Exogenous stimulation of meniscus cells for the purpose of tissue engineering the knee meniscus
Gunja, Najmuddin Juzer
Athanasiou, Kyriacos A.
Doctor of Philosophy
Injuries to avascular regions of menisci do not heal and result in significant discomfort to patients. Current treatments, such as partial meniscectomy, alleviate the symptoms, but lead to premature osteoarthritis due to reduced stability and changes in knee biomechanics. An alternative treatment to overcome these problems involves functional tissue engineering. This thesis examined several exogenous factors to enhance the capability of meniscus cells (MCs) to synthesize relevant ECM markers and improve the functionality of constructs in vitro. First, the effect of passage on the phenotype of MCs in monolayer was investigated, and rapid changes were observed in collagen I, collagen II, and COMP expression. Collagen I and aggrecan protein coatings assisted in reversing expression levels of certain ECM markers; however, collagen II expression could not be reversed. Next, 3D tissue engineering studies were conducted using a cell-scaffold approach with MCs seeded on PLLA meshes. Anabolic stimuli that aided in meniscus regeneration included (1) hypoxia and bFGF, which resulted in synergistic increases in the total glycosaminoglycan content and compressive properties of constructs; (2) 10 MPa static hydrostatic pressure (HP), which resulted in increases in collagen content and the relaxation modulus of constructs; and (3) 10 MPa static HP and TGF-beta1, which resulted in additive increases in collagen content, and synergistic increases in the compressive moduli of constructs. Finally, a self-assembly, scaffoldless approach was employed for meniscus regeneration using co-cultures of MCs and articular chondrocytes (ACs). A high density of cells were seeded on non-adherent agarose molds and allowed to coalesce into a construct without a scaffold. Different co-culture ratios of MCs and ACs resulted in a spectrum of fibrocartilages that recapitulated some biochemical and biomechanical properties of the rabbit meniscus. Cell culturing conditions were optimized with the identification of a smooth 1% agarose mold that resulted in geometrically-mimetic meniscus constructs. In conclusion, this thesis quantified phenotypic changes in MCs over passage, and used scaffold-based and scaffoldless approaches to regenerate constructs with biochemical and biomechanical properties in the range of native tissue values. Successful replacement of a damaged meniscus will improve the quality of patient life and reduce the risk of osteoarthritis.