratories) and

Miren Gratton (Centre for Bone and Periodontal Research).



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FIGURE LEGEND

Fig 1 X-ray analysis of joints at 4 and 12 months in Fgfr3+/+ and Fgfr3-/- mice

High resolution Faxitron x-rays of the knees (A, B, E, F left), shoulder joints (A, B, E, F right)

and paws (C, D, G, H) of Fgfr3+/+ and Fgfr3-/- mice at 4 months (A-D) and 9 months (E-H) of

age. Arrows and arrowheads show progressive joint destruction and asterisks show soft tissue

swelling in the Fgfr3-/- mice . Images captured at x2 magnification and are representative of 8-10

mice of each genotype at each age.

Fig 2 Quantitative microCT analysis of the shoulder joints of Fgfr+/+ and Fgfr-/- mice

Representative three dimensional reconstructions (A-D left hand panels) and mid-sagital two

dimensional sections (A-D right hand panels) of the shoulder joints of Fgfr3+/+ and Fgfr3-/- mice

at 4 months (A, B) and 9 months (C, D) of age were captured on a Skyscan®1072 instrument.

Significant deformation and mis-alignment of the joint was seen at 4 months and 9 months in

Fgfr3-/- mice, in association with a reduction in the joint space (F) and significant thinning of the

sub-chondral bone plate (G). Fgfr3+/+ (white bars) and Fgfr3-/- (black bars). Results in panels E

and F are expressed as the means ±SD of 3-5 measurements made on 6-8 mice of each genotype

at each age. * Significantly different from Fgfr3+/+ * p< 0.01

Fig 3 Histochemical and immunochemical staining for proteoglycan and type X collagen

The knee joints of Fgfr+/+ and Fgfr3-/- mice at 4 months (A-D) and 9 months (E-H) of age were

decalcified, embedded in paraffin and stained with Safranin O to identify proteoglycan (A, B, E,

F) or immunostained to identify type X collagen (C, D, G, H). Articular cartilage in the distal

femur and proximal tibia in Fgfr3-/- mice showed an increase in cell number and large lacunae at



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4 months (B) and a reduction in cell number and proteoglycan content at 9 months (F). Type X

collagen immunoreactivity was seen in the articular cartilage of Fgfr3-/- mice at 4 and 9 months

(D, H) but not in that of Fgfr3+/+ mice (C, G). Results are representative of those obtained from 5

mice of each genotype at each age. Magnification at source x20.

Fig 4 Immunochemical staining for neoepitopes of aggrecan and type II collagen

The knee joints of Fgfr3+/+ and Fgfr3-/- mice at 4 months (A-D) and 9 months (E-H)) of age were

decalcified, embedded in paraffin and stained with DIPEN, to identify aggrecan cleaved by

MMPs (A, B, E, F) or with an antibody that recognizes the ¾ C terminal fragment of type II

collagen cleaved by MMP13 (C, D, G, H). In Fgfr3-/- mice, aggrecan cleavage was increased at 9

months (F) compared with Fgfr3+/+ (E) mice. Type II collagen cleavage was significantly

increased at 4 months (D) and 9 months (H) compared with wild type control (C and G

respectively). Results are representative of those from 5 mice of each genotype at each age.

Magnification at source x20

Fig 5 Immunochemical staining for MMP13 and MMP9 in Fgfr3+/+ and Fgfr3-/- mice

The humerus of Fgfr+/+ and Fgfr3-/- mice at 4 months (A-D) and 9 months (E-H) of age were

decalcified, embedded in paraffin and immunostained with antisera that recognises MMP13 (A-

B, E-F) or MMP9 (C-D, G-H). Whereas MMP13 expression increased over time in both genetic

strains the staining was far more intense in Fgfr3-/- mice. Little difference was seen in MMP9

expression between Fgfr+/+ and Fgfr3-/.

Fig 6 Identification of un-calcified cartilage in the humerus of Fgfr3+/+ and Fgfr3-/- mice



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Specimens were prepared in the same manner as for Fig 5 and stained with Safranin O (A-B, E-

F) or Toluidine blue (C-D, G-H) to measure the depth of un-mineralized cartilage at the articular

surface. While the depth of unmineralized cartilage appeared greater in Fgfr3-/- mice compared

with Fgfr+/+, there was significantly less subchondral bone in the mutant mice at both ages.

Fig 7 Biomechanical properties of articular cartilage in Fgfr3+/+ and Fgfr3-/- mice

The freshly dissected heads of humeri from 4 month old Fgfr3+/+ and Fgfr3-/- mice were

subjected to loading by a flat tip indentor applied by an actuator moving at 5 m/s with a 0.1 µm

displacement precision (Mach-1® micro-mechanical tester). The load was measured

continuously and is shown at displacements of 4 m, 7 m and 10 m (A), while cartilage

stiffness was approximated as slopes of load vs displacement in the displacement intervals of 2.5

to 5.5 m (S1), 5.5 to 8.5 m (S2) and 8.5 to 11.5 μm (S3) (B). The response to loading (A) and

resulting stiffness (B) were significantly decreased in the Fgfr3-/- mice (hatched line) compared

with Fgfr3+/+ mice (solid line), as determined by MANOVA with Fgfr+/+ / Fgfr3-/- and

displacement as the predictors. The results are representative of 25 measurements made on n=13

FGFR3+/+ mice and 24 measurements on n=15 FGFR3-/- mice.



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TABLE 1 Serum levels of type II (C2C) and type I (CTX-1) collagen metabolites

C2C (ng/ml) CTX-1 (ng/ml)

Age

Fgfr3+/+ Fgfr3-/- Fgfr3+/+ Fgfr3-/-

2 mo 37.42±10.30 62.90±13.03* 18.98±1.26 34.54± 1.31*

4 mo 39.89± 8.87 54.80±10.10* 16.70±3.38 27.28±10.69*

6 mo 36.34± 6.17 46.24± 8.15* 14.22±3.20 24.11± 4.56*

9 mo 45.43± 7.30 55.75± 5.71* 16.90±3.96 35.69± 7.30*

Values are expressed as the mean ± SD of 20-25 serum samples from representative mice in each

group. Significantly different from Fgfr3+/+ *p< 0.01



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Defects in articular cartilage metabolism and early arthritis in fibroblast growth factor receptor 3...

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