Injuries to the fibrocartilage of the knee, the medial and lateral menisci, present a particular challenge:​ the tissue is incapable of self-repair and the long-term outcomes of meniscectomy are osteoarthritis and disability. A tissue engineered knee meniscus, recapitulating the extracellular matrix (ECM) and mechanical function of the native tissue, is a possible solution. It is hypothesized that by studying the application o f scaffoldless techniques to cultures o f fibrochondrocytes and co-cultures o f chondrocytes and fibrochondrocytes, then modifying the technique to increase ECM synthesis, biomechanically robust neofibrocartilage will be created. Having developed successful methodologies with primary cells, it is hypothesized that human embryonic stem cells (hESCs) can be differentiated to cells producing matrix similar to that found in native fibrocartilage and these differentiated cells can be used to tissue engineer fibrocartilage.

This thesis addresses these hypotheses through five specific aims:​ 1) examine self assembly of fibrochondrocytes and co-cultures of fibrochondrocytes and chondrocytes;​ 2) study the use of staurosporine, an actin-modifying agent, to increase ECM synthesis and decrease construct contraction;​ 3) modulate ECM synthesis and mechanical properties in fibrochondrocyte and co-culture constructs through TGF-β1 treatment and base medium serum content;​ 4) apply the self assembly technique to differentiated hESCs to create fibrocartilage constructs;​ and 5) evaluate two differentiation techniques with hESCs for fibrocartilaginous matrix production.

Specific Aims 1-3 resulted in the successful application of a scaffoldless technique to create fibrocartilage constructs. Co-cultures of chondrocytes and fibrochondrocytes resulted in robust constructs composed of glycosaminoglycans and collagen type I, II, and VI. Use of serum-free chondrogenic medium resulted in enhanced glycosaminoglycan density and compressive stiffness, such that these constructs successfully recapitulate many of the biochemical and mechanical characteristics of native tissue. Specific Aims 4-5 applied the self assembly technique to hESCs. Using the H9 hESC line and a differentiation time of 3 weeks, matrix production and function were optimized with respect to the BG01V hESC line. Further improvement in the differentiation phase was achieved through treatment with BMP-4 + TGF-β3. These experiments represent a significant achievement in fibrocartilage tissue engineering that lays critical foundations for future in vivo studies of this potential therapy.