Research Of Bone Regeneration Scaffold Material BMP-2/Collagen/Sr-HA

PartⅠEffect of bone morphogenetic protein-2 on Proliferation and Osteogenic Differentiation of Human umbilical cord mesenchymal stem cells in Vitro CultureObjective To study the effects of the bone morphogenetic protein-2(BMP-2)on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cell (hUCMSC) in vitro culture.Methods Surface markers (CD34,CD45,CD105,CD45,CD73,HLA-DR)of hUCMSC were tested by flow cytometry. hUCMSC were cultured with BMP-2(20ug/ml) in vitro. The proliferation effect of BMP-2 on hUCMSC was examined by MTT assay;the proportion of antigen-positive cells for STRO-1 was observed by Flow cytometry(FCM);the Osteopontin (OPN),alkaline phosphatase (ALP) and type 1 collagen(COL1) mRNA levels was detected by RT-PCR. The ALP activity was detected by ALP staining array.Von kossa staining method was used to study the calcification effects.Results The proliferation can be promoted after cultured with BMP-2 group and no BMP-2 group at 1,3,5,7 day,but was no statistical significance between two group (P>0.05).When the concentration of serum in the culture medium decreased from 10% to 2%, the proliferation of cells was just increasing 10%,and no different between BMP-2 group and no BMP-2 group (P>0.05).The percentages of STRO-1 positive cells was increasing from 25.1±4.0 to 51.1±6.4 after culture with BMP-2 (P<0.01).The BMP-2 also increased the ALP and COL 1 gene expression,induced the OPN gene expression.A lot of hUCMSC were positive in ALP staining array in BMP-2 medium.Many mineralized nodes was detected in the Von kossa staining method after culture 28 days in the BMP-2 medium.Conclusion BMP—2 can promote the osteogenic differentiation,and have a little effect on the prolferation of hUCMSC.PartⅡThe study of Human umbilical cord mesenchymal stem cells Osteogenic Differentiation induced by strontium chlorideObjective To investigate the roles ofβ-Catenin in Human umbilical cord mesenchymal stem cells Osteogenic Differentiation induced by strontium chloride in vitro culture.Methods hUCMSC were cultured in vitro,strontium chloride were added into culture fluid.Accoding to the different culture medium,three experimental groups were entered:blank group(10%FCS/DMEM),control group(10%FCS/DMEM+osteogenic medium),experiment group(10%FCS/DMEM+strontium chloride).Cell differentiation was examined by alkaline phosphatase(ALP) measurement kit.The Osteopontin(OPN),β-Catenin and type 1 collagen(COL1) mRNA levels was detected by RT-PCR. The expression ofβ-Catenin were detected by western bolt.p-Catenin expressing Human umbilical cord mesenchymal stem cells were detected by fluorescent microscopy.Von kossa staining method was used to study the calcification effects.Results Compared with the blank group and control group,the ALP activity in experiment group increased from 39.871±8.166 U/g.prot at 3 day to 70.317±14.231U/g.prot at 5day,even to 138.617±40.207U/g.prot at 14day,which was statistical significance contrast to the other group(P<0.05).At the experiment group, theβ-Catenin,COL 1 and OPN mRNA clearly expressed. On the contrary, OPN mRNA were weakly expression in the blank group(P<0.05).Clearβ-Catenin bands were observed in experiment group,but were weakly expressed in the blank group (P<0.05).The protein ofβ-Catenin localized in cytoplasm mainly after culture in the strontium chloride medium in fluorescent microscopy.Many mineralized nodes was detected in the Von kossa staining method after culture 28 days in the strontium chloride medium.Conclusion Strontium chloride can promote the osteogenic differentiation of hUCMSC through P-Catenin signaling.PartⅢThe Influence of bone morphogenetic proteins-2 and strontium chloride on the Human umbilical cord mesenchymal stem cells Proliferation and Osteogenic DifferentiationObjective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cell (hUCMSC) in vitro culture.Methods hUCMSC were cultured in vitro,BMP-2 and strontium chloride were added into culture fluid. Accoding to the different culture medium,three experimental groups were entered:blank group(10%FCS/DMEM), control group(10%FCS/DMEM+osteogenic medium), experiment group(10%FCS/DMEM+BMP-2+ strontium chloride).Cell proliferation were measured by methylthiazol tetrazolium(MTT) method.Cell differentiation was examined by alkaline phosphatase(ALP) measurement kit.The Osteopontin(OPN),alkaline phosphatase(ALP) and type 1 collagen(COL1) mRNA levels was detected by RT-PCR. The expression of smad1/5/8 and p38 were detected by western bolt.Von kossa staining method was used to study the calcification effects.Results Compared with the blank group and control group,the proliferation in experiment group was promoted after culture 5 day and 7day (P<0.05).The ALP activity increased from 33.443±9.061 U/g.prot at 3 day to 80.209±17.011 U/g.prot at 5day,even to 145.705±45.871 U/g.prot at 14day,which was statistical significance contrast to the other group (P<0.05).At the experiment group, the ALP,COL 1 and OPN mRNA clearly expressed.On the contrary, OPN mRNA were weakly expression in the blank group (P<0.05).Clear Smadl/5/8 and p38 bands were observed in experiment group,but were weakly expressed in the blank group (P<0.05).Many mineralized nodes was detected in the Von kossa staining method after culture 28 days in the BMP-2+ strontium chloride medium.Conclusion BMP-2 in combination with strontium chloride can promote the proliferation and osteogenic differentiation of hUCMSC.Part IV Repair of cranial bone defects with BMP-2/collagen/sr-HA scaffoldObjective To investigate the osteogenetic capacticy of BMP-2/collagen/sr-HA scaffold.Methods Preparation of collagen,collagen/HA,collagen/sr-HA,BMP-2/collagen/sr-HA scaffolds.And the scaffolds were tested by scann electron microscope.Cranial defects were created on 24 Sprague-Dawley rats.The Cranial defects were implanted the 4 scaffolds.Up to the 12 weeks,the rats were scanned by CT and the whole calvaria were scanned with 0.5mm in thickness. The rat calvaria from four groups were observed by histological staining and Immunohistochemistry.Results Three-dimensional reconstruction of computed tomography indicated that The Cranial defects of BMP-2/collagen/sr-HA group were almost repair completely.And the density of radiodense areas of BMP-2/collagen/sr-HA group were highest among all groups. Histological observation showed a large amount of new and mature bone had been observed in the BMP-2/collagen/sr-HA groups,but in the collagen/HA and collagen/sr-HA the number of new bone were less. Immunohistochemistry in collagen/HA and collagen/sr-HA group theβ-catenin and OPN signals appeared on some cells at the boundary of the formed bone, in BMP-2/collagen/sr-HA group more intense and concentrated P-catenin and OPN signals were observed on the cells surrounding the newly formed extracellular matrix.Conclusion The osteogenic capability of BMP-2/collagen/sr-HA scaffold is obviously superior to col/HA and col/sr-HA.BMP-2/collagen/sr-HA scaffold biomimetic scaffold material can be a potential repairing material for bone defects.