Decellularized skeletal muscle is a promising system to study the role of the extracellular matrix (ECM) in muscle regeneration. This study describes the development of a skeletal-muscle-specific-bioscaffold and its use as a substrate for myoblast proliferation and differentiation under serum free culture conditions. Three methods (Trypsin, SDS and Phospholipase A2) to decellularize skeletal muscle were compared with respect to efficacy of decellularization (no DNA) and retention of matrix proteins. A trypsin method effectively decellularized the tissue but it also removed antibody epitopes on fibronectin. An SDS method preserved most of the ECM components including perlecan and fibronectin, but it failed to preserve the small leucine rich proteoglycans biglycan and decorin. In addition, it did not completely remove cell debris as the decellularised matrix was smeared with DNA. The phospholipase (PLA2) method was the best with complete removal of cells and maximal retention of the ECM components including proteoglycans and glycosaminoglycans. Immunohistochemistry revealed good retention of epitopes on the ECM structural components (collagen I and collagen III) and basement membrane components (collagen IV, VI, fibronectin and laminin α-2 chain). Western blotting of fibronectin and collagen VI further supported the conclusion that the PLA2 best maintained ECM integrity.
Muscle matrix prepared after PLA2 decellularization was solubilized and used as a substrate for two dimensional (2D) culture of C2C12 mouse myoblasts under serum free culture conditions. This matrix supported cell proliferation and differentiaiton. Immunocytochemistry revealed that myoblasts differentiated into multinucleated myotubes on muscle matrix coated surfaces as evidenced by the expression of the differentiation marker, myosin. qPCR results showed a time dependent increase in the differentiation markers, myogenin and skeletal muscle actin 1, indicative of mature myotubes. Comparison of muscle derived matrix and type I collagen, modulates the differentiation markers differently. Decellularized whole muscle scaffolds (3D) also supported cell adhesion and spreading. Myoblasts became organised on the 3D matrix scaffolds suggesting that matrix proteins provide cues to direct cell adhesion. This is a first report of a solubilized muscle matrix promoting myoblast proliferation and differentiation in a serum free culture system.