(521d) Engineering 3D Silk Tissue Culture Platforms for Skeletal Muscle Repair

Authors: 
Cruz Quintero, R. G., University of Florida
Kaplan, D. L., Tufts University
Link, S. S., Tufts University
Stoppel, W. L., University of Florida
Black, L. D. III, Tufts University
Traumatic injuries caused by events such as vehicle accidents, surgeries, or compound bone fractures can cause extreme damage to muscle tissue. Although skeletal muscle is known to have an endogenous and robust healing response, grievous injury constituting Volumetric Muscle Loss (VML) can overwhelm the tissue’s natural regenerative ability and result in scar tissue formation and permanent loss of function. Advances in tissue engineering have employed the use of engineered muscle constructs to replace the lost tissue. However, a better understanding of the interactions that take place between the cells that compose the tissue and the engineered scaffold is still needed. To this end, we are developing an engineered skeletal muscle tissue using silk-based biomaterials as a base scaffold. One strategy we are employing focuses on probing the 3D parameters of the silk scaffold microenvironment, specifically composite silk scaffold formulation and extracellular matrix (ECM) composition, to elucidate compositional effects on cell proliferation and differentiation. In these in vitro skeletal muscle models, we applied uniaxial mechanical stimulation to the scaffolds to induce cell maturation using MechanoCulture T6® Bioreactors. Additionally, we analyzed changes in viscoelastic properties of the cell-free and cell-seeded scaffolds using dynamic mechanical analysis.

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 Young’s 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.