Experimental Study On Induced Osteogenesis Of The Bone Marrow Mesenchymal Stem Cells As Well Asits Influence On Denervation Of Fracture Healing

Objective:To investigate the induction effect of bone marrow mesenchyme stem cells (BMSCs) into osteoblast-like cells in in vitro assay, hence to provide a groundwork for the future transplantation of MSCs. Meanwhile, the influence of BMSCs on denervation of fracture healing was studied as well, in order to further explore the mechanism underlying this process.Method:this experiment was divided into two stages to study Bone marrow mesenchymal stem cell transplantation in fracture healing in the denervated Dominate area.(l)BMSCs were separated using the adherence sieve method, and then induced by the osteogenetic inducer for three weeks. The indexes of osteogenetic differentiation, including the expression of ALP, cell mineralization, collagen1and osteocalcin expression were then detected after7,14, and31days separately.3months old female SD rats were selected in the present study, and were divided into six groups randomly (n=8):group A is of left tibia fractures, group B is of T10spinal cord hengduan+left tibia fractures, group C is of T10spinal cord hengduan+left tibial fractures+autologous mesenchymal stem cell transplantation, group D is of Left tibial fractures+DMEM,group E is of T10spinal cord hengduan++DMEM left tibial fractures, group F extracted autologous MSCS,with Brdu marking, marked cells were transplanted into fracture stumps by the method of sectional injection two days after the models of group C and F were build, p E after the operation. Blood biochemical contain of ALP、 Ca、 P in each group were also tested1,2,3, and4weeks after the operaTARLOR grade was assessed among group B, group C and groution separately. Shoot left tibia DR, CT, detect its length, the biggest osteotylus cross section, take the left tibia materials meanwhile, make ues of electronic weighing scales weighed wet weight, did HE dyeing, to detect ALP and fracture healing, of each group with immunohistochemical method.Results:(1)The primary BMSCs which were cultured by the method of whole Bone marrow adherent presented spherical or megagon adherence growth while the BMSCs induced by osteogenetic inducers presented round or oval adherence growth, with strengthened ALP synthesis; alizarin red stain appeared positive Calcified tubercle; The protein expression level of collagen I was increased significantly as shown in western blotting. Besides, using ELISA, we found an enhanced expeks after transplantedand fluorescent marker was still existed.No significant differences in Serum Ca concentration was shown among group1,2,3,and4on postoperation(P>0.05) s. Serumression o osteocalcin.(2) The tovaersal graded of Group B,C and E all reached the experimental requirements. BMSCs marked by Brdu was still survived in tissues three weconcentration of P in group C and E were significantly higher than other groups one week after the operation (P<0.05). The serum content of ALP in group C have statistical significance compared with other groups one week after the operation (P<0.05). The serum content of ALP in group A and D increased obviously, but was decreased in group C compared with the former week. Statistical significance was shown in group A and D compared with other groups two weeks after the operation (P<0.05), while no significance difference can be found in the rest groups (P>0.05). ALP in group B and E was significantly higher than other groups three weeks after the operation. In the4th week, a decreasement of the ALP level was found in all treated groups, among which, group B and E were more evident than other groups, however, no statistical significance was shown among each group.Significant differences in the tibia callus width was shown among groupb,c,and E than other groups among1,2and3weeks after the operation (P<0.05). Callus width was decreased in group C compared with group B and E three weeks after the operation (P<0.05). Statistical significance diameter of section in The tibia callus CT scan was shown in group B,C and E compared with other groups1,2,and3weeks after the operation (P<0.05), In the4th week, a decreasement of Callus diameter in group B and E which have statistical significance compared with other groups (P<0.05). Tibial wet weight weighing in group B,C and E have statistical significance compared with other groups1,2,3, and4weeks after the operation separately (P<0.05).No significant differences in measurement of Tibial length was shown among group1,2,3,and4on postoperation(P>0.05). Bleeding edema, Inflammatory change,and a small amount of fiber callus formation were appeared one week one week after the operation by Histology Observation; chondrocyte was appeared at fracture, the quantity of fiber callus is more as well as Lacuna cells, Subperiosteal cartilage cells layer was incrassation two weeks after the operation; Cartilage callus can be seen so still can Fiber callus be, a little amount of bone appeared, Periosteum reaction was light, a little bit of fiber callus, based on the principle of intramembranous ossification which was coexist th entochondrostosis. ALP immunohistochemical stains showed that the fracture was filled with fibrocyte, cartilage cells appeared on bone stump, ALP positive particles was pale tan one week after the operation; mechanocyte and osteoblast which were inside of osteotylus presented dyeing positive in group B, Positive cells increases two weeks after the operation. ALP stain appeared positive in osteoblasts of the fracture in group C,D and E three weeks after the operation.Cartilage cells on bone stump were matured in group F, osseouscallus was increased whihe Cartilage callus was decreased,ALP express was positive in osteoblasts and poroma, Positive cells, decreased significantly. Conclusion:BMSCs cultured by the method of whole Bone marrow adherent is simple and practical, with osteolblastic morphology and bionomics presented after being induced by the osteogenetic nducer.Denervation affected the inductive potential of Bone Mesenchymal Stem Cells bone to osteogenic, as well as the function of osteoblast。The ability of induces Osteogenesis was increased in denervation BMSCs, and the fracture healing was accelerated through autoplastic transplantation。 the mechanism of that fracture healing was accelerated in denervation area probably was:the potential of BMSCs induced osteogenesis was changed by denervation, then Promoted the fracture healing.