| Background: At present, the skeletal muscle loss what caused by the trauma, operation and other reasons always be repaired by the muscle flap and musculo-cutaneous flap transplantation. Although they met many successes in clinic, the new defect must be occurred in the donor site inevitable, so, the scientists have been exploring the alterative solutions to instead of them. With the rapid development of the tissue engineering, more and more authors have devoted themselves on the researches of the reconstruction of the tissue engineering skeletal muscle.The aim of this study is to explore a feasible alteration to reconstruct the tissue engineering rat skeletal muscle fibers with ideal directiviy in vifro,thus, to provide exprerimental basis for the tissue engineering skeletal muscle what could be used in clinic . Part one:Quantification of CeU Adhesion with Material Using Centrifugation: Establishment and Preliminary ApplicationObjective: To introduce a simple and feasible method that was used to determine the adhesion between the cell and material.Methods: The establishment of this method based on the principle what the linear relationship exists in the centrifuge force and the radius under a definite angular velocity. Equipment was produced by using medical plaster to immobilize the petri dish firstly. And then, muscle satellite cell suspension of rat was dropped on the surface of material films(PLGA, PLGA +COLI,PLGA+COL I +FN), which covered the basis of the culture dish. The centrifugal apparatus was start with a definite angular velocity after a while. Finally, the radius of the cell patches, which was not shedding, was determined and the mean value was obtained.Results: The measured value of the radius were 7.96 15.1 26.10mm respectively. The significant difference exists between the three groups (p<0.001).Conclusion: The change of radius value could be used to indicate the alteration of cell adhesion with material. It is feasible that the determination of adhesion between the cell and material with centrifuge system. Especially, it has some advantage to the comparison of the adhesion between the same cell and the various materials. The usage of the type I collagen and fibronectin can gain the adhesion between the muscle satellite cell of rat and PLGA.Part two:Three-Dimensional Culture of Rat Skeletal Satellite Cells under Cyclic Mechanical StretchObjective: To explore the possibility how to reconstruct the rat tissue engineering skeletal muscle fiber with ideal directivity study through the three-dimensional culture of rat skeletal satellite cells under cyclic mechanical stretch. Methods: The poly vinyl alcohol (PVA) sponge, which is water-tolerant coated with Poly-DL -lactic -co-glycolic acid (PLGA) was used as scaffold in this study. Based on the apparatus for three-dimensional cell cultures under a cyclic mechanical strain, a specific stretch pattern (10% elongation, 10 stretches/mm for 10 min of each hour, 48 hours total) was applied to cell-scaffolding composites. The morphological change and arrangement of skeletal satellite cells were observed by the SEM. The proliferation and differentiation were studied by the cell counting and the detection of creatme phosphate (CPK) respectively .The m RNA changes and proteins synthesis of the functional proteins including the skeletal alpha-actin and myosin were explored by the semiquantitive RT-PCR and ELISArespectively.Results: Under the stretch pattern, the shape of satellite cells changed to oblate and spread to the direction of the stretch, the myotube also was observed in the strain group. On the contrary, the satellite cells spread to the all direction, and the formation of the myotube was not been found in the control group. Under cyclic mechanical condition, the cell number increased 14.6%, CPK increased 32%(P<0.001).The ratio of the skeletal alpha-actin and 18s RNA was increased from 0.45 to 0.54, the ratio of the skeletal MHC and 18s RNA was increased from 1.65 to 2.21. The ELISA data also indicated the...
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