| Objective:Removing the cells from natural periosteum(NP)and connecting the peptide(SKP)with the function of homing bone marrow mesenchymal stem cells to construct a functional membrane(DP-SKP).And study the membrane’s ability to promote the repair of bone defect.Methods:Getting the NP from the tibia of the rabbit after anesthesia.Then using physical and chemical elution steps to remove the osteoblasts and other cells contained in the NP to obtain decellularized periosteum(DP).Chemical reactions was used to connect SKP to the DP to obtain DP-SKP.H&E staining,Masson’s trichrome staining,DAPI staining,scanning electron microscope(SEM)observation,water imbibition measurement,collagen content analysis and glycosaminoglycan(GAG)content analysis were used to compare the difference of NP and DP.Rabbit bone marrow mesenchymal stem cells(BMSCs)were used in vitro cell experiments.Cytocompatibility and cell proliferation were assessed using live-dead staining and cell counting kit(CCK-8)for the DP group,the DP-SKP group,and blank control group.Osteogenesis of each group was assessed measuring alkaline phosphatase(ALP)activity,osteopontin(OPN)immunofluorescence staining and alizarin red staining.Rabbit skull defect model were used in vivo experiments.CD29 and CD90 immunofluorescence staining were used to compare the homing effect of BMSCs in the DP group and the DP-SKP group.Micro-CT Analysis,H&E staining,Masson’s trichrome staining were used to evaluate the ability of the DP group,DP-SKP group and Control group to promote bone defect repair in vivo.Results:DP was successfully obtained by decellularizing NP.DP eliminates immunogenicity and retains the double-layer structure of natural periosteum,collagen fibers and most other extracellular matrix.At the same time,DP-SKP was successfully prepared by connecting SKP with a peptide bond on DP.In vitro cell experiments,the result of the live-dead staining shows that the number of live cells in the DP-SKP group was greater than the other groups without significant dead cells.The CCK-8 optic density(OD)value was also higher than the other groups.It indicates that DP-SKP has good compatibility with rabbit BMSCs And plays a certain role in promoting proliferation.In the in vitro osteogenesis test,the ALP activity,OPN immunofluorescence staining,and alizarin red stained calcium nodules were higher in the DP-SKP group than the other groups.It indicates that it has a good promotion effect on osteogenesis.In the rabbit skull defect model,the DP-SKP group had more MSCs than the DP group,It indicates the homing effect of DP-SKP on MSCs.Moreover,the surface reconstruction of the bone defect area,data analysis of bone tissue and trabecular bone of Micro-CT,and H&E staining and Masson’s trichrome staining showed that the number and quality of new bone tissue in DP-SKP group were higher than those in other groups.This shows that DP-SKP can significantly promote bone defect repair.Conclusion:DP-SKP was successfully processed.In vitro cell experiments,DP-SKP has good cell compatibility and can promote cell proliferation.And it plays an important role in promoting the differentiation of osteogenesis in vitro.In vivo experiments,it has obvious homing effect on MSCs in rabbits.And it has excellent bone formation performance for rabbit skull defect repair. |
