Hybrid Muscular Tissues: Preparation of Skeletal Muscle Cell-Incorporated Collagen Gels

We prepared three different types of hybrid muscular tissues in which C2C12 cells (skeletal muscle myoblast cell line) were incorporated in type I collagen gels and then differentiated to myotubes upon culture: a disctype, a polyester mesh-reinforced sheet-type, and a tubular type. A cold mixed solution of the cells and type I collagen was poured into three different types of molds and was kept at 37°C in an incubator to form C2C12 cell-incorporated gels. A polyester mesh was incorporated into a gel to form the sheet-type tissue. The tubular hybrid tissue was prepared by pouring a mixed solution into the interstitial space of a tubular mold consisting of an outer sheath and a mandrel and subsequently culturing after removal of the outer sheath. Hybrid tissues were incubated in a growth medium (20% fetal bovine serum medium) for the first 4 days and then in a differentiation medium (2% horse serum medium) to induce formation of myotubes. Transparent fragile gels shrank with time to form opaque gels, irrespective of type, resulting in the formation of quite dense hybrid tissues. On day 14 of incubation, myoblasts fused and differentiated to form multinucleated myotubes. For a tubular type hybrid tissue, both cells and collagen fiber bundles became circumferentially oriented with incubation time. Periodic mechanical stress loading to a mesh-reinforced hybrid tissue accelerated the cellular orientation along the axis of the stretch. The potential applications for use as living tissue substitutes in damaged and diseased skeletal and cardiac muscle and as vascular grafts are discussed.