<p>Human joints are able to withstand millions of loading cycles with loads regularly more than 3 times an individual's body weight in large part due to the unique bearing properties of articular cartilage, a strong, slippery tissue that covers the ends of long bones. PRG4 is a boundary lubricating glycoprotein present on the cartilage surface and in the synovial fluid surrounding it. While evidence that PRG4 lubricates and preserves normal joint function is strong, little is known of its effect on cartilage surface properties, the mechanism by which it lubricates, or its postulated role of preventing wear on joints. The effect of PRG4 on cartilage friction, wear, structure, morphology, and the mechanisms by which it mediates these factors are studied here. Methods to study these parameters at the microscale using atomic force microscopy are also developed. </p><p>Cartilage of mice with the Prg4 gene (which expresses PRG4) deleted is shown to be different in a number of ways from wild type cartilage. The uppermost layer is thicker and less uniform and the surface is rougher and softer. There is also a loss of proteoglycans, structural components of cartilage, from the underlying superficial tissue, and apparent tissue damage in some cases. Wear in the presence of PRG4 in shown to be significantly lower than in its absence, a finding which may have direct implications for prevention and treatment of osteoarthritis. It appears that PRG4 needs to be present in solution, not merely on the cartilage surface to have this effect, indicating that adsorption properties are important for wear prevention.</p> / Dissertation

Skeletal development begins when mesenchymal stem cells migrate, condensate and differentiate into chondrocytes. The chondrocytes differentiate in one of two ways. Either the cells form the cartilaginous template for endochondral ossification or they form the articular cartilage which express proteoglycan 4. The underlying mechanisms for articular cartilage formation are poorly understood. The purpose of this study was to assess the effect of different fractions of synoviocyte-conditioned medium on chondrocyte differentiation. We show evidence that Synovial-like fluid contains a protein which promotes chondrocytes to express proteoglycan 4, thus promoting articular cartilage formation. The synovial-like fluid was fractionized by size exclusion chromatography and reversed phase chromatography and thus, with that method, this manuscript lays the foundations for further research to identify the putative factor. Because of this study, we are now closer in identifying the proteins that promote articular cartilage formation.

Lubricin, or proteoglycan 4 (PRG4), is a secreted, glycosylated protein that binds to cartilage surfaces, which functions as a boundary lubricant. The loss of lubricin from cartilage is identified as a major pathogenic factor in post-traumatic osteoarthritis (PTOA) that has now been the aim of therapeutic intervention. Intra-articular injection of PRG4 protein provides short-term benefits that might be extended using sustained delivery methods such as in gene therapy. Here we describe the development and testing of such therapy using adeno-associated virus (AAV) as a vector for the transfer of PRG4-green fluorescent protein (GFP) fusion gene. Our recombinant PRG4 gene produces a PRG4-GFP fusion protein to facilitate tracking of its expression and distribution on joint surfaces. We hypothesized that PRG4-GFP is fully functional as a cartilage lubricant and that PRG4-GFP produced in vivo is expressed by synoviocytes and other joint cells, and cartilage surfaces remained coated for several weeks up to months after intra-articular injection of the virus. PRG4-GFP showed lubricin-like cartilage binding in vitro, and lubrication immunoblot analysis confirmed that purified PRG4-GFP from cultured media conditioned by PRG4-GFP-transduced synoviocytes was heavily glycosylated, while confocal microscopy revealed binding of the fluorescent fusion protein to cartilage surfaces. Metal-on-cartilage friction tests showed that PRG4-GFP reduced friction coefficients to a degree comparable to that of synovial fluid and had strong chondro-protective effects in explanted cartilage exposed to shear loading. The chondrocyte viability after shear loading showed that PRG4-GFP had a strong chondro-protective effect on par with that of the synovial fluid. Confocal microscopy and immunohistology confirmed that cartilage surfaces in the stifle joints of mice injected with viruses were coated with PRG4-GFP for up to 2 or 4 weeks after the treatment. The overexpression of PRG4-GFP and coating of cartilage surfaces in the stifle joints of mice injected with Adeno-Associated Virus for the transfer of PRG4-GFP fusion gene (AAV-PRG4-GFP) was confirmed by confocal microscopy and immunohistology for up to 2 or 4 weeks post-injection. The μCT imaging and immunohistology in AAV-PRG4-GFP injected rabbit knees showed stronger inhibition in degeneration of damaged tissues than in AAV-GFP injected control group. Collectively these findings indicate that AAV-PRG4-GFP transduction is a valuable new tool for evaluating the effects of long-term lubricant supplementation on PTOA in animal models.

Osteoarthritis is a joint disease characterized by the breakdown of articular cartilage. The field of tissue engineering is interested in developing methods to produce biological alternatives to current orthopedic procedures. Lubricin is a molecule which is important in the proper lubrication of articular cartilage. It is a challenge in the field of tissue engineering to produce cartilage with sufficient lubricin expression. Developing a reporter cell for lubricin allowed for a more efficient investigation of the conditions wh­­­ich may influence its expression. By comparing "optimized" and traditional media solutions, it was determined that the use of a previously reported type II collagen optimized media would negatively affect the expression of lubricin. This information indicates the need to further evaluate the conditions which are conducive to producing cartilage with both sufficient types of type II collagen and lubricin.