Study On Biological Characteristics Of Vascular Muscle Flap As A Carrier Of Bone Morphogenetic Protein To Repair Bone Defect

Objective (1) To investigate the effect and changes of skeletal muscle during vascular muscle flap as a carrier of bone morphogenetic protein to repair bone defect. (2) To investigate the proliferation and differentiation characteristics of skeletal muscle satellite cells in vitro. (3) To investigate the effects of bone morphogenetic protein on adhension, proliferation and differentiation of skeletal muscle satellite cells so as to interpret the biological mechanisms of vascular muscle flap as a carrier of bone morphogenetic protein to repair bone defect. (4) To detect the expression of hBMP7 in skeletal muscle satellite cells transferred with retroviral vector mediated hBMP7 gene.Methods (1) 15 mm radius bone defect was made. Denerved flexor digitorum profundus muscle flap with BMP was implanted to repair the bone defect. The osteogenesis and changes of skeletal muscle were investigated by gross, X-ray, histology, electro-microscope and TUNEL observation. (2) Rat triceps brachii muscle was acquired to separate skeletal muscle satellite cells with the two-step method of collegenase-1 and trypsin and were cultured and subcultured in vitro. Morphological observation, growth curve and rate of myotube formation were investigated to evaluate the proliferation and differentiation characteristics of skeletal muscle satellite cells. The cells were identified with immunochemical stain. (3) 0, 50, 100, 500, 1000 u g/L BMP were used to induce skeletal muscle satellite cells. The attached cells were calculated with fluorescence method at Ih after seeding. The lanimin of cells were measured with radio-immunology analysis. Cell proliferation wereassayed with methabenzthiazuron. The rate of myotube formation was calculated. Syntheses of collagen were assessed with 3H-proline incorporation test. Alkaline phosphatase activity and osteocalcin were also detected using NPP methods and radio-immunity methods. The mRNA of ALP, collegan-1, osteocalcin and osteopontin in skeletal muscle satellite cells were determined with RT-PCR. (4) Skeletal muscle satellite cells were infected with the replication-deficient retrovirus granules expressing human BMP7. The mRNA of hBMP7 gene in transferred cells was determined by RT-PCR.Results (1) The bone defect was filled up with new bone tissue formed in the muscle flap at 2 weeks. The new bone tissue bridged the bone defect at 6 weeks. Histological examination showed that a large amount of cartilage was formed in the gaps of muscles at 2 weeks. The cartilage was absorbed and replaced by normal bone containing hematopoietic bone marrow at 6 weeks with obvious muscle cell atrophy. Histological examination showed a portion of nuclei collapsed and apoptosis bodies were found with the atrophy of skeletal muscle. Apoptotic nuclei were found by TUNEL. Standard apoptotic changes were also observation by electro-microscope. (2) The two-step method of collegenase- I and trypsin is fit to acquire skeletal muscle satellite cells. The cells showed strong proliferation ability in the proliferation media and could form myotubes in differentiation media. Skeletal muscle satellite cells were faint positive, while myotubes were strong positive with immunochemical stain with a -sarcometric actin and myosin. (3) The number of attached cells increased after the cells were induced with BMP when the concentration below 500 u g/L, and reached the highest number when 500 u g/L BMP. The laminin of cells increased after the cells were induced with BMP, and reached the highest when 500 u g/L BMP. BMP promoted cell proliferation and reduced the rate myotube formation. Collagen syntheses were promoted when skeletal muscle satellite cells were induced with BMP which concentration were above of 500 u g/L. And with the increase of concentration this effect became stronger. Alkalinephosphatase activity and osteocalcin were also increased after skeletal muscle satellite cells were induced with BMP. The skeletal muscle satellite cells induced with BMP expressed abundant mRNA of collegan-1, osteocalcin and osteopontin in...