Transcriptional profiling and biochemical analysis of mechanically induced cartilaginous tissues in a rat model.

Articular cartilage injuries and degenerative joint diseases such as osteoarthritis pose significant treatment challenges due to the insufficiency of the natural repair response in damaged joint tissues. In recent years, techniques such as microfracture, osteochondral transplantation, and autologous chondrocyte implantation have shown promising clinical results but typically only in patients under age 40 (1–4). Moreover, concerns about the long-term durability of the repair tissue remain (2–5). One alternate approach for developing regenerative strategies for cartilage repair is to first elucidate the key cellular and molecular processes involved in the formation of joint tissues, either in utero or post-natally. These processes would then be logical targets in identifying possible new treatment regimens. Prior investigations of the molecular processes of joint development in utero have established multiple, characteristic spatiotemporal patterns of gene expression and have suggested the importance of mechanical factors for formation and maintenance of articular cartilage. For example, uridine diphosphoglucose dehydrogenase (Ugdh), an essential enzyme in hyaluronan synthesis, and Cd44, the main cell surface receptor for hyaluronate, are upregulated in or near the presumptive joint surfaces just prior to segmentation (6). Following segmentation, collagen expression (types I, II, III, V, IX, X, and XI) varies with position within the joint but is relatively constant over time (7, 8), while wide variations in expression are seen both temporally and spatially for many proteoglycans (aggrecan, biglycan, decorin, fibromodulin), matrix metalloproteinases (Mmp2, Mmp9, and Mmp13), and matrix components such as cartilage oligomeric matrix protein (Comp) and matrillin 1 (9–11). Articular cartilage in utero does not initially exhibit the zonal variations in collagen fiber orientation that are the hallmark of the mature tissue architecture; rather the fibers are initially parallel to the joint surface throughout the depth of the chondral layer (12). It has been suggested that the transformation to zonal variations is modulated by mechanical loading of the joint in utero (12). Embryonic immobilization studies also indicate the importance of mechanical loading for joint development as demonstrated by reduced glycosaminoglycan (GAG) and collagen contents, reduced instantaneous compressive modulus (13), and a “ragged” surface of the immobilized articular cartilage (14), secondary fusion in cavitated joints, and failure to cavitate in uncavitated joints (14–16). Consistent with these findings, immobilization of diarthrodial joints post-natally results in decreased GAG content, increased proteoglycan proteolysis and MMP activity, accelerated advancement of the tidemark, and loss of tissue stiffness (reviewed in (17)). In parallel with these in vivo observations, many studies have used an in vitro approach to determine the effects of mechanical stimulation on molecular expression during hyaline cartilage formation. Cyclic tensile strain increases hyaluronan synthesis, UGDH activity, and Cd44 expression (18). Compression and hydrostatic pressure result in increased expression of collagen type II, SRY (sex determining region Y)-box 9 (Sox9), and aggrecan, as well as increased proteoglycan synthesis and chondrogenic matrix deposition, by chondrocytes (e.g., (19, 20)) and mesenchymal stem cells (e.g., (21–23)). Thus, there is strong evidence that mechanical stimuli affect hyaline cartilage formation not just in utero but also post-natally. By extension, these data suggest an experimental approach in which mechanical loading is used to promote cartilage formation in the mature or aged skeleton, as a means of studying the underlying molecular processes and of the possibilities for improving the long-term viability and durability of the cartilage that forms. Bone fracture healing provides a viable model for this type of approach for two reasons. First, prior studies have demonstrated that altering the mechanical environment of a healing fracture, such as by applying increased compressive, shear, or bending movements at the fracture site, can result in increased amounts of cartilage in the fracture callus (24–28). In particular, an oscillatory bending motion applied at the fracture site promotes formation of cartilage with abundant type II collagen expression and zonal variations in collagen fiber orientation similar to those found in articular cartilage (26, 29). Second, fracture healing is a regenerative process that in many ways recapitulates aspects of skeletal development (30–32). Study of the molecular events involved in mechanically induced chondrogenesis during fracture healing therefore may offer insight into key processes necessary for post-natal cartilage formation, repair, and regeneration. The overall goal of this study was to profile the mRNA expression patterns that occur during mechanically induced chondrogenesis in a fracture healing model. Building upon prior work that investigated how application of a bending motion to a healing fracture alters expression of a small set of cartilage- and bone-related genes (29), this study investigated the effects on a broader set of genes via transcriptional profiling with a custom microarray. These analyses were complemented with measurements of glycosaminoglycan content in order to provide a functional assessment of the newly formed cartilage tissue.