Characterization of the Temporomandibular Joint Disc and Fibrocartilage Engineering using Human Embryonic Stem Cells
Willard, Vincent P.
Athanasiou, Kyriacos A.
Doctor of Philosophy
Fibrocartilages in the body, including the temporomandibular joint (TMJ) disc and knee meniscus, lack intrinsic healing capacity following trauma or disease. Current treatments only address the symptoms of fibrocartilage damage and do nothing to prevent further degradation of the joint. A tissue engineered replacement, with biochemical and biomechanical properties approaching those of native tissue, could provide a solution. This thesis investigates two components critical to the generation of a tissue engineered TMJ disc: 1) characterization of the native disc to identify a suitable animal model and create design parameters, and 2) development of approaches to use human embryonic stem cells (hESCs) in fibrocartilage tissue engineering. The first step to achieving this goal was to identify an animal model for the human TMJ disc based on quantitative biochemical and biomechanical properties. To this end, rabbit, goat, pig, cow, and human discs were analyzed, and the pig disc was shown to possess properties most similar to the human. The next step was to further characterize the pig TMJ, as many aspects of the joint were still poorly understood. Though the TMJ disc is anchored to the surrounding bony tissue on all sides by discal attachments, little was known about their properties. Biochemical and histological analysis was performed on these attachments and indicated that they are similar to the disc but possess distinct regional matrix content related to joint biomechanics. Finally, though the contribution of collagen to the mechanical properties of the TMJ disc was well characterized, the contribution of the glycosaminoglycans (GAGs) was unknown. By removing sulfated GAGs with chondroitinase ABC, it was found that these molecules contribute to the viscoelastic compressive properties of the disc, but only in regions with the highest native GAG content. The second aspect of this thesis involved producing fibrocartilage tissue from hESCs. The pluripotency and unlimited self-renewal of these cells makes them ideally suited for producing fibrocartilages that contain a spectrum of matrix components. This work began by investigating what factors are necessary for fibrochondrogenic differentiation of hESCs in embryoid bodies (EBs). Growth factors and co-cultures with primary fibrochondrocytes were both shown to be potent modulators of fibrochondrogenesis, although differentiation of hESCs consistently produced a heterogeneous cell population. To purify populations of fibrochondrocytes differentiated form hESCs, two inexpensive and novel techniques were investigated. First, density gradient separation was the first technique attempted. This technique was able to isolate distinct subpopulations of cells, some of which were mechanically similar to native chondrocytes. Second, a chondrogenic tuning technique was applied to differentiated hESCs. Following fibrochondrogenesis in EBs, cells were expanded in monolayer in chondrocyte specific media before being used for tissue engineering. Chondrogenic tuning produced several distinct cell populations during expansion, and, as a result, a spectrum of different cartilaginous tissues was achieved for tissue engineering. Three of the cell populations produced tissues similar to the native TMJ disc, outer meniscus, and inner meniscus. Overall, this thesis identified an animal model for TMJ characterization and in vivo studies, furthered understanding of structure-function relationships of the TMJ disc and its attachments, and developed a technique for producing a spectrum of engineered fibrocartilages from hESCs.