Thermoresponsive material for muscle cell sheet engineering

Loss of functional skeletal muscle caused by traumatic injury, congenital defects or functional damage due to a variety of myopathies produces a physiological deficit for which there is still no effective clinical treatment. Transplantation of exogenous myogenic cells (satellite cells and myoblasts) has been proposed to increase the regenerative capacity of skeletal muscle, but it is hampered by numerous limitations, including significant cell loss due to primary hypoxia or cell wash-out, difficulty to control the location of the grafted cells and poor cell migration. Current interest in regenerative medicine have prompted the development of living cells and polymeric materials (scaffold), that can cause immunological reaction, low bioactivity and biocompatibility of the exogenous materials. As an attempt to overcome these limitations, we proposed a cell sheet-based tissue engineering, approach, which involves stacking confluently cultured cells (two-dimensional), for muscle regeneration. We considered the characteristic of smart hydrogels to support muscle cells proliferation in vitro and to allow the detachment of a contiguous cell sheet, keeping its naturally formed networks. Smart hydrogels, in fact, have the ability of responding to some external stimuli, such as temperature, pH, electric and photo fields. Among numerous environmentally sensitive polymers, poly(N-isopropylacrylamide) (PNIPAAm) has drawn more interest because of its thermo-responsive properties. PNIPAAm is known to exhibit a conformational transition in an aqueous solution around 32°C. In order to obtain a biocompatible and thermoresponsive substrate we prepared a series of poly (N - isopropylacrylamide – co – 2 - hydroxyethylmethacrylate) based hydrogels. These hydrogels were synthesized by free-radical polymerization of the two co-monomers with a redox initiator. The biological evaluation was focused on viability and proliferation of either murine myoblasts cells line (C2C12) and human muscle derived stem cells (MSH). In vitro experiments confirmed C2C12 and human muscle cells adhesion and proliferation (MTT assay and Live Dead fluorescence staining), establishing hydrogel biocompatibility. Moreover, once achieved 80% cell confluence on the surfaces, we performed the sheet detachment test, decreasing the temperature under 32°C. The cellular sheet obtained was undamaged and contiguous in both conditions, either with murine and human stem cells, as confirmed by immunostaining. Testing several polymeric ratios, we observed a strong correlation between stem cell behaviour and hydrogel structure, such as bulk polymer density, monomers and solvents composition. Starting from these results, we are performing further experiments modifying hydrogel properties, in order to direct stem cells fate, focusing on proliferation and myogenic differentiation. Further experiments are also planned to verify the possibility to produce a viable multilayer cell construct, and test its regenerative potential in an animal model of muscle degeneration. After multiple cell sheet transplantation, it could be possible to achieve muscle regeneration and restore muscle function, thanks to the promotion of new blood vessel formation, inhibition of fibrosis and apoptosis through the continued secretion of cytokines from the viable transplanted cells and good cell migration to the muscle compound. Using cell sheet engineering approaches, we speculate to develop advanced applications for the treatment of several muscle disease through the understanding of the mechanisms to improved cell survival and migration after the transplantation