Synergistic Effect Of Deproteinized Bovine Bone Matrix And Periosteal-derived Cells On Activity Of M1 Phenotype Macrophage

Objective: Bone defect caused by trauma,tumor resection or periodontitis is one of the most common oral diseases in clinic.Therefore,repairing bone defects has become one of the urgentest problems in clinic.Autologous bone transplantation and guided bone regeneration are common bone augmentation methods in clinic,in both of which bone graft biomaterials are required.The implanted bone regeneration materials firstly regulate the immune response and inflammatory response of M1 macrophages.It is widely demonstrated that biomaterials can regulate the affection of bone marrow mesenchymal cells(BMSCs)on M1 or M2 polarization to provide an osteogenic environment.Periosteal-derived cells(PDCs)which belong to mesenchymal stem cells,play an important role in bone regeneration.However,it is not known whether PDCs can regulate inflammatory response of macrophage under the effect of bone replacement materials.The purpose of this study was to investigate whether deproteinized bovine bone matrix(DBBM,Bio-Oss)widely used in clinical practice can affect the regulation of the M1 phenotype of macrophages by PDCs and thus regulate the immune response of the first stage.Methods: PDCs were collected from 6-week-old female Kunming mice.The expression of CD90,CD29,CD45 and CD34 on the cell membrane and their differentiation ability were detected.All of these results proved that the cells extracted were PDCs.Treating PDCs with Lipopolysaccharide(LPS)from low concentration to high concentration(0,1,5,10 and 20 ng/m L)for 24 h respectively,to determine the most appropriate concentration of inflammatory priming PDCs model based on CCK-8 and q RT-PCR results.Then,inflammatory PDCs were cultured in conventional medium and extracted medium of DBBM(Bio-Oss)for 24 h,they were changed to DMEM medium for further culture for 24 h,and the supernatants were denoted as liquid A and liquid B,respectively.The mouse macrophage line RAW 264.7 was selected as the cell model,according to q RT-PCR results,the M1 macrophage was induced by LPS.The above conditional culture medium was added respectively: the control group was added with liquid A,and the DBBM group was added with liquid B.The proliferation activity and migration ability of M1 cells was measured by CCK8 after 24 h culture.The expression of M1-type macrophage-related genes(IL-1β,i NOS,TNF-α)at gene level,protein level and secretion level were detected by q RT-PCR,immunofluorescence and ELISA.Results: We observed that the positive rates of CD90 and CD29 were 99% and negative for CD34/45,and Oil red O,alcian blue and alizarin red staining were all positive,which indicated that the cells extracted were PDCs.In the induction experiment of inflammatory model,CCK-8 and q RT-PCR results showed that 10 ng/ m L LPS could successfully induce the inflammatory priming PDCs,and q RT-PCR results showed that M1 macrophages were successfully induced.CCK-8 results showed that the proliferation activity of M1 macrophages could be increased in the experimental group.The migration ability of M1 macrophages in DBBM group could be better than control group,as the result of transwell migration experiment illustrated.Compared to control group,the expression of Inos、Tnfa and Il1 b were down-regulated in the experimental group on m RNA expression levels.The INOS expression in the cytoplasm of M1 macrophage in DBBM group was lower as assayed by immunofluorescence.ELISA exhibited that PDCs of DBBM group could downregulate both of the secretion of TNFA and IL1B(P<0.05).Conclusions: DBBM can inhibit the inflammatory expression of M1 macrophage instead of proliferation and migration by affecting the paracrine of periosteal-derived cells in vitro to regulate the initial immune response.