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Developmental and osteoarthritic changes in Col6a1-knockout miceBiomechanics of type VI collagen in the cartilage pericellular matrix.

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Vol. 60, No. 3, March 2009, pp 771–779
DOI 10.1002/art.24293
© 2009, American College of Rheumatology
Developmental and Osteoarthritic Changes in
Col6a1-Knockout Mice
Biomechanics of Type VI Collagen in the Cartilage Pericellular Matrix
Leonidas G. Alexopoulos,1 Inchan Youn,1 Paolo Bonaldo,2 and Farshid Guilak1
Objective. Chondrocytes, the sole cell type in
articular cartilage, maintain the extracellular matrix
(ECM) through a homeostatic balance of anabolic and
catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as
mechanical stress. Chondrocytes are encapsulated by a
narrow tissue region termed the “pericellular matrix”
(PCM), which in normal cartilage is defined by the
exclusive presence of type VI collagen. Because the PCM
completely surrounds each cell, it has been hypothesized
that it serves as a filter or transducer for biochemical
and/or biomechanical signals from the cartilage ECM.
The present study was undertaken to investigate
whether lack of type VI collagen may affect the development and biomechanical function of the PCM and
alter the mechanical environment of chondrocytes during joint loading.
Methods. Col6a1ⴚ/ⴚ mice, which lack type VI
collagen in their organs, were generated for use in these
studies. At ages 1, 3, 6, and 11 months, bone mineral
density (BMD) was measured, and osteoarthritic (OA)
and developmental changes in the femoral head were
evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1-monthold Col6a1ⴚ/ⴚ, Col6a1ⴙ/ⴚ, and Col6a1ⴙ/ⴙ mice were
assessed using an electromechanical test system, and
mechanical properties of the PCM were measured using
the micropipette aspiration technique.
Results. In Col6a1ⴚ/ⴚ and Col6a1ⴙ/ⴚ mice the
PCM was structurally intact, but exhibited significantly
reduced mechanical properties as compared with wildtype controls. With age, Col6a1ⴚ/ⴚ mice showed accelerated development of OA joint degeneration, as well as
other musculoskeletal abnormalities such as delayed
secondary ossification and reduced BMD.
Conclusion. These findings suggest that type VI
collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence
that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or
Col6a1ⴚ/ⴚ-derived joint laxity, can lead to progression
of OA.
Articular cartilage is the tissue that lines the
surfaces of diarthrodial joints and serves as the resilient,
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 of anabolic and catabolic activities. The micromechanical environment of chondrocytes, in conjunction
with biochemical factors (e.g., growth factors, cytokines)
and genetic factors, plays an important 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 termed the “pericellular matrix” (PCM), which, together with the enclosed
chondrocyte, has been termed the “chondron” (4–7).
The PCM is characterized primarily as being the exclusive location of type VI collagen in normal cartilage, but
proteoglycans, fibronectin, and types II and IX collagen
are also present in high concentrations in the PCM (4,8).
Supported by NIH grants AG-15768, AR-48182, AR-48852,
and AR-50245, and by Italian Telethon Foundation grant GGP04113.
Leonidas G. Alexopoulos, PhD (current address: National
Technical University of Athens, Athens, Greece), Inchan Youn, PhD,
Farshid Guilak, PhD: Duke University Medical Center, Durham,
North Carolina; 2Paolo Bonaldo, PhD: University of Padua, Padua,
Address correspondence and reprint requests to Farshid
Guilak, PhD, Duke University Medical Center, 375 Medical Sciences
Building, Box 3093 Medical Center, Durham, NC 27710. E-mail:
Submitted for publication March 28, 2008; accepted in revised
form November 3, 2008.
The functional role of the PCM in articular
cartilage is still unknown, although the fact that it
completely surrounds the cell suggests that it regulates
the biomechanical, biophysical, and biochemical signals
that the chondrocyte perceives (9). For example, interactions between cell surface receptors and the ECM
significantly influence matrix metabolism, gene expression, and response to growth factors (10–12). Furthermore, cytokines and growth factors that interact with the
chondrocyte surface traverse the pericellular environment, where they may be retained and modified (13,14).
From a biomechanical standpoint, there has been considerable speculation that the PCM plays a critical role
in either protecting the cells or serving as a “filter” or
transducer of physical signals in the ECM (4,6,9,15,16),
potentially through an interaction of type VI collagen
with integrins or other cell surface receptors (17–20).
Indirect evidence in support of these hypotheses is
provided by experimental data showing that a newly
formed PCM augments the cellular metabolic response
to biomechanical loading (21).
Type VI collagen serves as the defining boundary
of the PCM in articular cartilage, but it is also found in
the ECM in many connective tissues (22). It has a
characteristic beaded filamentous structure of tetrameric units consisting of 3 different ␣-chains, ␣1(VI),
␣2(VI), and ␣3(VI). Type VI collagen has high affinity
with numerous ECM components (i.e., biglycan,
decorin, hyaluronan, fibronectin, perlecan, and heparin)
as well as with the cell membrane (21,23–26). Thus, it
has been hypothesized that type VI collagen plays
important roles in mediating cell–matrix interactions as
well as intermolecular interactions in various tissues and
cell cultures (27–31). In articular cartilage, type VI
collagen forms a network that anchors the chondrocyte
to the PCM (32–35) through its interaction with hyaluronan (21,36), decorin (24), and fibronectin (37).
The goal of this study was to examine the hypothesis that lack of type VI collagen alters the biomechanical
properties of the PCM and ECM of articular cartilage.
Type VI collagen–deficient mice were generated by
targeted disruption of the Col6a1 gene (38). Histologic
analysis and dual x-ray absorptiometry (DXA) were
used to examine differences in skeletal development,
bone mineral density (BMD), and progression of osteoarthritic (OA) joint degeneration in wild-type and type
VI collagen–deficient mice. In addition, micromechanical testing was performed on the articular cartilage and
on isolated chondrons, using microindentation and micropipette aspiration techniques, respectively, to deter-
mine the role of type VI collagen in the elastic properties of the articular cartilage ECM and PCM.
Type VI collagen–knockout mice. All procedures were
approved by the Duke University Institutional Animal Care
and Use Committee. Type VI collagen–knockout mice were
generated on a CD1 genetic background by targeted disruption
of the Col6a1 gene, which is responsible for the production of
the ␣1(VI)-chain (38). The elimination of the ␣1(VI)-chain
resulted in the absence of triple-helical type VI collagen
molecules in the ECM (38). Mice were killed at age 1, 3, 6, or
11 months.
Fluorescence immunohistochemistry. Type VI collagen immunostaining was performed using a polyclonal anti–
type VI collagen antibody raised against a peptide mapping
near the amino-terminus of the murine ␣1(VI)-chain (Santa
Cruz Biotechnology, Santa Cruz, CA). Cryostat sections from
the coronal plane were obtained from decalcified femoral
heads, using standard histologic methods.
Skeletal staining. One-month-old mice were killed,
skinned, and eviscerated. Alcian blue and alizarin red were
used to stain the cartilage and bone, respectively, by standard
skeletal staining techniques.
Histologic analysis and morphometric grading. Fixed
cryostat sections were stained with toluidine blue and hematoxylin and eosin, using standard histologic techniques. Osteoarthritic and developmental changes in the femoral head of 60
mice (ages 1, 3, 6, and 11 months) were assessed using a
quantitative histomorphometric grading system (39,40). OA
was graded based on the sum score of surface fibrillation
(0–4), intensity of toluidine blue staining (0–3), and fibrocartilage presence (0–2) (maximum possible total sum score 9)
and was reported as a percentage of the 0–9 scale, with 0%
corresponding to no histologic signs of OA and 100% corresponding to the most severe changes (grade 9). Specimens
were classified as non-OA (score 0–1), mild OA (⬎1–5), or
severe OA (⬎5–9).
Developmental changes were assessed histologically
based on the presence of the growth plate and the extension of
the secondary ossification center (40), which was graded from
0 to 5 (0 corresponding to findings in normal 1-month-old
mice, and 5 corresponding to findings in normal 11-month-old
mice). The percentage of ossified area relative to the total area
was graded as follows: 0 ⫽ cartilage with growth plate and no
area of secondary ossification present, 1 ⫽ area of secondary
ossification ⬍10%, 2 ⫽ area of secondary ossification ⬎10%
and ⬍50%, 3 ⫽ area of secondary ossification ⬎50% and
⬍90%, 4 ⫽ area of secondary ossification ⬎90%, and 5 ⫽ no
growth plate present. Secondary ossification was reported as a
percentage of the 0–5 scale, with 0% corresponding to grade 0
(no secondary ossification) and 100% corresponding to the
fully developed femoral head (grade 5).
BMD measurement. BMD was measured by DXA
(PIXImus; Lunar, Madison, WI) (41). The mice were weighed
and placed in the DXA unit in a supine position, and the whole
body except the skull was measured. A total of 82 mice were
analyzed, at age 1, 3, 6, or 11 months.
Figure 1. Immunostaining for type VI collagen in representative
mouse cartilage specimens. Type VI collagen was present in a pericellular distribution in the cartilage of 1-month-old Col6a1⫹/⫹ and
Col6a1⫹/⫺ mice (A and B) and was also abundant in the ossification
area in Col6a1⫹/⫹ mice (A). No type VI collagen was present in
1-month-old Col6a1⫺/⫺ mice (C). The pericellular distribution of type
VI collagen was also observed in 11-month-old Col6a1⫹/⫹ and
Col6a1⫹/⫺ mice (D and E), and not in 11-month-old Col6a1⫺/⫺ mice
(F). (Original magnification ⫻ 20.)
Mechanical testing of articular cartilage. A total of 18
right hip joints from 1-month-old mice (7 Col6a1⫺/⫺, 7
Col6a1⫹/⫹, 4 Col6a1⫹/⫺) were tested in indentation using an
electromechanical test system (ELF 3200; EnduraTEC,
Minnetonka, MN) instrumented with a low-capacity load cell
(250 gm; Sensotec, Columbus, OH) and extensometer (1 mm;
Epsilon, Jackson, WY) (42). Plane-ended microindenters were
machined from glass fibers (110 ␮m diameter; Thorlabs,
Newton, NJ). A dual-angle camera system was used to optically
align the indenter tip perpendicular to the cartilage surface.
After applying a tare load of 0.3 gm force and allowing it to
equilibrate, 4 consecutive indentation displacements (5 ␮m per
step with a ramping speed of 1 ␮m per second) were applied to
the cartilage surface and allowed to equilibrate for 200 seconds
per step. The time, reaction force, and displacement data were
collected at 1 Hz throughout the test. The equilibrium force
versus displacement curve was obtained from the linear region
of the curve. After mechanical testing, the thickness of cartilage from the tissue surface to the calcified cartilage was
measured at a site adjacent to the test site, using routine
histologic procedures (5-␮m sections labeled with Safranin O
and fast green). The Young’s modulus of mouse cartilage was
calculated using an elastic indentation model (43) with an
assumed Poisson’s ratio of 0.25 (42).
Mechanical testing of the PCM. Chondrons were
mechanically isolated from the femoral articular cartilage of
1-month-old mice (93 chondrons from 26 mice) with a custombuilt “microaspirator,” which applies suction pressure to the
cartilage surface with a modified syringe, as described previ-
ously (44). The micropipette aspiration technique (45–47) was
used to measure the mechanical properties of the PCM, as
described previously (44,48,49). With this technique, the surface of the PCM is aspirated into a glass micropipette (12 ␮m
diameter) by the application of a series of controlled pressures
up to 18 kPa, and the ensuing equilibrated aspiration length is
measured using video microscopy. The Young’s modulus of
the PCM was determined using a theoretical model that
represents the chondron as an elastic, compressible layer (i.e.,
PCM) overlying an elastic half-space (i.e., chondrocyte) (44).
Statistical analysis. Statistical analysis was performed
using a multifactorial analysis of variance setup in Statistica
(StatSoft; Tulsa, OK). The only categorical predictors considered were age (1, 3, 6, 9, or 11 months) and genotype (⫹/⫹,
⫹/⫺, or ⫺/⫺). We assumed that these variables would be able
to predict 4 dependent measurements, i.e., weight, OA score,
BMD, and ossification score. Full factorial design revealed that
both age, genotype, and the age ⫻ genotype effects were
significant contributors; thus, post hoc comparison was performed using Fisher’s least significant difference method. Age
effects were significant for all 4 measurements, as expected.
Genotype effects between Col6a1⫹/⫹ and Col6a1⫺/⫺ mice
Figure 2. A and B, Representative images showing skeletal analysis of
1-month-old Col6a1⫹/⫹ (A) and Col6a1⫺/⫺ (B) mice. Cartilage and
bone were stained with Alcian blue and alizarin red, respectively.
Knockout mice were smaller than wild-type animals. C–F, Images of
the upper (C and D) and lower (E and F) extremities, corresponding to
the arrows in A and B. Ossification progress was slower in the
Col6a1⫺/⫺ mice.
labeling for type VI collagen, whereas type VI collagen
was absent in the knockout mice (Figure 1). Intense
labeling for type VI collagen was also observed in the
growth plate of 1-month-old wild-type mice (Figure 1A).
Staining of skeletal bone and cartilage from
1-month-old mice (Figure 2) indicated that Col6a1⫺/⫺
mice were smaller and exhibited a slower ossification
process in the upper extremities (Figures 2C and D) and
lower extremities (Figures 2E and F) than their wildtype counterparts. The smaller size was also consistent
with a trend toward lower body weight in 1-month-old
Col6a1 ⫺/⫺ mice compared with Col6a1 ⫹/⫹ mice
Figure 3. Delayed growth and ossification due to lack of type VI
collagen. A–C, Toluidine blue staining of femoral head specimens from
representative 3-month-old mice. In wild-type mice, the secondary
ossification process was almost complete by 3 months (A), while
Col6a1⫹/⫺ mice showed a delay, with ⬃50% ossification at this age (B).
Col6a1⫺/⫺ mice exhibited delayed ossification (C). Bars ⫽ 100 ␮m.
D, Quantitative assessment of secondary ossification of the femoral
head, showing that the extent of ossification depends significantly on
age and genotype (P ⬍ 0.001). Col6a1⫹/⫹, Col6a1⫹/⫺, and Col6a1⫺/⫺
mice exhibited similar secondary ossification at very early (1 month)
and late (11 months) stages of life. However, the rate of ossification
was slower in mice lacking type VI collagen, with a significant
difference evident at age 3 months. Values are the mean and SD. Color
figure can be viewed in the online issue, which is available at
were also significant for all 4 measurements. Genotype effects
between Col6a1⫹/⫹, Col6a1⫹/⫺, and Col6a1⫺/⫺ mice were
significant only for OA and ossification scores. Age ⫻ genotype effects within the same age groups were not significant for
OA. For ossification scores, a significant difference between
Col6a1⫹/⫹ and Col6a1⫺/⫺ mice was observed at 3 months only.
For BMD, a significant difference between Col6a1⫹/⫹ and
Col6a1⫺/⫺ mice was observed at all ages except 1 month.
Histologic findings. Mice lacking type VI collagen exhibited no apparent abnormalities, and all animals
survived the full 11 months of the study unless killed
earlier per the protocol. Articular cartilage of wild-type
and heterozygous mice showed extensive pericellular
Figure 4. Age-related osteoarthritis (OA) in the hip due to lack of
type VI collagen. A–C, Hematoxylin and eosin (H&E) staining of
femoral cartilage specimens from representative 11-month-old mice.
There was more significant progression of OA in the Col6a1⫺/⫺ mice
compared with their wild-type counterparts. Bars ⫽ 100 ␮m. D,
Semiquantitative scoring of histologic sections stained with Safranin O
and H&E, showing OA degeneration in Col6a1⫺/⫺ mice compared
with Col6a1⫹/⫺ and Col6a1⫹/⫹ mice. No significant differences were
found between the wild-type and heterozygous groups. Values are the
mean and SD. Color figure can be viewed in the online issue, which is
available at
(mean ⫾ SD 16.5 ⫾ 2.4 gm versus 18.3 ⫾ 1.99 gm; P ⫽
0.11 by 2-tailed t-test).
To better evaluate the developmental process, we
measured the secondary ossification process in the femoral head (Figures 3A–C). Col6a1⫺/⫺ mice showed
significantly delayed ossification at 3 months, compared
with Col6a1⫹/⫹ mice (mean ⫾ SD grade 2.2 ⫾ 1.8 versus
4.1 ⫾ 0.2; P ⬍ 0.02) (Figure 3D). Among 3-month-old
animals, only 1 of 6 Col6a1⫺/⫺ mice had an ossification
grade of ⬎ 4, whereas the ossification grade was ⱖ4 in 3
of 3 Col6a1⫹/⫹ mice. In all mice, the ossification process
was complete after 6 months.
Semiquantitative histologic analysis of cartilage
degeneration revealed significant age-dependent osteoarthritic changes in the Col6a1⫺/⫺ mice. OA changes
depended on age (P ⬍ 0.001) and genotype (P ⬍ 0.05)
(Figure 4). Among 6–11-month-old animals, only 2 of 12
Col6a1⫹/⫹ mice, 3 of 7 Col6a1⫹/⫺ mice, and 11 of 16
Col6a1⫺/⫺ mice scored ⬎1 and were thus characterized
as having OA (either mild or severe; see Materials and
Methods). However, scores were ⬎5, i.e., representing
severe OA, in 0 of 12, 0 of 7, and 2 of 16 of the
Col6a1⫹/⫹, Col6a1⫹/⫺, and Col6a1⫺/⫺ mice, respectively.
BMD. BMD, as measured by DXA, was significantly higher in wild-type mice than their type VI
collagen–knockout counterparts at ages 3–6 months
(P ⬍ 0.001). These differences were no longer evident at
11 months (Figure 5).
Mechanical properties of articular cartilage and
PCM. The PCM exhibited linear elastic behavior, and
the Young’s modulus of the PCM of chondrons isolated
Figure 6. Mechanical properties of articular cartilage and pericellular
matrix (PCM). A, Microindentation system comprising a plane-ended
glass indenter, used to assess the mechanical properties of murine
articular cartilage. B, Micropipette aspiration system, used to measure
the mechanical properties of the PCM of isolated chondrons. C and D,
Young’s moduli of PCM and cartilage specimens from Col6a1⫹/⫹,
Col6a1⫹/⫺, and Col6a1⫺/⫺ mice (n ⫽ 7, 4, and 7, respectively). The
Young’s modulus of the PCM was measured using the micropipette
technique, and significant differences among all 3 groups were observed (C). The Young’s modulus of the articular cartilage extracellular matrix was measured using the microindentation technique, and no
differences between groups were found (D). Values are the mean ⫾
from Col6a1⫹/⫹ mice was significantly higher than that
of the PCM of chondrons from Col6a1⫹/⫺ mice. This
was further reduced in the knockout Col6a1⫺/⫺ mice
(Figure 6C). Microindentation tests revealed no significant differences between Col6a1⫹/⫹, Col6a1⫹/⫺, and
Col6a1⫺/⫺ mice in terms of mechanical properties of
femoral head articular cartilage (Figure 6D).
Figure 5. Bone mineral density in Col6a1⫹/⫹, Col6a1⫹/⫺, and
Col6a1⫺/⫺ mice, as measured by micro–dual x-ray absorptiometry.
Bone mineral density depended on age (P ⬍ 0.001) and was significantly lower in mice that lacked type VI collagen. Values are the mean
and SD.
This report presents new evidence of significant
musculoskeletal changes in Col6a1⫺/⫺ mice. Primarily,
our findings show that mice lacking type VI collagen
exhibit accelerated development of hip osteoarthritis, as
well as a delayed secondary ossification process and
reduced BMD. Lack of type VI collagen resulted in a
loss of the stiffness of the articular cartilage PCM
(decreased modulus), prior to the occurrence of any
detectable histologic changes. However, no differences
in ECM properties were observed. These findings provide indirect evidence of a role of type VI collagen in
regulating the physiology of the articular cartilage chondrocyte, potentially via alterations in the biologic and
mechanical environment of chondrocytes due to changes
in biomechanical properties of the PCM or increased
joint laxity associated with a type VI collagen deficiency.
The mechanical environment of the chondrocytes
is one of several environmental factors that influence the
normal balance between the synthesis and breakdown of
articular cartilage, and it is an important participant in
the etiopathogenesis of OA (1–3,50,51). Thus, changes
in the mechanical interactions between the cell and the
ECM may have a significant influence on the regulatory
response of the chondrocyte.
A biomechanical function of the PCM has long
been hypothesized (5,6,9), and there is growing evidence
from both theoretical modeling and experimental studies that the PCM plays a significant role in regulating the
biomechanical signals perceived by the chondrocyte
(16,52,53). The mechanical properties of the PCM are
significantly altered in OA, exhibiting reduced stiffness
and increased fluid permeability (44,49). These changes
occur throughout the thickness of the articular cartilage,
affecting the properties of the PCM in the superficial
and middle/deep zones in a similar manner (48). The
PCM appears to function by providing a relatively
uniform cellular microenvironment despite a great lack
of homogeneity in local tissue strain (16,54). Thus, a
compromised PCM could significantly affect the mechanical environment of the chondrocytes in articular
cartilage, leading to increased strain at the cellular level
(53), which may affect catabolic responses at the level of
single cells (55). In other tissues such as bone, however,
the pericellular region (i.e., the glycocalyx) can serve as
a strain amplifier by coupling fluid drag forces to the
actin cytoskeleton within the processes of osteocytes
(56–58). In the present study, Col6a1⫺/⫺ mice showed
significantly reduced PCM stiffness at 1 month of age,
prior to the appearance of any histologic or biomechanical changes in the overall articular cartilage. With age,
these mice exhibited accelerated development of OA.
These findings provide indirect evidence that early alterations in the mechanical properties of the PCM are
associated with the progression of OA.
In normal articular cartilage, type VI collagen is
present exclusively in the PCM, and it has been charac-
terized as being a discrete marker of chondron anatomy
(34). For this reason, we hypothesized that type VI
collagen is necessary for providing the structural integrity and mechanical properties of the PCM. Contrary to
our hypotheses, though, Col6a1⫺/⫺ mice exhibited intact
chondrons that could be isolated despite the lack of type
VI collagen. This finding suggests that proteins other
than type VI collagen provide some of the structural
integrity of cartilage PCM. Nonetheless, the Young’s
modulus (stiffness) of the PCM in Col6a1⫺/⫺ mice was
dramatically decreased (approximately one-third that of
the PCM in wild-type controls), illustrating the important role of type VI collagen in the properties of the
An important issue that must be considered is the
link between type VI collagen deficiency and changes in
muscle physiology displayed by Col6a1⫺/⫺ mice (38).
Such a link has also been observed in humans, with
studies showing that mutations of type VI collagen genes
play a causal role in two inherited disorders of muscle:
Bethlem myopathy and Ullrich congenital muscular
dystrophy (UCMD) (59,60). It is possible that some
features of UCMD, particularly joint laxity or predisposition to hip dislocation, may also contribute to the
accelerated hip degeneration observed in Col6a1⫺/⫺
mice. Since joint laxity and mechanical alterations of the
PCM are heritably coupled in Col6a1⫺/⫺ mice and both
lead to an altered mechanical environment in chondrocytes, both factors could contribute to the development
of OA.
While the present findings clearly demonstrate
an association between Col6a1 deficiency and OA, presumably via mechanical alterations caused by joint laxity
or altered PCM properties, further studies aimed at
developing and characterizing conditional or tissuespecific knockout animals may be needed to fully understand the mechanisms by which Col6a1 deficiency leads
to OA. Nonetheless, our results are consistent with the
hypothesized role of type VI collagen as an integrating
molecule in the structure of cells and tissues; downregulation of type VI collagen is associated with tissue
laxity and wasting (e.g., Bethlem myopathy, UCMD,
joint hyperlaxity), whereas up-regulation of type VI
collagen results in increased fibrosis and tissue stiffness
(e.g., bullous keratopathy, scleroderma) (38,61–68).
Our studies revealed no gross morphologic differences between wild-type and type VI collagen–
knockout mouse chondrons, other than reduced skeletal
size of Col6a1⫺/⫺ mice. Skeletal changes were apparent
as a retardation of the developmental process until 11
months of age. During development, histogenesis of
long bones occurs via endochondral ossification of cartilage tissue. During this process, chondrocytes in the
epiphyseal plate differentiate into mature hypertrophic
cells and finally are eliminated from the growth plate
(69). The hypertrophic cell lacunae are invaded by
vessels carrying mesenchymal and osteogenic cells that
differentiate into osteoclasts and synthesize a bony
matrix. A similar procedure, known as secondary ossification, takes place at the end of the bone, where the
formation of the bony epiphysis occurs.
The present results provide evidence of a slowing
of secondary ossification changes and decreased BMD
in Col6a1⫺/⫺ mice. While there is no known mechanism
directly linking type VI collagen deficiency with endochondral ossification, type VI collagen may provide a
scaffold for osteoblasts, preosteoblasts, and chondrocytes to proceed to osteochondral ossification (70). In
addition, type VI collagen has been linked to the early
events of chondrocyte differentiation (71), the regulation of mesenchymal cell proliferation in vitro (72), and
ECM stabilization during development (34). It has also
been hypothesized that type VI collagen is important for
chondrocyte proliferation and hypertrophy in cartilage.
Taken together with the findings of these previous
studies (34,73,74), our observation of the ubiquitous
presence of type VI collagen in the growth plate (Figure
1A), supports the notion that type VI collagen deficiency
may delay cell differentiation and proliferation, resulting
in delayed development and reduced bone formation.
Interestingly, the COL6A1 gene was recently
identified as the locus for ossification of the posterior
longitudinal ligament of the spine (70) and has also been
associated with increased systemic BMD and diffuse
idiopathic skeletal hyperostosis (75). These findings
provide evidence of a role of type VI collagen in diseases
associated with high bone-mass, consistent with our
observation of decreased BMD in Col6a1⫺/⫺ mice.
While it was beyond the scope of the present study to
analyze the mechanisms resulting in altered BMD, these
changes may also be biomechanical in origin, since type
VI collagen deficiency causes muscular dystrophy
(38,63), which can lead to abnormal mechanical loading
of the musculoskeletal system.
In conclusion, results of the present study, in
which the effect of an abnormal mechanical environment on chondrocytes was investigated using type VI
collagen–knockout mice, suggest that type VI collagen
plays a major role in the mechanical properties of the
PCM, and thus, in the mechanical environment of
chondrocytes. Col6a1⫺/⫺ mice showed accelerated development of osteoarthritis that may be “biomechanical”
in nature, via either altered properties of the PCM or
heritable joint laxity. In addition, our findings provide
direct evidence that type VI collagen might have a
significant role in the osteochondral ossification process,
by modulating chondrocyte and mesenchymal cell differentiation and proliferation activities. This model may
provide a valuable tool for better understanding of the
way changes in the mechanical environment of chondrocytes may lead to abnormal skeletal development and
development of OA.
The authors would like to thank Dr. David Birk for
valuable advice and Gregory Williams and Jason Perera for
assistance with the project.
Dr. Guilak had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Alexopoulos, Bonaldo, Guilak.
Acquisition of data. Alexopoulos, Youn.
Analysis and interpretation of data. Alexopoulos, Youn, Bonaldo,
Manuscript preparation. Alexopoulos, Bonaldo, Guilak.
Statistical analysis. Alexopoulos, Guilak.
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development, matrix, pericellular, change, osteoarthritis, typed, cartilage, knockout, collagen, col6a1, micebiomechanics
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