Tissue engineering of skeletal muscle. Highly dense, highly oriented hybrid muscular tissues biomimicking native tissues.

A highly dense, highly oriented hybrid muscular tissue was devised using C2C12 cells (skeletal muscle myoblast cell line) and Type I collagen. A cold mixture of C2C12 cells suspended in DMEM (Dulbecco's modified Eagle's medium; Gibco Lab Inc., Grand Island, NY) and Type I collagen solution was poured into capillary tube molds of two different sizes (inner diameters: 0.90 mm and 0.53 mm, respectively) sealed at each end. After centrifugation (1000 RPM, 5 min) and subsequent thermal gelation, a rod shaped gel was formed. The resultant gel shrank to become a highly dense tissue after incubation on an agarose gel coated dish. Small diameter rod shaped tissues were composed of numerous multi-nucleated myotubes and a few necrotic cells. On the other hand, a ring shaped tissue fabricated by centrifugation with a specially devised agarose gel mold was subjected to cyclic stretching at 60 RPM. The resultant highly dense, highly oriented hybrid muscular tissue involved both densely accumulated cells and collagen fiber bundles, which tended to be aligned in the direction of stretching. Sequential procedures of a centrifugal cell packing method and a mechanical stress loading method facilitated fabrication of hybrid muscular tissues similar to native muscular tissues in terms of cell density and orientation.