Characterization of chondrocytic differentiation and optimization of a self-assembling process for tissue engineering of articular cartilage
Revell, Christopher Morton
Athanasiou, Kyriacos A.
Doctor of Philosophy
Articular cartilage degeneration, which occurs due to trauma or disease, results in the formation of tissue with inferior structural and mechanical properties. Tissue engineering has been proposed as a method to aid in the repair of damaged tissue. This thesis describes advancements in our laboratory's articular cartilage tissue engineering approach performed in three specific aims. One major limitation in cartilage tissue engineering is the paucity of donor tissue. To address this concern, dermal fibroblasts were investigated for their potential use in cartilage tissue engineering. Specifically, cartilage-specific proteins were examined for their ability to modulate the morphological characteristics of dermal fibroblasts toward the characteristically spherical morphology of articular chondrocytes. Optimal coating conditions were identified for stimulating fibroblasts into a more chondrocytic morphology with significantly increased cell height and lower surface area-volume ratios. Another limitation of cartilage tissue engineering is the production of tissue with sufficient biochemical and biomechanical properties. To this end, a self-assembling process was used to engineer neotissue with articular chondrocytes. Optimization of various parameters of this process was performed to increase this method's functionality toward clinical applicability and translatability. Three studies were performed: (1) Removal of serum was achieved, an essential requirement toward translatability, with a concomitant increase in functionality as evidenced by a 5-fold increase in compressive stiffness over serum-containing controls. (2) Temporal assessment identified 4 wks as an optimal time for both in vitro culture and the application of external stimuli to assist in enhancing functional properties. (3) Optimized parameters were then combined to examine the minimum quantity of cells required for the production of self-assembled constructs. It was found that the number of cells could be reduced by 32% while maintaining construct salient properties. To increase the functionality of self-assembled constructs, exogenous stimulation was investigated in this aim. Treatment of developing constructs with Chondroitinase ABC resulted in a more functional biochemical network which led to a 50% increase in tensile stiffness. Finally, a direct compression bioreactor was used to examine the effects of mechanical stimulation on self-assembled constructs. Properties of self-assembled constructs indicated that certain compression regimens imparted an immediate increase in GAG which contributed to increased compressive properties. Ultimately, direct compression with 17% strain applied at 0.1 Hz allowed constructs to reach 12% GAG/ww and an aggregate modulus of 290 kPa. The results of this thesis in toto advance the field of articular cartilage tissue engineering through optimizing conditions toward the use of an alternative cell source and its significant contributions to the understanding and progression of the self-assembling process. Moreover, successes of this thesis have led to the production of neocartilage constructs with ECM and compressive stiffness values elevated above native immature bovine cartilage.