Myoblast adhesion, proliferation and differentiation on Human Elastin-Like Polypeptide (HELP) hydrogels

Purpose: The biochemical, mechanical and topographic properties of extracellular matrix are crucially involved in determining skeletal muscle cells morphogenesis, proliferation and differentiation. Human elastin-like polypeptides (HELPs) are recombinant biomimetic proteins designed to mimicking some properties of the native matrix protein; when employed as myoblasts adhesion substrates they stimulate in vitro myogenesis. Given the consequences that biophysical properties of extracellular matrix exert on skeletal muscle cells, the aim of this work was to investigate the effects of HELP hydrogels on myoblasts viability and functions. Methods: We recently synthesized a novel polypeptide, HELPc, by fusing the elastin-like backbone to a 41aa stretch present in the α2 chain of type IV collagen, containing two RGD motifs. To obtain hydrogels, the enzymatic cross-linking of the HELPc was accomplished by transglutaminase. Here, we employed both non cross-linked HELPc glass coatings and cross-linked HELPc hydrogels at different monomer density as adhesion substrates for C2C12 cells, used as myoblasts model. Results: By comparing cell adhesion, proliferation and differentiation, we revealed several striking differences. Depending on support rigidity, adhesion to HELPc substrates dictates cell morphology, spreading, focal adhesions formation and cytoskeletal organization. Hydrogels greatly stimulated cell proliferation, particularly in low serum-medium, and partially inhibited myogenic differentiation. Conclusions: In the whole, the results underline the potentiality of these genetically engineered polypeptides as a tool for dissecting crucial steps in myogenesis.