(521d) Engineering 3D Silk Tissue Culture Platforms for Skeletal Muscle Repair
To assess the proliferative capacity of seeded human skeletal muscle myoblasts, we quantified the number of cells present in the scaffold by staining their nuclei with DAPI, via fluorescent microscopic analysis and correlating images with both DNA quantification assays and metabolic activity. For maturation of these myoblasts in culture, we used fluorescent microscopy to visualize mature muscle marker expression, shown as an increase in desmin and myogenin. ECM protein deposition by Human Skeletal Muscle Myoblasts seeded within the constructs was evaluated via Western Blot analysis, quantifying differences in key ECM and ECM binding proteins such as collagen I, fibronectin, and laminin along with key integrins such as integrin beta 1, alpha 5, alpha V, and integrin linked ILK, which are all key components of skeletal muscle and its microenvironment. We also quantified changes in skeletal muscle markers via western blot to confirm results seen via microscopy. These analyses confirm that, in our model system of skeletal muscle maturation, scaffold compositions, addition of cells, and length of time in culture can influence Youngâs modulus. This skeletal muscle platform enables us to correlate initial material composition and structure with the role of time-dependent ECM protein deposition by the cells present within muscle tissue undergoing mechanical stimulation in a bioreactor system.