The application's long-term objective is to use a comprehensive bioengineering approach to successfully address articular cartilage regeneration. To this end, a self-assembling process was recently developed to tissue engineer articular cartilage using high density seeding of native chondrocytes in molds made of agarose. The study's main hypothesis is that the self-assembling process can be enhanced synergistically by the addition of growth factors and mechanical stimulation. To test this hypothesis, we propose the following specific aims: 1) To determine optimal growth factor conditions for the self-assembling process for in vitro articular cartilage regeneration. 2) To enhance this process using mechanical stimuli. 3) To engineer articular cartilage combining optimal growth factor and mechanical conditions for the self-assembling process. Application of four growth factors (TGF-(31, TGF-(33, BMP-2, and IGF-I) will be implemented in the self-assembling process at varying concentrations and dosage frequencies. The most favorable concentrations and dosage frequencies of each growth factor will be determined and combinations of these will be examined to select the optimal growth factor conditions. The effects of hydrostatic pressure and direct compression will also be examined at varying magnitudes, frequencies, and application times. The best conditions for each mechanical stimulus and their combinations will be selected from the multiple treatments. Finally, the effects of interactions between the optimized growth factor conditions combined with the optimal mechanical stimulation conditions on the self-assembling process will be assessed. At various time points, morphological, biochemical, and biomechanical properties will be quantified and compared to native tissue values, enabling the selection of optimal conditions. More specific hypotheses include: 1) The combination of growth factors will exhibit a synergistic effect by improving the self-assembled constructs' functional properties approaching two-thirds of native tissue. 2) Interactions between hydrostatic pressure and direct compression will enhance the regeneration process when compared to unloaded controls or constructs under a single mechanical stimulus. 3) The synergistic effects of combined growth factor and mechanical stimulation conditions will yield articular cartilage constructs with combined functional properties approaching those of native articular cartilage. ? ? ?
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