A comparative analysis of different biomaterials in the engineering of skeletal muscle using C2C12 cells in vitro

Tissue engineering represents a possible approach to replace the lost or defective muscle. The purpose of this study was to assess a new scaffold design for muscle tissue engineering, by comparing the growth of C2C12 mouse cells on different polymers. Polymeric substrata were prepared with a soft lithography technique (replica molding) from three different masters (a milled aluminium plate, a laser-processed alumina sheet, and a microgrooved modeling paste mould obtained from a home-made fibers array). Each master imprinted with an elastomeric material (Elastosilw RT601 Wacker), was in turn used as a mould to prepare the microgrooved polymeric substrata by solvent-casting. Polymer selected was a medical grade biodegradable poly(L-lactic acid) /trimethylencarbonate (PLLA/TMC 68:32, Boerhinger Ingelheim). Four different groups based on fiber spacing (30–35, 50–55, 70–75, and 90–95 mm) were evaluated. We compared 3-week growth of C2C12 cells cultured on scaffolds alone, or supplementing the scaffold with two key cytokines involved in muscle regeneration, bFGF(30G120 pM) and HGF(70G280 pM). Both methods facilitated cell attachment, growth, and viability. The cells lined the inner and outer surfaces of the scaffold, filling the pores, as demonstrated by scanning electron microscopy and histology. BrdUrd incorporation confirmed high levels of cellular proliferation. Yet, the combination of both cytokines showed a strong synergistic stimulation of C2C12 myoblast proliferation and chemotactic activity. After 21 days in the HGF-treated samples large areas of well-formed and aligned myotube fascicles were present which appeared to be more frequent than in the scaffold-alone samples and to show higher expression of differentiation-associated proteins. Our results show that the combination of PLLA/TMC polymer as a vehicle for myoblasts and exogenous HGF as a proliferative stimulator is a good candidate for the generation of skeletal muscle in vitro.