The Research Of The Biocompatibility And Osteogenesis Ability Of Demineralized Dentin Matrix Surface Decorated By BFP-1

Objective: In this study,carboxymethyl chitosan(CMC)was used as the medium,and bone forming peptide 1(BFP-1)was used to decorate the demineralized dentin matrix(DDM)after the carboxyl group was activated by1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride/N-Hydro xysuccinimide(EDC/NHS).At the same time,the biocompatibility of the composite material and its ability to promote osteogenic differentiation of rat bone marrow mesenchymal stem cells(r BMSCs)were discussed.Methods: DDM was obtained by pulverizing extracted human teeth that had been systematically demineralized.This study evaluated three groups of materials: DDM,DDM/CMC,DDM/CMC/BFP-1,and added a Blank group(only containing r BMSCs)as a control.Firstly,Fourier transform infrared spectroscopy(FT-IR),X-ray photoelectron spectroscopy(XPS)and fluorescence localization were used to characterize the material to determine whether BFP-1 modified DDM success.At the same time,r BMSCs were isolated and cultured.it was identified by oil red O staining,after inducing adipocyte differentiation of r BMSCs.Alkaline phosphatase staining and Alizarin red staining were used to identify,after inducing osteogenic differentiation of r BMSCs.The surface markers of r BMSCs were detected by flow cytometry.Then,Cell counting kit-8 assay(CCK8)and scanning electron microscope(SEM)imaging were used to evaluate the the proliferation and adhesion of r BMSCs when co-cultured with materials.Finally,alizarin red staining,alkaline phosphatase qualitative and quantitative experiments,and real-time quantitative polymerase chain reaction(RT-q PCR)were used to analyze the materials’ ability to promote osteogenic differentiation of r BMSCs.Results: The FT-IR results showed that compared with the DDM group and the DDM/CMC group,new peaks appeared on the surface of the DDM/CMC/BFP-1 group,representing the amide bond and the carboxyl group of the peptide,respectively.XPS results showed that carbon,oxygen,calcium,phosphorus and nitrogen were visible on the surface of the three groups of materials,but the amount of nitrogen on the surface of DDM/CMC/BFP-1 materials has increased significantly.Under the fluorescence microscope,red fluorescent substance could be seen on the surface of DDM/CMC/BFP-1 material,but the control groups had no such phenomenon.The primary r BMSCs were successfully isolated and cultured,and the adipogenic and osteogenic induction medium were used to induce the differentiation of r BMSCs.The results were confirmed by oil red O staining,alkaline phosphatase staining and alizarin red staining.Compared with the control group,the experimental group Red-stained lipid droplets were formed,a deeper blue-stained area and red-stained calcium nodules appeared.The results of flow cytometry showed that CD29,CD90,CD31 and CD45 could be detected on the surface of the cells cultured.The expression of CD29 and CD90 were positive,and the positive rates were96.02% and 95.40%,while the expression of CD31 and CD45 were negative.The CCK8 assay was used to screen the appropriate concentration of co-cultured materials with r BMSCs.The results showed that compared with 100 mg/m L and 50 mg/m L,when the material concentration was 10mg/m L,the material had less effect on cell growth.The CCK8 assay was used to detect the effects of DDM,DDM/CMC,DDM/CMC/BFP-1 on the proliferation of r BMSCs,the results showed that compared with the DDM group and DDM/CMC group,the DDM/CMC/BFP-1 group had a promoting effect on the proliferation of r BMSCs.At the same time,cell proliferation was BFP-1 concentration-dependent,that is,as the concentration of BFP-1 increases from 0.1 mg/m L to 0.5 mg/m L,cell proliferation activity increases.However,when the concentration was increased to 1 mg/m L,cell proliferation was inhibited,and the difference was statistically significant(P < 0.05).Scanning electron microscopy results showed that compared with the DDM and the DDM/CMC,cells adhered most densely on DDM/CMC/BFP-1 materials.In the experiment of the various materials’ ability to promote osteogenic differentiation in vitro,the alkaline phosphatase staining of the DDM/CMC/BFP-1 group showed deeper color,the alkaline phosphatase activity was higher(P <0.05),and there were more red-stained calcium nodules.The expression of osteogenic genes in the DDM/CMC/BFP-1 group were also the highest(P< 0.05).Conclusion: In this study,BFP-1 was successfully used to decorate DDM.Compared with DDM and DDM/CMC,DDM/CMC/BFP-1composite material has better biocompatibility and the ability to promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro.The composite material initially shown that it has the potential to be used as a scaffold material for bone augmentation.