Controlling myoblast phenotype with RGD-modified alginate matrices

New strategies are being developed to grow tissues and organs for transplantation from cells and biomaterials. Biomaterials are critical components of engineered tissues, as they act as scaffolds for new tissues development. To date, however, engineered tissues do not have the complete structure or function of native tissues they are to replace. It is important to provide cells within these tissues with biological signals to help guide cell function during engineered tissue development. We hypothesized that the phenotype of cells adherent to a biomaterial could be regulated by controlling the mechanism of cell adhesion to the biomaterial, and we developed a model biomaterial system based on alginate hydrogels to address this hypothesis.; Alginates are hydrophilic polysaccharides composed of mannuronic (M) and guluronic (G) acid monomers that gel in the presence of divalent cations such as Ca+2. We modified the alginates with RGD-peptides using carbodiimide chemistry. Ligand type and density may be varied on alginates of varying M:G over several orders of magnitude, with incorporation efficiency typically >60%. Myoblasts adhered, proliferated and differentiated on RGD-alginate hydrogels with a surface density of 10 fmols/cm2. Myoblast adhesion specificity was demonstrated, as soluble RGD (1mM) completely inhibited adhesion to the substrates, while RGE-peptides (1mM) had no effect. Myoblast function was controlled by varying ligand type, ligand density, and M:G of the alginate. Myoblast proliferation increased on RGD vs. YIGSR peptide ligands (density = 10 fmols/cm2), and proliferation and fusion increased by increasing RGD-density from 1–100 fmols/cm2. Furthermore, myoblast proliferation and fusion, but not muscle-specific gene expression, were dependent on the M:G ratio of alginates. Varying M:G ratio from 30:70 to 65:35 increased myoblast proliferation and fusion, but did not alter the expression of MyoD or myogenin (myoblast transcription factors) or the activity levels of creatine kinase. This effect was caused by calcium calcium ions released from different alginate substrates. In conclusion, myoblast phenotype may be controlled by varying ligand type and density at the material surface, and by modulating local calcium concentrations using different alginate types.