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).
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 α-chains, α1(VI), α2(VI), and α3(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 determine the role of type VI collagen in the elastic properties of the articular cartilage ECM and PCM.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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 characterized 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 PCM.
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 tissue-specific 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; down-regulation 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.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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, Guilak.
Manuscript preparation. Alexopoulos, Bonaldo, Guilak.
Statistical analysis. Alexopoulos, Guilak.