Muscular tissue engineering: capillary-incorporated hybrid muscular tissues in vivo tissue culture

Requirements for a functional hybrid muscular tissue are 1) a high density of multinucleated cells, 2) a high degree of cellular orientation, and 3) the presence of a capillary network in the hybrid tissue. Rod-shaped hybrid muscular tissues composed of C2C12 cells (skeletal muscle myoblast cell line) and type I collagen, which were prepared using the centrifugal cell-packing method reported in our previous article, were implanted into nude mice. The grafts, comprised three hybrid tissues (each dimension, diameter, approximately 0.3 mm, length, approximately 1 mm, respectively), were inserted into the subcutaneous spaces on the backs of nude mice. All nude mice that survived the implantation were sacrificed at 1, 2, and 4 wk after the implantation. The grafts were easily distinguishable from the subcutaneous tissues of host mice with implantation time. The grafts increased in size with time after implantation, and capillary networks were formed in the vicinities and on the surfaces of the grafts. One week after implantation, many capillaries formed in the vicinities of the grafts. In the central portion of the graft, few capillaries and necrotic cells were observed. Mononucleated myoblasts were densely distributed and a low number of multinucleated myotubes were scattered. Two weeks after implantation, the formation of a capillary network was induced, resulting in the surfaces of the grafts being covered by capillaries. Numerous elongated multinucleated myotubes and mononucleated myoblasts were densely distributed and numerous capillaries were observed throughout the grafts. Four weeks after implantation a dense capillary network was formed in the vicinities and on the surfaces of the grafts. In the peripheral portion of the graft, multinucleated myotubes in the vicinities of the rich capillaries were observed. Thus, hybrid muscular tissues in vitro preconstructed was remodeled in vivo, which resulted in facilitating the incorporation of capillary networks into the tissues.