Articular cartilage lines the ends of the long bones in diarthrodial joints, providing a low friction, low wear, and weight bearing surface for pain free mobility. Although articular cartilage is extremely durable, the lack of blood vessels and relatively low cellularity causes it to be extremely recalcitrant to self-repair once it is damaged. Damaged articular cartilage can lead to osteoarthritis (OA), which hinders mobility, causes pain, and is the leading cause of disability in the United States. Tissue engineering has continually strived to overcome the shortcomings of current surgical strategies to treat focal defects in articular cartilage by engineering replacement tissues with sufficient mechanical and lubrication properties to function under in vivo loading. Originally thought to be a simple tissue due to its low cellularity and lack of blood vessels, articular cartilage is rather complex. It is a biphasic tissue composed mostly of water and an extracellular matrix (ECM) that is heterogeneous, anisotropic, and depth-dependent.
Although this highly structured and organized ECM has not been successfully recapitulated in engineered tissues, constructs with bulk compressive and tensile properties nearing native tissue levels have been achieved. A scaffold-free, self-assembly method is able to form robust articular cartilage constructs with high glycosaminoglycan and collagen content and compressive mechanical properties nearing a third of native tissue levels. In combination with growth factors and mechanical stimuli, the compressive and tensile properties of the self-assembled articular cartilage can be further improved. Although this is an incredible feat, as the bulk mechanical properties reach sub-par native tissue levels, it is critical to explore the surface lubrication properties of engineered tissues. In order for engineered cartilage to function in vivo and endure a lifetime of use, they must also possess the superior wear and lubrication properties as seen in native articular cartilage.
Lubrication of articular cartilage is provided by fluid contained in the tissue and surrounding synovial fluid as well as boundary lubricants adsorbed onto the cartilage surfaces. Boundary lubricants are essential in protecting the surfaces from excessive wear and damage under loading conditions when a thick fluid film cannot be maintained to separate the cartilage surfaces. Chapter 2 and 3 review the important cartilage boundary lubricant, superficial zone protein (SZP), also known as lubricin or PRG4. Chapter 2 is a comprehensive overview of its history. The discovery, isolation, and purification of SZP, its role in vivo and in OA animal models, and its future perspectives are discussed in the review. Chapter 3 reviews the enzyme-linked immunosorbent assay (ELISA) method which is commonly used in many research laboratories to determine and quantify SZP.
SZP was first identified in synovial fluid and later discovered to be secreted by superficial zone articular chondrocytes and synoviocytes. Middle zone chondrocytes secrete virtually zero SZP. While SZP is mostly secreted and accumulates into the surrounding synovial fluid in vivo, localization of SZP at the surfaces of tissues suggests its critical role in surface interactions. SZP localizes on different tissues of the knee joint, such as the meniscus, tendons, and ligaments. Chapter 4 aims at characterizing the frictional properties of tissues from different compartments of the diarthrodial joint using a juvenile bovine stifle joint. Immunolocalization of SZP was investigated and compared for each tissue. Results of this study demonstrated increased SZP immunostaining in tissues with lower friction coefficients, bolstering the evidence that SZP plays a role in reducing friction in knee/stifle joint tissues.
Since SZP is synthesized by superficial zone chondrocytes and not by middle zone chondrocytes, Chapter 5 aims to engineer self-assembled articular cartilage constructs with high levels of SZP and low friction coefficients by increasing the proportion of superficial zone chondrocytes in self-assembly. Increasing the proportion of superficial zone chondrocytes had the intended effect of increasing SZP synthesis, but did not reduce the friction coefficients. This paradoxical finding suggested that additional factors such as SZP binding macromolecules, surface roughness, and adhesion need to be examined to modulate the friction properties of engineered cartilage.
Chapter 6 is an extension to the proceeding chapter and investigates the role of superficial zone ECM macromolecules on SZP binding and retention in engineered cartilage. Superficial zone ECM macromolecules were extracted from bovine femoral condyles and added directly into the culture medium of self-assembled tissues. Treatment with ECM did reduce its friction coefficient and improve bulk mechanical properties of engineered cartilage. However, the exact mechanism of the reduction in friction cannot be fully explained by this investigation alone. Chapter 7 discusses the perspectives and future directions of engineering functional lubrication in self-assembled tissues.
These data suggest that additional factors other than SZP need to be considered in engineering functional lubrication in tissue engineered cartilage. Although SZP synthesis was increased in engineered cartilage by increasing the proportion of superficial zone chondrocytes, it had no effect on its friction coefficient. This was hypothesized to be due to the lack of SZP retention in the ECM. Therefore, the role of ECM molecules to aid in SZP retention in engineered cartilage was investigated. ECM molecules did not affect SZP synthesis but did improve friction. Immunohistochemistry revealed more intense SZP staining in ECM treated constructs. However, the biochemistry data show that there was greater SZP retention in constructs without ECM treatment. Since the biochemical assay cannot distinguish surface and bulk retention of SZP, it is hypothesized that the ECM molecules may be presenting more SZP at the surface of engineered cartilage rather than in the bulk matrix. However, the data cannot conclusively determine the exact mechanism in which the ECM molecules are contributing to the reduction in friction and further investigation is required. Further investigations include examining the exact constituents of the extracted superficial zone ECM as well as additional factors, such as the effects of surface roughness and adhesion on the friction coefficient of selfassembled articular cartilage constructs.
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