Experimental Study Of Human Tooth Bone Graft Material Promotes Proliferation And Differentiation Of Mouse Preosteoblast MC3T3-E1

Objective To investigate the effects of human tooth bone graft material(HTBGM)on the proliferation and osteogenic differentiation of mouse pre-osteoblast MC3T3-E1,and to understand the biocompatibility and mechanism to induce osteogenesis of HTBGMMethods HTBGMs were collected and compared with the commonly used bone graft OSTEONⅡ in clinical practice.Both of them were co-cultured with mouse preosteoblasts MC3T3-E1.The experiments were divided into 4 groups.Which were Group A(blank control group),Group B(Untreated human tooth particles),Group C(OSTEONⅡ),Group D HTBGM(human tooth particles with complex acid treatment).After 7 days of co-cultivation,cell morphology was observed under the inverted microscope,and cell adhesion was observed on the material under the immunofluorescence microscope,use MTT assay was used to determine cell proliferation after co-culture after 1d,3d,5d,and 7d,and measure alkali Phosphatase activity was measured after 7d co-culture,and mineralized nodules were found with Alizarin red experiment,protein expressions of BMP2,BMP7,type 1 collagen(COL-I)were detectd using Western blot,m RNA expression of BMP2,BMP7,COL-I and alkaline phosphate were mersured by real time PCR.Results After 7d co-culture,the cells in each group were observed to have good morphology,and the cells in Group D had obvious spindle-shaped differentiation.Immunofluorescence microscopy showed that pre-osteoblasts MC3T3-E1 had a large amount of adhesion on the Group C and Grough D materials,obvious differentiation was found in Group D.The MTT results showed that there was no significant difference in the OD values of the four groups of cells on the 1d and 3d co-cultures.On 5d,the MTT value in Group C was the highest,the MTT value both in Group C and Group D were higher than Group A and Group B.The results of Group C and Group D were significantly higher than Group A and Group B on 7d.The results of ALP,alizarin red staining,COL-I,BMP7,BMP2 protein expression and m RNA expression showed that HBTGM group was at the highest level.Conclusions HTBGM can promote the proliferation and osteogenic differentiation of preosteoblast MC3T3-E1 cells,increase the expression of ALP,alizarin red staining,COL-I,BMP7,BMP2 proteins and genes,HTBGM has the potential advantages of bone graft replacement materials,can be used as bone graft materials to promote the repair and reconstruction of bone defects in the clinic.