Download - Developmental and osteoarthritic changes in Col6a1 -knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrix

DEVELOPMENTAL AND OSTEOARTHRITIC CHANGES IN Col6a1KNOCKOUT MICE: THE BIOMECHANICS OF COLLAGEN VI INTHE CARTILAGE PERICELLULAR MATRIX

Leonidas G. Alexopoulos1, Inchan Youn1, Paolo Bonaldo2, and Farshid Guilak1

1Departments of Surgery and Biomedical Engineering, Duke University Medical Center, Durham,NC 27710, USA2Department of Histology, Microbiology and Medical Biotechnologies, University of Padova,35121 Padova, Italy

AbstractChondrocytes are the sole cell type in articular cartilage and maintain the extracellular matrixthrough a homeostatic balance of anabolic and catabolic activities that are influenced by geneticfactors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes areencapsulated by a narrow tissue region termed the “pericellular matrix”, which in normal cartilageis defined by the exclusive presence of type VI collagen. Because the pericellular matrixcompletely surrounds each cell, it is hypothesized to serve as a filter or transducer for biochemicaland/or biomechanical signals from the cartilage extracellular matrix. In this study, we usedCol6a1−/− mice to investigate whether the lack of collagen VI may affect the development andbiomechanical function of the pericellular matrix and alter the mechanical environment of thechondrocytes during joint loading. Col6a1−/− and Col6a1+/− mice possessed structurally intactpericellular matrices, but with significantly reduced mechanical properties as compared to wild-type controls. With age, Col6a1−/− showed accelerated development of osteoarthritic jointdegeneration, as well as other musculoskeletal abnormalities such as delayed secondaryossification process and reduced bone mineral density. These findings suggest an important rolefor type VI collagen in regulating the physiology of the synovial joint, and provide indirectevidence that alterations in the mechanical environment of the chondrocytes, either due to loss ofpericellular matrix properties or Col6α1−/− derived joint laxity, can lead to the progression ofosteoarthritis.

Keywordsarthritis; collagen VI; proteoglycan; pericellular matrix; chondron; chondrocyte

IntroductionArticular cartilage is the tissue that lines the surfaces of diarthrodial joints and serves as theresilient, low-friction, load-bearing material for joint motion. A sparse population of cells –chondrocytes - maintains the extracellular matrix (ECM) of this tissue through a balance ofanabolic and catabolic activities. The micromechanical environment of chondrocytes, in

Corresponding author: Farshid Guilak, Ph.D., Duke University Medical Center, 375 MSRB, Box 3093, Durham, NC 27710 USA,(919) 684-2521, Fax (919) 681-8490, [email protected] address for Dr. Alexopoulos: Department of Mechanical Engineering, National Technical University of Athens, Athens,15780, Greece

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Published in final edited form as:Arthritis Rheum. 2009 March ; 60(3): 771–779. doi:10.1002/art.24293.

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conjunction with biochemical (e.g., growth factors, cytokines) and genetic factors, plays animportant role in cartilage homeostasis and, as a consequence, the health of the joint (1–3).Chondrocytes in articular cartilage are enclosed by a narrow region of tissue, the pericellularmatrix (PCM), which together with the enclosed chondrocyte has been termed the chondron(4–7). The PCM is primarily characterized by the exclusive presence of type VI collagen innormal cartilage, but it also possesses a high concentration of proteoglycans, fibronectin,and types II and IX collagen (4,8).

The functional role of the PCM in articular cartilage is still unknown, although the fact thatit completely surrounds the cell suggests that it regulates the biomechanical, biophysical,and biochemical signals that the chondrocyte perceives (9). For example, interactionsbetween cell surface receptors and the ECM significantly influence matrix metabolism, geneexpression, and response to growth factors (10–12). Furthermore, cytokines and growthfactors that interact with the chondrocyte surface traverse the pericellular environment,where they may be retained and modified (13,14). From a biomechanical standpoint, therehas been considerable speculation that the PCM plays a critical biomechanical role in eitherin protecting the cells or serving as a “filter” or transducer of physical signals in the ECM(6,9,15–17), potentially through an interaction of type VI collagen with integrins or othercell surface receptors (18–21). Indirect evidence in support of these hypotheses is providedby experimental data showing that a newly formed PCM augments cellular metabolicresponse to biomechanical loading (22).

Type VI collagen serves as the defining boundary of the PCM in articular cartilage, but it isalso found in the ECM of many connective tissues (23). It has a characteristic beadedfilamentous structure of tetrameric units that consists of three different α-chains, α1(VI),α2(VI), and α3(VI). Collagen VI has high affinity with numerous ECM components (i.e.,biglycan, decorin, hyaluronan, fibronectin, perlecan, and heparin) as well as with the cellmembrane (24–28). Thus, it has been hypothesized that collagen VI plays important roles inmediating cell-matrix interactions as well as intermolecular interactions in various tissues aswell as cell cultures (29–33). In articular cartilage, collagen type VI forms a network thatanchors the chondrocyte to the PCM (34–37) through its interaction with hyaluronan(28,38), decorin (25), and fibronectin (39).

The goal of this study was to examine the hypothesis that lack of type VI collagen alters thebiomechanical properties of the PCM and ECM of articular cartilage. Collagen VI deficientmice were generated by targeted gene disruption of the Col6a1 gene (40). Histologicalanalysis and dual energy x-ray absorptiometry (DXA) were used to examine differences inskeletal development, bone mineral density, and progression of osteoarthritic jointdegeneration in wild-type and collagen VI deficient mice. In addition, micromechanicaltesting was performed on the articular cartilage and on isolated chondrons usingmicroindentation and micropipette aspiration techniques, respectively to determine the roleof type VI collagen on the elastic properties of the ECM and PCM of articular cartilage.

Materials and MethodsCollagen VI knockout mice

Collagen VI knockout mice were generated on a CD1 genetic background by targeted genedisruption of the Col6a1 gene, which is responsible for the production of α1(VI) chain (40).The elimination of the α1(VI) chain resulted in the absence of triple helical collagen VImolecules in the ECM (40). Mice were sacrificed at 1, 3, 6, and 11 months time points. Allprocedures had been approved by the Duke University Institutional Animal Care and UseCommittee.

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Fluorescence immunohistochemistryCollagen VI immunostaining was performed using a polyclonal anti-collagen VI antibodyraised against a peptide mapping near the amino terminus of murine α1(VI) chain (SantaCruz Biotechnology, CA). Cryostat sections of the coronal plane were obtained fromdecalcified femoral heads of mice using standard histological methods.

Skeletal StainingOne-month-old mice were sacrificed, skinned, and eviscerated. Alcian blue and alizarin redwere used to stain the cartilage and bone respectively using standard skeletal stainingtechniques.

Histological analysis and morphometric gradingFixed cryostat sections were stained with Toluidine blue and Hematoxylin and Eosin (H&E)using standard histological techniques. Osteoarthritic and developmental changes wereassessed using quantitative histomorphometric grading schemes on the femoral head of 60mice at ages of 1, 3, 6, and 11 months (41,42). Osteoarthritic grading was based on the sumscore of surface fibrillation (0 to 4), toluidine blue intensity staining (0 to 3), andfibrocartilage presence (0 to 2), which was reported as percentage of the 0 to 9 scale where0% corresponds to no histological signs of osteoarthritis and 100% corresponds to the mostsevere changes (grade 9). Specimens were classified as non-OA (score 0–1), mild OA (1–5),and severe OA (5 to 9).

For developmental changes, the histological grading scale was based on the presence of thegrowth plate and the extension of the secondary ossification center (42), which was gradedfrom 0 to 5 (0 corresponded to normal 1-month-old mice and 5 corresponded to normal 11-month-old mice). The grading scale was based on the assessment of the percentage of theossified area relative to the total area: cartilage with growth plate and no secondaryossification area present (grade 0), area of secondary ossification less than 10% (grade 1),area of secondary ossification more than 10% and less than 50% (grade 2), area ofsecondary ossification more than 50% and less than 90% (grade 3), area of secondaryossification more than 90% (grade 4), no presence of growth plate (grade 5). The secondaryossification was reported as a percentage of the 0 to 5 scale where 0% corresponds to grade0 (no secondary ossification) and 100% corresponds to the fully developed femoral head(grade 5).

Bone Mineral DensityDual energy X-ray absorptiometry (DXA, PIXImus, Lunar Corp, Madison, WI) was usedfor measuring bone mineral density (43). The mice were weighed and placed in supineposition in the DXA unit and the whole body except the skull was measured. A total of 82mice were analyzed at ages of 1, 3, 6, and 11 months.

Mechanical testing of articular cartilageA total of 18 right hip joints from one-month-old mice (Col6a1−/−: n=7, Col6a1+/+: n=7,Col6a1+/−: n=4) were tested in indentation using an electromechanical test system (ELF3200, EnduraTEC, Minnetonka, MN) instrumented with a low capacity load-cell (250g,Sensotec, Columbus, OH) and extensometer (1mm, Epsilon, Jackson, WY) (44). Plane-ended microindenters were machined from glass fibers (diameter: 110 µm, Thorlabs,Newton, NJ). A dual-angle camera system was used to optically align the indenter tipperpendicular to the cartilage surface (Fig. 8). After applying a tare load of 0.3 grams-forceand allowing it to equilibrate, four consecutive indentation displacements (5 µm/step with aramping speed of 1 µm/sec) were applied to the cartilage surface and allowed to equilibrate

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for 200 sec per step. The time, reaction force, and displacement data were collectedthroughout the test at 1 Hz. The equilibrium force vs. displacement curve was obtained fromthe linear region of the curve. After mechanical tests, the thickness of cartilage from thetissue surface to the calcified cartilage was measured at a site adjacent to the test site usingroutine histology (5 µm sections labeled with Safranin-O and fast green). The Young’smodulus of mouse cartilage was calculated using an elastic indentation model (45) withassumed Poisson’s ratio of 0.25 (44).

Mechanical testing of the pericellular matrixChondrons were mechanically isolated (n=93 from 26 donors) from the femoral articularcartilage of 1-month-old mice as described previously with a custom-built “microaspirator”,which applies suction pressure to the cartilage surface with a modified syringe (46). Themicropipette aspiration technique (47–49) was used to measure the mechanical properties ofthe PCM, as described previously (50–52). With this technique, the surface of the PCM isaspirated into a glass micropipette (12 µm diameter) by the application of a series ofcontrolled pressures up to 18 kPa, and the ensuing equilibrated aspiration length is measuredusing video microscopy (Fig. 8). The Young’s modulus of the PCM was determined using atheoretical model that represents the chondron as an elastic, compressible layer (i.e., PCM)overlying an elastic half-space (i.e., chondrocyte) (46).

Statistical analysisStatistical analysis was performed using a multi factorial Analysis of Variance (ANOVA)setup in Statistica (StatSoft, Tulsa, OK, USA). Categorical predictors were considered onlyAGE (1, 3, 6, 9, and 11) and GENOTYPE (+/+, +/−, and −/−). We assumed those variablewere able to predict 4 dependent measurements namely weight, OA score, BMD, andossification score. Full factorial design revealed that both AGE, GENOTYPE and theAGE*GENOTYPE effects were significant contributors, and thus post hoc comparison wasperformed using the Fisher LSD method. AGE effects were significant for all 4measurements as expected. GENOTYPE effects between Col6a1+/+ and Col6a1−/− weresignificant for all 4 measurements (Figure 3, Figure 4, and Figure 5). GENOTYPE effectsbetween Col6a1+/+, Col6a1+/−, and Col6a1−/− were significant only for OA and ossificationscores. AGE*GENOTYPE effects within the same age groups were not significant for OA.For ossification scored, significant difference was observed between Col6a1+/+ andCol6a1−/− at 3 months only. For BMD, a significant difference was observed betweenCol6a1+/+ and Col6a1−/− for all ages except for 1 month.

ResultsHistological Evaluation

Mice lacking collagen VI exhibited no apparent abnormalities and all animals survived untilsacrifice at 11 months. Wild type and heterozygous mice showed extensive pericellularlabeling for type VI collagen in the articular cartilage, whereas knockout mice revealed nopresence of type VI collagen (Fig. 1). Intense labeling of type VI collagen was also observedin the growth plate of one-month-old wild type mice (Fig. 1a).

Skeletal staining of bone and cartilage (Fig. 2) in one-month-old mice indicated thatCol6a1−/− mice are smaller in size and exhibit a slower ossification process of the upper(Fig. 2 C,D) and lower (Fig. 2 E,F) extremities that the wild type counterparts. The smallersize was also consistent with a trend toward lower body weight of one month old Col6a1−/−

mice (16.5±2.4 g) as compared to Col6a1+/+ mice (versus the 18.3±1.99 g) (p=0.11, two-tailed t-test).

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To better evaluate the developmental process, we measured the secondary ossificationprocess of the femoral head (Fig. 3A–C). Col6a1−/− mice showed significantly delayedossification at 3 months (grade 2.2±1.8) as compared to Col6a1+/+ mice (grade 4.1±0.2)(p<0.02) (Fig. 3D) (see also Materials and Methods for grading scale and statisticalanalysis). For the 3 months old mice, only one out of 6 Col6a1−/− mice showed ossificationgrade above 4, whereas 3 out of 3 Col6a1+/+ showed an ossification grade above or equal to4. The ossification process was complete for all mice after the 6th month.

Semi-quantitative histological analysis of cartilage degeneration revealed significant age-dependent osteoarthritic changes in the Col6a1−/− mice. Osteoarthritic changes depended onage (p<0.001) and genotype (p<0.05) (Fig. 4). For the 6- to 11-month-old mice, only 2/12 ofthe Col6a1+/+, 3/7 of the Col6a1+/−, and 11/16 Col6a1−/− scored above 1 and arecharacterized with OA (either mild or severe, see Materials and Methods for grading scale).However, 0/12 of the Col6a1+/+, 0/7 of the Col6a1+/−, and just 2/16 Col6a1−/− scored above5 and were characterized as exhibiting severe OA.

Bone Mineral DensityDXA revealed that wild type mice have significantly higher bone mineral density than theknockout counterparts at 3 and 6 months (p<0.001), although these differences were nolonger present by 11 months (Fig. 5)

Mechanical properties of articular cartilage and PCMThe PCM exhibited linear elastic behavior, and the Young’s modulus of the PCM ofchondrons isolated from Col6a1+/+ mice was significantly higher than those of heterozygousCol6a1+/− mice, which was further reduced in the knockout Col6a1−/− mice (Fig. 6C).Microindentation tests revealed no significant differences in the mechanical properties of thefemoral head articular cartilage Col6a1+/+, Col6a1+/−, or Col6a1−/− mice (Fig. 6D).

DiscussionThe findings of this study provide new evidence of significant musculoskeletal changes inCol6a1−/− mice. Primarily, our findings show that mice lacking collagen VI exhibitaccelerated development of hip osteoarthritis, as well as a delayed secondary ossificationprocess and lower bone mineral density. Lack of type VI collagen resulted in a loss of thestiffness (decreased modulus) of the PCM of the articular cartilage prior to any detectablehistological changes. However, no differences in ECM properties were observed. Thesefindings provide indirect evidence of a role for type VI collagen in regulating the physiologyof the chondrocyte, potentially due to alterations in the biological and mechanicalenvironment of the chondrocytes in articular cartilage due to changes in biomechanicalproperties of the PCM or due to increased joint laxity associated with a deficiency in type VIcollagen.

The mechanical environment of the chondrocytes is one of several environmental factorsthat influence the normal balance between the synthesis and breakdown of articular cartilageand is an important factor in etiopathogenesis of osteoarthritis (1–3,53,54). Thus, changes inthe mechanical interactions between the cell and ECM may have a significant influence onthe regulatory response of the chondrocyte. While a biomechanical function for the PCMhas long been hypothesized (5,6,9), there is growing evidence from both theoreticalmodeling and experimental studies that the PCM plays a significant role in regulating thebiomechanical signals perceived by the chondrocyte (17,55,56). In normal cartilage, themechanical properties of the PCM are relatively uniform with depth (57) but aresignificantly altered with osteoarthritis, exhibiting reduced stiffness and increased fluid

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permeability (46,58). The PCM appears to function by providing a relatively uniformcellular microenvironment despite large inhomogeneities in local tissue strain (17,59). Thus,a compromised PCM could significantly affect the mechanical environment of thechondrocytes in articular cartilage, leading to increased strain at the cellular level (56),which may affect catabolic responses at the level of single cells (60). In other tissues such asbone, however, the pericellular region (i.e., the glycocalyx) can serve as a strain amplifier bycoupling fluid drag forces to the actin cytoskeleton within the processes of osteocytes (61–63). In the present study, Col6a1−/− mice showed significantly reduced PCM stiffness at 1month of age, preceding any histological or biomechanical changes in the overall articularcartilage. With age, these mice exhibited accelerated development of osteoarthritis. Thesefindings provide indirect evidence that early alterations in the mechanical properties of thePCM are associated to the progression of osteoarthritis.

In normal articular cartilage, type VI collagen is exclusively present in the PCM and it hasbeen characterized as a discrete marker of chondron anatomy (36). For this reason, it hasbeen hypothesized that type VI collagen is necessary for providing the structural integrityand mechanical properties of the PCM. Contrary to our hypotheses, though, Col6a1−/− miceexhibited intact chondrons that could be isolated despite the lack of type VI collagen. Thisfinding suggests that proteins other than collagen VI provide some of the structural integrityof cartilage PCM. Nonetheless, the Young’s modulus (stiffness) of the PCM of Col6a1−/−

mice was dramatically decreased to nearly one-third of the wild-type controls indicating theimportant role of type VI collagen in the properties of the PCM.

An important issue that must be considered is the link between collagen VI deficiency andchanges in muscle physiology displayed by Col6a1−/− mice (40). Such link has also beenobserved in humans, where mutations of collagen VI genes have been shown to play acausal role in two inherited disorders of muscle, Bethlem myopathy and Ullrich congenitalmuscular dystrophy (UCMD) (64,65). It is possible that some features of UCMD,particularly joint laxity or predisposition to hip dislocation, may also contribute to theaccelerated hip degeneration observed in Col6a1−/− mice. Since joint laxity and PCMmechanical alterations are both inheritably coupled in Col6a−/− mice and both lead toaltered mechanical environment in chondrocytes, both factors can contribute to thedevelopment of OA. While the present study clearly shows an association between Col6a1deficiency and OA, presumably via mechanical alterations caused by joint laxity or alteredPCM properties, further studies aimed at developing and characterizing conditional ortissue-specific knockouts may be required to fully understand the mechanisms by whichCol6a1 deficiency leads to OA. Nonetheless, our results are consistent with thehypothesized role of type VI collagen as an integrating molecule in the structure of cells andtissues; downregulation of collagen VI is associated with tissue laxity and wasting (e.g.,Bethlem myopathy, UCMD, joint hyperlaxity), whereas collagen VI upregulation results inincreased fibrosis and tissue stiffness (e.g., Bullous keratopathy, scleroderma) (40,66–73).

In our experiments we found no gross morphologic differences between wild type andcollagen VI knockout chondrons other than reduced skeletal size of Col6a1−/− mice.Skeletal changes were apparent as a retardation of the developmental process until 11months of age. During development, histogenesis of long bones occurs via endochondralossification of cartilage tissue. During this process, chondrocytes in the epiphyseal platedifferentiate into mature hypertrophic cells and finally are eliminated from the growth plate(74). The hypertrophic cell lacunae are invaded by vessels carrying mesenchymal andosteogenic cells that differentiate into osteoclasts and synthesize a bony matrix. A similarprocedure, known as secondary ossification, takes place at the end of the bone where theformation of the bony epiphysis occurs. Our experimental results point to a slowing ofsecondary ossification changes and decreased bone mineral density in the Col6a1−/− mice.

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While there is no known direct mechanism coupling type VI collagen deficiency toendochondral ossification, type VI collagen may provide a scaffold for osteoblasts,preosteoblasts and chondrocytes to proceed to osteochondral ossification (75). In addition,type VI collagen has been linked to the early events of chondrocyte differentiation (76), tothe regulation of mesenchymal cell proliferation in vitro (77), and to ECM stabilizationduring development (36). It has also been hypothesized that collagen VI is important forchondrocyte proliferation and hypertrophy in cartilage (36,78,79). These studies inconjunction with our observation of the ubiquitous presence of type VI collagen in thegrowth plate (Fig. 1A) support the hypothesis that collagen VI deficiency may delay celldifferentiation and proliferation, resulting in delayed development and decreased boneformation. Interestingly, the COL6A1 gene was recently identified as the locus forossification of the posterior longitudinal ligament of the spine (75) and has been alsoassociated with increased systemic bone mineral density and diffuse idiopathic skeletalhyperostosis (80). These findings point to a role for collagen VI in diseases associated withhigh-bone-mass, consistent with the lower bone mineral density we observed in Col6a1−/−

mice. While it is beyond the scope of the present study to analyze the mechanisms resultingin altered bone mineral density, these changes may also be biomechanical in origin, ascollagen VI deficiency causes muscular dystrophy (40,68), which can lead to abnormalmechanical loading regime of the musculoskeletal systems.

In this study, the role of an abnormal mechanical environment on chondrocytes wasinvestigated by using collagen type VI knockout mice. Our findings suggest that collagen VIplays a major role in the mechanical properties of the PCM, and thus, the mechanicalenvironment of the chondrocytes. Col6a1−/− mice showed accelerated development ofosteoarthritis that may be “biomechanical” in nature, either via altered properties of thePCM or inheritable joint laxity. In addition, our findings provide direct evidence thatcollagen type VI might have a significant role on the osteochondral ossification process bymodulating the chondrocyte and mesenchymal cell differentiation and proliferationactivities. This model may provide a valuable tool to better understand how changes in themechanical environment of the chondrocytes may lead to abnormal skeletal developmentand development of osteoarthritis.

AcknowledgmentsThe authors would like to thank Dr. David Birk for his important advice and Gregory Williams and Jason Perera fortheir assistance with the project. This study was supported by the National Institutes of Health grants AG15768,AR48182, AR48852, and AR50245, and by Telethon grant GGP04113.

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Figure 1.Immunostaining revealed the pericellular distribution of type VI collagen in the cartilage ofone-month-old Col6a1+/+ and Col6a1+/− mice (panel A and B). Collagen VI was alsoabundant in the ossification area (A). No type VI collagen was present in Col6a1−/− mice(C). The pericellular distribution of type VI collagen was also present in 11-month oldCol6a1+/+ and Col6a1+/− mice but not in Col6a1−/− mice (D, E, and F).

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Figure 2.Skeletal analysis of one-month-old Col6a1+/+ (A) and Col6a1−/− (B) mice using alcian blue(cartilage) and alizarin red (bone) staining. Homozygous mutants are smaller, with slowerossification progress in the upper (C–D) and lower (E–F) extremities (black arrows in A andB).

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Figure 3.Lack of collagen VI results in delayed growth and ossification. (A–C) Toluidine bluestaining of the femoral head of 3-month-old mice. (A) In the wild type mice, the secondaryossification process is almost complete by 3 months, while (B) Col6a1+/− mice showed adelay with approximately 50% ossification at this age. (C) Col6a1−/− mice exihbiteddelayed ossification Bars: 100 µm. (D) Quantitative measures of secondary ossification ofthe femoral heads showed that the extent of ossification depends significantly on the age andgenotype (p<0.001). Col6a1+/+, Col6a1+/−, and Col6a1−/− mice show similar secondaryossification at very early (1 month) and late stages (11 months) of their life. However, therate of ossification is slower in mice lacking collagen VI.

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Figure 4.Lack of collagen VI results in age-related osteoarthritis in the hip. (A–C) Histologicalsections of the femoral cartilage of 11-month-old mice stained with hematoxylin-eosinrevealed significant progression of osteoarthritis in the knockout mice (C) when comparedwith their wild type counterparts (panel A). Scale bars = 100 µm. (D). Semi-quantitativescoring of histologic sections stained with Safranin O and H&E staining showingosteoarthritic degeneration of Col6a1−/− mice as compared to Col6a1+/− and Col6a1+/+

mice. No significant differences were found for the heterozygous counterparts.

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Figure 5.Bone mineral density of Col6a1+/+, Col6a1+/−, and Col6a1−/− mice, as measured by micro-DXA. Bone mineral density depended on age (p<0.001) and was significantly lower in micethat lack type VI collagen (p<0.001).

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Figure 6.Mechanical testing of articular cartilage and PCM. (A) A microindentation systemcomprised by a plane-ended glass indenter was employed to assess the mechanicalproperties of murine articular cartilage. (B) The micropipette aspiration technique was usedto measure the mechanical properties of the PCM of isolated chondrons. (C) Young’smoduli of the PCM and cartilage specimens in Col6a1+/+, Col6a1+/−, and Col6a1−/− mice.The Young’s moduli of the PCM was measured using the micropipette technique andsignificant differences were observed among all three groups. (D) Young’s moduli of theECM of the cartilage. No differences were found in the Young’s modulus of the articularcartilage ECM as measured by the microindentation technique (n=18).

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