Culture of satellite cells in an electrically-stimulated three-dimensional scaffold for the generation of skeletal muscle engineered graft

The production of skeletal muscle engineered grafts holds promising implications for treatment of a variety of muscle diseases using methods of regenerative medicine, reconstructive surgery and cell therapy. In the present work, we studied a biomimetic approach on 3D culture of satellite cells, in order to obtain a functional engineered skeletal muscle graft suitable for tissue regeneration and cell delivery applications. Satellite cells were isolated from single muscle fibers of flexor digitorum brevis and extensor digitorum longus of wild type and GFP-positive mice and they were seeded and cultured on three-dimensional high-porous collagen sponges. In addition, in order to enhance satellite differentiation and myofiber functionality, electrical square pulses of 70mV amplitude and 3ms duration were applied to 3D scaffold mimicking physiological contraction. After one week of culture analyses of cell viability, histology, protein expression and NOx release, which is a signaling molecule implicated in satellite stem cell differentiation pathway, were performed. Both electrically-stimulated and non-stimulated 3D scaffold, seeded with GFP-positive satellite cells, were implanted in tibialis anterioris muscle of syngenic GFP-negative mice. Cell viability was investigated through immunofluorescence analysis at different time point: 10, 24, 34 and 62 days. In order to evaluate the effectiveness of myogenic-cell delivery for treatment of muscle dystrophies, 3D scaffold seeded with wild type satellite cells were implanted in tibialis anterioris and quadriceps femoralis muscles of aged mdx mice and analyzed using immunofluorescence technique. In our study, satellite cell culture within 3D scaffold in static conditions shows a good cell viability and protein expression, demonstrating that collagen creates a suitable “niche” for satellite cells survival and proliferation. On the other hand, histological analysis show preferential cell growth on the seeding side of the scaffold; an uniform cell seeding was achieved using a dynamic cell culture in perfusion bioreactor. Electrical-physiological stimuli do not significantly affect cell viability. On the other hand, they can enhance the satellite cell differentiation process. The measurement of the NOx concentration in the culture media shows faster (about 60%) NOx release rate in electrically stimulated cultures compared with non-stimulated cultures. The enhancement of NOx release from satellite cells by electrical stimulation was demonstrated culturing the satellite cells with a non-specific inhibitor of NO-synthase (L-NAME); when it was used, the release of NOx was depleted in both electrically stimulated and non-stimulated cultures. In vivo results show that GFP-positive cells were still present on the 3D scaffolds in long term implants of 62 days. In addition, in vivo analysis on mdx aged mice show a cluster of dystrophin-positive myofibers after 20 days of implant on the interface between the scaffold and the native muscle. Our findings demonstrate that 3D satellite cell cultures coupled with electrical stimulation can be a new strategy for the generation of a skeletal-muscle engineered graft and for the development of a new cell delivery protocol.