Study On The Mechanism Of Induced Membrane To Improve Vascularization And Bone Formation Of Bone Grafts By Promoting Stem Cell Migration

BackgroundLarge bone defects caused by infections,traumas,and tumors have been one of the serious complications in the field of trauma surgery,and also a difficult problem for orthopedic surgeons.Since Masquelet first reported the induced membrane technique in 2000,it has attracted increasing attention from scholars and is increasingly used in clinical research.Recent studies have shown that the induced membrane is a fibrous blood-rich structure around bone cement,which has the effects of reducing bone resorption,promoting angiogenesis and cancellous bone corticalization.The induced membrane technique have acquired is stable and reliable effects as a mothed of treating large bone defects.Bone is grafted in the defect when the bone cement is removed in the second phase.The selection of autogenous bone graft is the gold standard for the treatment of bone defect.The limited source of autogenous bone is the main bottleneck restricting the repair of large bone defects in the induced membrane technology,and there are complications such as pain in the donor area,insufficient osteogenic ability,scars,infection,and blood loss.So scholars seek to use autogenous bone substitutes to make up for the shortage of autogenous bone.It is currently believed that autologous cancellous bone graft can be partially replaced by allogeneic cancellous bone,but should not exceed 1/3 of the total amount,because the graft does not contain stem cells and growth factors.Therefore,whether it is possible to increase the proportion of bone substitutes,and how to promote the osteogenesis and vascularization of bone substitutes in induced membrane technology through migration of stem cells will be an important research direction.In this study,we constructed a rat model of induced membrane with bone defect firstly,then artificial material was implanted in the membrane.The regulatory effect of the induced membrane on the expression of osteogenic genes of implanted material was studied to explore the possibility of increasing the proportion of artificial material in the future.Secondly,to repair bone defects with artificial graft implantation under induced membrane,the establishment of an effective blood supply was requires.The recruitment of EPCs at the site of bone defects can promote angiogenesis,improve the survival rate of artificial bone grafts.The Induced membranehas rich blood vessels on the inner surface,and can secrete multiple growth factors.EPCs have an important physiological role in maintaining the stability of the vascular environment and regulating angiogenesis.Therefore,we have studied osteoprotectin(OPG)to improve the activity of EPCs in bone defect sites,enhance the migration function of EPCs,and promote the formation of new blood vessels,which providea new idea for the vascularization and osteogenesis of bone grafts.Thirdly,BMSCs play an important role in the bone formation process of transplanted bone with induced membrane technology.Some scholars have confirmed that MSCs combined with EPCs can significantly promote the bone healing process in animal models,but the mechanism that initiates the migration of host BMSCs to the defect site is not clear.Previous studies have shown that the expression of stromal cell-derived factor-1(SDF-1)in the induced membrane is significantly increased.SDF-1 is a well-known chemotactic protein secreted by bone marrow stromal cells and other stromal cells.Membrane receptor,expressed on the surface of mesenchymal stem cells(MSCs)and BMSCs,SDF-1 and its receptor CXCR4 play an important role in the activation of hematopoietic stem cells and the regulation of BMSCS migration through the gradient of SDF-1 concentration in tissues and blood effect.Therefore,this research intends to study the migration effect of SDF-1 on BMSCs during the process of induced membrane formation and repairing bone defects through Transwell and animal experiments in order to understand the mechanism of induced membrane formation and the role of BMSCs in bone transplantation after induc ed membrane formation.In addition,osteoblasts play an important role in the formation of new bone in transplanted bone.It has been found that some LncRNAs can regulate the cellular function of bone tissue through various mechanisms and participate in certain pathologic al processes of bone diseases in the field of bone biology.Up to now,the research on the role of LncRNA in bone formation and reconstruction has mainly focused on BMSC and osteoblasts.Therefore,we studied its effect on the proliferation of osteoblasts and the expression of osteogenic genes by intervening with LncRNA UCA1(urina ry epithelial cancer-related 1),The role of Lnc RNA UCA1 and regulatory mechanisms in the repair of bone defects were also studied.The purpose is to provide a new strategy for creating the bone formation microenvironment and preventing bone resorption after bone substitute transplantation.Methods:1.Study on Induced Membrane Technique Combined with Different Biomaterials to Promote Osteoblast Gene ExpressionMaking BCP,OSA-SA,OSA-SA-HA scaffolds.A rat induced membrane model was constructed,and the induced membrane was observed by HE and Masson staining.The femoral bone was drilled under the induced membrane,and the BCP,OSA-SA,and OSA-SA-HA scaffold materials were implanted.Runx2,BMP2,Col-I,ALP,and OCN osteogenic gene expression were detected by PCR test.Western blotting was used to detect the expression of p-ERK and ERK,and the results of p-ERK/ERK were analyzed quantitatively.2.Study on the mechanism of promoting vascularization of bone graft compound with induced membrane technologyThe EPCs extracted from human peripheral blood were cultured in vitro and the expression of CXCR4 and its downstream p-AKT was monitored by the Western blot analysis after OPG treatment.Using the scratch wound healing test and Transwell assay,we assessed the variables influencing the effect of OPG on EPCs after pretreatment with CXCR4 blocker(AMD3100)and PI3 K blocker(Ly294002).After 4 weeks,the bone defect repair condition was estimated via micro-CT and staining with HE and Masson trichrome.Then,immunofluorescence staining was performed to assess angiogenesis in bone defects,while the expression of EPC marker and vascular endothelial growth factor receptor 2(VEGFR2)was detected by immunohistochemical staining.3.Study on Mechanism of Promoting Migration of BMSCs in Induced Membrane TechnologyRat BMSCs were cultured in vitro,and the optimal concentration of BMSCs induced bySDF-1 in vitro migration was studied by Transwell experiment,and the effect of AMD3100,a CXCR4 receptor blocker on BMSCS migration was studied.Induced membrane model was creation through animal experiments.After the induced membrane formation,artificial bone was implanted in the membrane,BMSCs labele d with DIR were injected into the animal model simultaneously.To explore the migrated ability of BMSCs to the surgical area and the possible mechanisms of induced membrane formation and osteogenesis,the trace of transplanted BMSCs labeled with DIR was ob served.4.Partial mechanism of promoting proliferation and differentiation on the osteoblastsPlasma samples were collected from 52 osteoporosis patients treated in our hospital and 30 healthy subjects receiving a physical examination,respectively.The expression level of lncRNA UCA1 in OST patients and healthy subjects were detected via Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Furthermore,osteoblast MC3T3-E1 cell lines with a stable knockout of UCA1 in mice were constructed using small-interfering RNA(si RNA).The influence of UCA1 knockout on the proliferation of osteoblasts was detected using cell counting kit-8(CCK-8)assay.Meanwhile,the proportion of EdU-positive cells in osteoblasts of the control group and UCA1 knockout group w as detected using Ed U staining.Moreover,the messenger RNA(mRNA)levels of differentiation-related genes,including Runt-related transcription factor 2(Runx2),Collagen1?1,osteoclast(OC),osteoprotegerin(OPG),osteopontin(OPN)and Osterix(OSX),were detected via RT-PCR.The protein expression level of Runx2 was detected via Western blotting.In addition,osteoblasts were cultured with a bone-derived medium for 14 d.Then,the differentiation status was detected via alizarin red staining and alkaline phosphatase staining.Finally,the expression of bone morphogenetic protein-2(BMP-2)/(Smad1/5/8)signaling pathway was analyzed using Western blotting.Results:1.Induced membrane animal models can be successfully constructed.Different materials were implanted in the induced membrane.The results of PCR detection showed that the expression of Runx2 in the BCP group increased significantly at week 1,and the highest expression of BMP-2 at week 1 was observed in the OSA-SA-HA group.In terms of COL-I expression,the expression of COL-I in the BCP group was highest at all three time points;in terms of ALP expression,the ALP of both the BCP group and the OSA-SA group reached the highest value at week 6;At 9 weeks,the expression of ALP in the three groups was higher than that in the Blank group,especially in the OSA-SA group;in terms of OCN expression,the BCP group reached its maximum at the 6th week.2.The EPCs treated with OPG had increased levels of CXCR4 and p-AKT.Moreover,the difference in EPC levels among groups in the scratch wound healing experiment and migration experiment indicated that the OPG treatment promoted cell migration and AMD3100 and LY294002 inhibited the function of OPG.In addition,OPG promoted angiogenesis and repair of bone defect in rats,and these effects were abolished by AMD3100 and LY294002 administration.3.When cultured in vitro at 100 ng / m L SDF-1 concentration,the migration effect of BMSCS cells was most significant.After adding the SDF-1 inhibitor AMD3100,the number of migrating cells was significantly reduced.A rat induced membrane model was constructed.BMSCs were labeled with DIR iodide.In vivo imaging showed that the labeled BMSCS can migrated to the site area in the bone cement group(induced membrane formation group)and the bone graft group.Only a few cells were traced to the site area in the groups treated with AMD3100.4.The expression of plasma lncRNA UCA1 was significantly increased in osteoporosis patients(p<0.05).Cell experiments revealed that UCA1 si RNA intervention could significantly promote the proliferation and differentiation of osteoblast MC3T3-E1 cell lines.In addition,Western blotting showed that the pro-apoptotic effect of UCA1 might be mediated by the BMP-2/(Smad1/5/8)signaling pathway in osteoblasts.Conclusions:In the presence of the induced membrane,it can show better osteoinductive activity on BCP,OSA-SA and OSA-SA-HA scaffold,and play a role in enhancing osteogenesis.OPG enhanced the proliferation and migration of EPCs through the CXCR4 pathway and promoted angiogenesis and bone formation at bone defect sites.SDF-1 can induce the migration of BMSCs.After bone cement implantation and bone grafting in the induced membranes,BMSCs can migrate to the surgical area in large numbers,which indicated that SDF-1 induces the migration of BMSCs through the SDF-1/CXCR4 axis,This effect can be observed during the formation of induced membrane,as well as the procedure of bone graft in the induced membrane formated six weeks later.Inhibiting lncRNA UCA1 can promote the proliferation and differentiation of osteoblasts by activating the BMP-2/(Smad1/5/8)signaling pathway in osteoblasts.Therefore,UCA1 is expected to become a new therapeutic target to promote bone formation and inhibit bone resorption after bone transplantation.