Mimicking the Tendon Microenvironment to Enhance Skeletal Muscle Adhesion and Longevity in a Functional...

Microcantilever platforms are functional models for studying skeletal muscle force dynamics in vitro. However, the contractile force generated by the myotubes can cause them to detach from the cantilevers, especially during long-term experiments, thus impeding the chronic investigations of skeletal muscles for drug efficacy and toxicity. To improve the integration of myotubes with microcantilevers, we drew inspiration from the elastomeric proteins, elastin and resilin, that are present in the animal and insect worlds, respectively. The spring action of these proteins plays a critical role in force dampening in vivo. In animals, elastin is present in the collagenous matrix of the tendon which is the attachment point of muscles to bones. The tendon microenvironment consists of elastin, collagen, and an aqueous jelly-like mass of proteoglycans. In an attempt to mimic this tendon microenvironment, elastin, collagen, heparan sulfate proteoglycan, and hyaluronic acid were deposited on a positively charged silane substrate. This enabled the long-term survival of mechanically active myotubes on glass and silicon microcantilevers for over 28 days. The skeletal muscle cultures were derived from both primary and induced pluripotent stem cell (iPSC)-derived human skeletal muscles. Both types of myoblasts formed myotubes which survived for five weeks. Primary skeletal muscles and iPSC-derived skeletal muscles also showed a similar trend in fatigue index values. Upon integration with the microcantilever system, the primary muscle and iPSC-derived myotubes were tested successively over a one month period, thus paving the way for long-term chronic experiments on these systems for both drug efficacy and toxicity studies.