Carbon Nanotube Functionalization and Scaffold Topography Guide Differentiation of Myoblasts

Recreating native microenvironment and providing appropriate guidance cues are instrumental in the design of regenerative scaffolds. Despite decades of research in skeletal muscle tissue engineering, there is limited clinical success in developing such regenerative scaffolds. This may be attributed to limited success in building complex features of native skeletal muscle tissue in synthetic scaffolds. These complex features include electroactivity of skeletal muscle cells, nano-(myofibrils) to macro-scale (muscle fiber bundles) hierarchical architecture along with aligned fibrous structure to provide contact guidance. Although scaffolds possessing each of these features have been built, combining multiple such features into a single scaffold still remains a challenge. Electrically conductive carbon-based scaffolds can be processed to possess multi-scale hierarchy along with porous structure and different geometries making them a potential candidate for skeletal muscle tissue engineering. Carbon-based materials have been explored mostly for their application in energy storage and electronics; however, few studies have evaluated their applications in tissue engineering. In this study, we investigated potential of carbon-based scaffolds with different geometries (interconnected porous vs aligned fibrous structure) and nanoscale surface functionalization (carbon nanotube) for skeletal muscle tissue engineering. We engineered multiscale hierarchical scaffolds based on carbon materials, namely, highly porous carbon foams and highly aligned carbon fibers. Both porous foam scaffolds and aligned fibrous scaffolds were nano-functionalized with carbon nanotube (CNT) and silica-CNT (only foams) coatings on their surfaces. It was hypothesized that aligned fibrous structure and nanoscale CNT coatings will provide synergistic guidance cues to enhance myoblast differentiation. Additionally, immobilization of CNTs on the scaffold surface will obviate the cytotoxicity issues. The results showed combined role of surface modification (CNT functionalization) and alignment cues to facilitate cell adhesion, growth and differentiation. Surface functionalization of porous carbon foams with CNT and silica-CNT provided physico-chemical cues for skeletal muscle cells to differentiate into myocytes; however, failed to promote their fusion into functional multinucleated myotubes. More interestingly, CNT nano-functionalization coupled with alignment cues in the form of aligned carbon fibers were able to overcome this limitation leading to formation of continuous multinucleated myotubes. In summary, CNT functionalization and aligned scaffold architecture improved cell-material interaction and promoted the process of myogenesis