SYNERGISTIC ROLES OF CHEMICAL AND MECHANICAL STIMULI IN IMPROVING THE ELASTICITIES OF ENGINEERED ARTICULAR CARTILAGE TISSUES THROUGH MECHANOTRANSDUCTION PATHWAYS
Quisenberry, Chrystal Rae
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Articular cartilage (AC) acts as a bearing surface to transmit forces within a joint. Because AC is avascular and has a low cellular density, it has a limited ability for self-repair upon injury. Current clinical treatments are incapable of restoring the functionality of the tissue. Tissue engineering of AC offers a promising alternative to current therapies. As such, the overall objective of this dissertation is to better understand the factors that improve the mechanical function of engineered AC tissues. Toward this end, we examine the synergistic effects of oscillating hydrostatic pressure (OHP) and transforming growth factor beta 3 (TGF-β3) on the elastic moduli of engineered AC from human adipose-derived stem cells (hASCs). Tissues grown with OHP were compared with static cultures in the form of micromass and pellet. Using atomic force microscopy (AFM) to quantify the elastic modulus of the engineered tissues, we found that OHP induces a 44-fold increase in the elastic modulus compared to the micromass and pellet samples. Supplementation with TGF-β3 further increased the elastic modulus in OHP samples by 1.86-fold. Because N-cadherins are important in the initial steps of mesenchymal stem cell chondrogenic differentiation, single molecule force spectroscopy (SMFS) was used to quantify the expression of N-cadherins on the surfaces of hASCs undergoing chondrogenesis. Tissues grown in the bioreactor with or without OHP exhibited a positive correlation between SOX9 mRNA expression and N-cadherin expression and TGF-β3 supplementation increased SOX9 and N-cadherin expression. These results suggest that TGF-β3 supplementation in the CBR improves chondrogenic differentiation through a path dependent on N-cadherin expression. Since OHP significantly increased the elastic modulus of engineered tissues, we believe that the mechanical stimulation is being transduced into the cell via mechanotransduction. To investigate this mechanism, we probed for the mechanoreceptor β1-integrin because of its frequency in chondrocytes and function in cell adhesion. From this exploration, we found that tissues with a less robust extracellular matrix (ECM), lower elastic modulus, had a lower expression of β1-integrins on their cell surfaces. With OHP, TGF-β3 increased the elastic modulus by 1.9-fold and decreased the β1-integrin count by 1.1-fold.