We prepared highly dense, highly oriented hybrid muscular tissues that are composed of C2C12 cells (skeletal muscle myoblast cell line) and type I collagen. A cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into capillary tube molds of two different sizes (inner diameters; 0.90 and 0.53 mm, respectively). One end of each mold was sealed. Upon centrifugation (1000 rpm, 5 min) and subsequent thermal gelation, a rod-shaped gel was obtained. It was cultured in an agarose gel-coated dish for 7 days (first for 3 days in a growth medium and then for 4 days in a differentiation medium), during which time it shrank to become a highly dense tissue. Small-diameter rod-shaped, highly dense cellular assemblages with multinucleated myotubes were formed and only few necrotic cells at the core of the tissue were observed. On the other hand, a ring-shaped tissue prepared using a specially devised agarose gel mold was subjected to cyclic stretching at 60 rpm, resulting in the formation of a highly dense, highly oriented hybrid muscular tissue in which both densely accumulated cells and collagen fiber bundles tended to be aligned in the direction of stretching. The hybrid muscular tissues that were prepared using via sequential procedures of a centrifugal cell packing method and a mechanical stress-loading method became closer to native muscular tissues in terms of cell density and orientation.
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.
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