Regeneration of striated muscle complex by the transplantation of myoblast in rats

Abstract

Objective To investigate the feasibility of myoblast transplantation in the regeneration of striated muscle complex, explore a potential treatment strategy to improve the pelvic floor muscle in anorectal malformations. Methods Skeletal muscle satellite cells obtained from the hindlimbs of neonatal Wistar rats were purified and cultured. eGFP labeled myoblasts were seeded into the acellular matrices, obtained by hypotonic-detergent treatment of striated muscle complex. After 2 d culture in vitro, myoblast-seeded matrices were implanted in situ into the pelvic floor. 4w after surgery, the matrices were harvested and observed with microscopy and immunofluorescence. Results Cultured cells were characterized as myoblasts by the presence of specific antigens Desmin and Myosin with immunocytochemical staining. Two subpopulations were identified in myoblasts, the earlier preplated cells proliferated and differentiated quickly, terminal differentiated into myotube rapidly, with limited passage ability; while the later preplated subclone still remain stem cell-like properties, positively expressed Pax7, a satellite cell marker, and divided slowly. 98. 6% cells were viral integrated 24h after transfeetion of eGFP-adenovirus as detected by flow cytometry. The acellular matrices mainly composed of basal membrane tube which were Laminin positive; at 48h from seeding, matrices showed many round myoblasts attached; the cultures remained viable until the 7th day, and fused myoblasts were seen. 4w after transplantation, muscle bundles can be seen behind M. bulbocarvernosus, but failed to form a sling-like structure of SMC. The myoblasts differentiated into myofibers, which were GFP and Myosin double positive staining, neovascularization were evidenced as vWF positive expressing vascular endothelial cells were observed, and Pax7 positive satellite cells were founded outside the sarcolemma of muscle fibers. Conclusions The satellite cells, after being implanted into the pelvic floor, showed strong proliferation and differentiation ability, they arefit to construct the cell bank of tissue engineering in rernolding the maldeveloped pelvic floor muscle. Further researches are needed to identify strategies to improve and maintain the structural and functional integrity of implants. Key words: Anus, abnormalities; Rectum, abnormalities; Skeletal muscle satellite cell; Transplantation