A Study Onthe Effect Of Osteoimmunomodulatory On Bone Defect Repair Regulated By CGRP-Releasing Borosilicate Bioactive Glass Scaffold

Objective: To study the effect of releasing Calcitonin gene-related peptide(CGRP)of borosilicate bioactive glass scaffold(BBG)on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)after promoting proliferation and polarization of macrophages,and the effect of regulating bone immunity on bone defect repair.Methods:(1)First of all,the material BBG-CGRP or BBG and DMEM medium were used to prepare the extract for 1g/10 ml according to the ratio of solid to liquid.Experimental groups: blank control group,BBG group,BBG-CGRP group.The material extract was co-cultured with Mouse peritoneal macrophages.The proliferation of macrophage was detected by CCK8 method,the adhesion morphology of macrophage was observed by SEM scan,the surface antigen expression of M1/M2 was detected by flow cytometry and immunohistochemical fluorescence staining,The secretion of inflammatory cytokines(TNF-α,IL-1β,IL-6,IL-10 and IL-1ra)was detected by ELISA.RT-PCR was used to detect the expression of related genes(inflammatory genes:IL-1β,IL-6,IL-10 and IL-1ra;related genes: TGF-β1,TGF-β3,VEGF,BMP-2).(2)the conditioned medium of macrophages was co-cultured with mouse bone marrow mesenchymal stem cells(m BMSCs).Alkaline phosphatase activity,alkaline phosphatase staining and alizarin red staining were used to detect the effect of cell conditioned medium on osteogenic differentiation and extracellular matrix mineralization of m BMSCs.The expression of osteogenesis related genes such as alkaline phosphatase(ALP),Collagen I(COLL-1),osteocalcin(OCN)and osteoprotegerin(OPG)was detected by RT-PCR,and the expression of osteogenic related proteins such as ALP/OPN was detected by Wstern Blot.Animal experiment in vivo: Experimental groups: BBG group,BBG-CGRP group,autogenous bone group.The rabbit model of large segmental tibial bone defect was established,and BBG-CGRP glass stent,BBG glass stent and in situ autogenous bone were implanted into the bone defect area respectively.X-ray examination,HE staining and methylene blue acid fuchsin staining were performed at 1,3 and 6 months after operation to detect the growth and material degradation of new bone.Results:(1)Cytology experiment in vitro: SEM scans showed that the groups of macrophages were round or elliptical.CCK8 experiment confirmed that BBG-CGRP could promote the proliferation of macrophages compared with control group and BBG group,and the difference was statistically significant(P<0.05).Flow cytometry,immunohistochemical fluorescence staining and inflammation-related gene expression assay confirmed that BBG and BBG-CGRP could promote the polarization of macrophages towards M2 macrophages,and the promoting effect of BBG-CGRP was more obvious than that of BBG,and the difference was statistically significant.ALP activity,ALP staining,alizarin red staining,osteogenic gene and Wstern Blot experiments showed that the macrophage culture medium of BBG and BBG-CGRP could promote the osteogenic differentiation of m BMSCs,and the osteogenic effect of macrophage culture medium of BBG-CGRP was more significant,the difference was statistically significant,indicating that the cytokines contained in the macrophage culture medium of BBG-CGRP were beneficial to the directional osteogenic differentiation of m BMSCs.(2)According to zoological experiments of postoperative X-ray,HE staining and methylene blue acid fuchsin staining,it was found that 3 months and 6 months after operation,more new bone tissue formed around BBG-CGRP was basically consistent with the osteogenic ability of autogenous bone,and the osteogenic ability was better than that of BBG group,indicating that BBG-CGRP has a better bone repair effect than BBG.It also shows that BBG-CGRP can promote bone regeneration and repair in vivo.Conclusion: CGRP loaded on BBG can regulate the polarization of macrophages towards M2 direction and regulate the release of inflammatory factors,thus creating a good microenvironment for osteogenesis and promoting bone regeneration.BBG-CGRP may be one of the directions of future research on Osteoimmunomodulatory.