| Objective: (1) To explore tissue engineering muscles which were constructed by combining allogeneic myoblasts with small instestinal submucosa(SIS) in rabbits repair defect of skeletal muscles. (2) To observe expression of allogeneic myoblasts which are cultured in vitro in skeletal muscles of rabbits by local muscle injection. (3) To observe distribution in vivo and effect of rats after transplantation of allogeneic myoblasts which were cultured in vitro by injection of rat caudal vein.Methods: (1) A large purified number of myoblasts could be obtained with multiprocedure digestion and repeated attachment method from skeletal muscles by taking from extremities of immature rabbits which were 7 days after they were born. The myoblasts were labeled by 5-bromo-2-deoxyuri dine(BrdU) ,then combined to SIS to construct tissue engineering muscles. This kind of tissue engineering muscles was grafted into the gastrocnemius muscle defect which was 1.5 cm in length, 1.0 cm in width of fifteen rabbits as the experimental group. The SIS was grafted into the same position in the control group and the skeletal defect without repairing as the blank control group. The animals were sacrificed on 4, 6, 8 weeks after operation. The tissue engineering muscles were evaluated by macroscopic , histological and immuno histo- -chemical observations, and quantitative analysis of localimmunocyte in the grafting site. (2) A large purified number of myoblasts could be obtained with multiprocedure digestion and repeated attachment method from skeletal muscles by taking from extremities of immature rabbits which were 7 days after they were born. The myoblasts were labeled by 5-bromo-2-deoxyuridine (BrdU) ,then making 1 ml cell suspension injected the middle piece of gastrocnemius muscle with multipoint and symmetrical methods. The control groups injected saline. The animals were sacrificed on 3 weeks after operation. The injection muscles were evaluated by macroscopic histological and immunohistochernical(BrdU) observations. (3) A large purified number of myoblasts could be obtained with multiprocedure digestion and repeated attachment method from skeletal muscles by taking from extremities of immature rats which were 3 days after they were born. The myoblasts were labeled by 125I-UdR. The 1ml labeled myoblasts were injected by rat caudal vein in the experimental group and DMEM injection in the control group. Every one week injection, total three times, the rats were sacrificed after three weeks. The blood routin, liver function, kidney function and value of γ counting were detected and observed pathologic alteration of transplantation immunity reaction and condition of blood vessel embolism.Results: (1) Allogeneic myoblasts with SIS were combined perfectly in vitro. The SIS was connected tightly to surrounding skeletal muscles and inflammation response was obvious on 4 weeks after grafting. On 6, 8 weeks of operation, the SIS began to break down and inflammation response became slight. Quantitative analysis of local immunocyte showed that there was statistic significance compared 8 weeks with 4, 6 weeks in the experimental and control groups respectively. The new formed muscle tissue were found around SIS in the experimental group on 4, 6, 8 weeks. Expression of BrdU and myosin immunohistochemical staining werepositive in the experimental group and negative in the control group. The blank control group was repaired by scar tissue. (2) There is no change by macroscopic observation in the control group and experimental. The histological observation showed that the few lymphocytes infiltrated and the experimental group have more myocytes nucleus than the control group in the injection muscle. Expression of BrdU immunohistochemical staining was positive in the experimental group and negative in the control group.The staining positive myoblasts distribute two sides of muscle fibre. (3) The value of y counting in the skeletal muscles are obviously higher than others organs in the experimental groups after injected allogeneic myoblasts which were cultured in vitro by rat caudal vein. There are statistic difference of lymphocyte count, monocyte count and kidney function in the experimental and control groups. There are no changes of pathologic alteration of transplantation immunity reaction, condition of blood vessel embolism and tissue necrosis in the experimental groups.Conclusion: (1) Tissue engineering muscles of rabbit which are constructed by combining allogeneic myoblasts with SIS can live, proliferate and repair defect of skeletal muscle.(2) Allogeneic myoblasts which were cultured in vitro as a satisfactory carrier can fuse with myocytes and normal growth in the receptor after local muscle injection. The local muscle injection myoblasts can be one of gene treatment methods. (3) The allogeneic myoblasts which were cultured in vitro had chemotaxis and distributed in the skeletal muscles Though this experimental methods had a little influence on the experimental animals, it may be a new clinical treatment toward myopathic disease.
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