Repair Of Bone Defects By Composite Of BMP9 Induced Bone Marrow Mesenchymal Stem Cells And P3HB4HB

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Pluripotent differentiation of rat bone marrow mesenchymal stem cells and biocompatibility of P3HB4HB1.1 Study aims BMP9 is a potential osteogenic inducer,BMSCs are excellent seed cells,P3HB4 HB is a good scaffold material.We propose that the composite of BMP9-induced BMSCs and P3HB4 HB can be used to repair bone defects.We first evaluate the osteogenic differentiation of BMSCs induced by BMP9 on P3HB4 HB material.1.2 Materials and methods Bone marrow mesenchymal stem cells(BMSCs)were isolated from 2-week-old SD rats and induced into adipocytes which were examined using Oil red O staining.The osteogenic differentiation was examined by alizarin red staining and ALP staining.BMSCs were co-cultured with P3HB4 HB and were examined in terms of growth and morphological changes of BMSCs on the surface of P3HB4 HB.CCK8 was used to examine the effect of P3HB4 HB on the proliferation of BMSCs.Flow cytometry was used to examine the effect of P3HB4 HB on the cell cycle of BMSCs.1.3 Results We have susscessfully isolated and culutured rat primary BMSCs.These cells can undergoadipogenic differentiation as revealed by the presence of lipid droplets and oil red O staining in the induction group.These cells can also undergo osteogenic differentiation as revealed by the presence of calcium nodules and Alizarin red staining as well as alkaline phosphatase(ALP)staining in the induction group.The results of co culture of BMSCs and P3HB4 HB showed that BMSCs stably attached to the surface of P3HB4 HB,and the cells grew and were morphologically changed.CCK8 assay showed that P3HB4 HB enhanced cell growth and viability of occulted BMSCs.Flow cytometry results showed that the proportion of cells in G1 phase was significantly reduced,and the proportion of cells in S phase was significantly increased,while the proportion of cells in G2 phase was not significantly different in the BMSCs and P3HB4 HB co-culuture group.There is no obvious change inn the normal cell group.1.4 Conclusion Rat bone marrow mesenchymal stem cells(rBMSCs)had the capability of adipogenic and osteogenic differentiation and were well compatible with P3HB4 HB materials.P3HB4 HB promoted the stable proliferation and the G1/S transition of BMSCs.Part ? BMP9 induces the osteogenic differentiation of rat bone marrow mesenchymal stem cells on P3HB4HB1.1 Study aims To evaluate the osteogenic differentiation of rBMSCs induced by BMP9 and to evaluate the osteogenic differentiation of rBMSCs induced by BMP9 on P3HB4 HB.1.2 Materials and methods Adenovirus vector containing BMP9 and the control vector were construced and used to infect rBMSCs cells.Fluorescence microscopy was used to observe the expression of BMP9 and the efficiency of adenovirus infecting target cells was estimated by examining GFP expression.QPCR was used to examine the expression of BMP9 m RNA.The rBMSCs cells were infected with the BMP9 adenovirus vector and the control vector and treated with the osteogenic differentiation media.QPCR used to examine the m RNA levels of osteogenic factors Runx2,OCN,OPN and OSX.Alizarin red and ALP staining were used to evaluate the degree of osteogenic differentiation.The rBMSCs cells were also infected with the BMP9 adenovirus vector and the control vector and cultured on P3HB4 HB treated with the osteogenic differentiation media.The levels of osteogenic factors were then examined by q PCR.The cability of osteogenic differentiation was assessed by alizarin red staining and ALP staining.The expression of osteogenic factors Runx2,OCN,OPN and OSX were determined by Western blotting.1.3 Results The BMP9 adenovirus vector has been successfully constructed.The relative level of BMP9 m RNA in BMSCs infected with the BMP9 adenovirus vector was 10 times higher than that in BMSCs transfected with the control vector.CCK8 assay showed that BMP9-adenovirus infection enhanced growth of rat BMSCs.Alizarin red staining showed that there were more and larger red calcified nodules in the cells infected with the BMP9-adenovirus vector,and ALP staining showed that the area of blue purple ALP staining was larger,the color was darker,the expression of Runx2,OCN,OPN,OSX m RNA was increased 4,7,7,4 fold on the first day of infection,and maintained 4-7 fold of increases on the seventh day,compared with those of the control vector-infected cells.In the BMSCs infected with the BMP9 adenovirus vector and cocultured with P3HB4 HB,Alizarin red staining showed that the deposition of red calcified nodulewas larger and more,alkaline phosphatase(ALP)staining were larger and darker,the expression of Runx2 and OCN m RNA was higher,the protein levels of Runx2,OCN,OPN and OSX protein in BMSCs infected with the BMP9 adenovirus vector were further increased than those of in the group infected with the control vector.1.4 Conclusion BMP9 is stably overexpressed in BMSCs infected with BMP9 adenovirus vector,which promoted the high expression of osteogenic factors Runx2,OCN,OPN and OSX,cell proliferation,calcium deposition and ALP activity.BMP9 adenovirus infection promoted the calcium deposition and ALP expression of BMSCs on P3HB4 HB composite,and the levels of osteogenic factors such as Runx2,OCN,OPN and OSX were continually increased.BMP9 adenovirus infection promoted the osteogenic differentiation of BMSCs,and promoted the osteogenic differentiation of BMSCs on P3HB4 HB.Part ? Repair of skull defects with BMP9-BMSCs-P3HB4 HB composite1.1 Study aims To evaluate the effect and mechanism of BMP9-BMSCs-P3HB4 HB composite materials on skull defect repair using animal models.1.2 Materials and methods SPF male SD rats,6-8 weeks old were divided into four groups: the sham operation group(A),the model group(B)where the skull defect model was made,the CNTL-BMSCs-P3HB4 H group(C)where BMSCs were infected with the control vector,and the BMP9-BMSCs-P3HB4 HB group(D)where BMSCs were infected with the BMP9 virus vector,and both were co-cultured with P3HB4 HB composite material and were then used to treat the skull defect models.There are 4 rats in each group.For the the sham operation group,only the scalp was opened,and no defect was made.At 4 weeks after operation,the skull of the injured area was removed and examined using micro CT detection to observe the repair of the defect,hematoxylin eosin staining to observe the pathological changes of the defect,by Masson staining to observe the changes of collagen fibers in the defect,the immunohistochemistry staining was used to determine the expression of osteogenic factors OSX,OCN,OPN and Runx2.1.3 Results The results of micro CT showed that CNTL-BMSCs-P3HB4 H had certain repair cability,while BMP9-BMSCs-P3HB4 HB had stronger repair cability.The results of HE staining showed that the repaired bone in the CNTL-BMSCs-P3HB4 H group was histologically good,and the BMP9-BMSCs-P3HB4 HB group was histologically better.The results of Masson staining showed that compared with the model group,the collagen fiber content of the CNTL-BMSCs-P3HB4 H group was increased to a certain extent,and that of the BMP9-BMSCs-P3HB4 HB group was increased more significantly.The results of immunohistochemistry showed that Runx2 staining in sham operation group was lighter than that in model group.Compared with the model group,the staining of the CNTL-BMSCsP3HB4 H group was dark,and that of the BMP9-BMSCs-P3HB4 HB group was even darker.OCN,OPN and OSX also had the same expression changes.The results showed that compared with the model group,the protein levels of Runx2,OCN,OPN and OSX in the CNTL-BMSCsP3HB4 H group were increased,and the protein levels of Runx2,OCN,OPN and OSX in the BMP9-BMSCs-P3HB4 HB group were increased more significantly.1.4 Conclusion BMP9-BMSCs-P3HB4 HB composite material has a good effect on repairing bone defects,which made the repair of bone defects have better histological changes,promoted the formation of collagen fibers,the expression of osteogenic factors Runx2,OCN,OPN,OSX protein in the repair site of bone defects.Therefore,BMP9-BMSCs-P3HB4 HB composite is a promising tool for bone defect repair.Summary In this project,the biocompatibility of BMSCs and P3HB4 HB,the osteogenic differentiation of BMSCs induced by BMP9 on P3HB4 HB,and the effect of BMP9-BMSCs-P3HB4 HB composite on repairing skull defects were evaluated.It was found that BMSCs could be successfully cultured in vitro,and differentiated into adipocytes and osteogenic cells.BMSCs and P3HB4 HB had good compatibility.P3HB4 HB promoted cell proliferation and osteogenic differentiation.BMP9 could promote the osteogenic differentiation of BMSCs on P3HB4 HB material.BMP9-BMSCs-P3HB4 HB composite material has a good effect on the repair of rat skull defects.The results showed that BMP9 induced BMSCs and P3HB4 HB could be used to repair bone defects.