| PART ONE Study on migration of BMSC improve fracture healing in murinemodel.ObjectiveOn the basis of establish closed femoral fracture in murine model, to study on thephenomenon and its possible mechanism of homing of BMSCs to fracture site, on theoutcome and function of BMSCs for fracture healing. To study on the optimal time toinject bone mesenchymal stem cells for fracture healing in murine model, provide areference for injection BMSCs to fracture healing.Materials and Methods1、Design and establish closed femoral fracture in murine model.2、Detect the change over time of mRNA expression of chemokine after fracture byusing Real-Time-Polymerase chain reaction(RT-PCR).3、Test the chemotaxis of callus tissue homogenate for the migration of BMSCs byusing transwell cell invasion model, test the function of SDF-1on improving migrationof BMSCs by using AMD3100, the specific blocker of SDF-1/CXCR4axis.4、BMSCs transfected with red fluorescent protein (RFP-BMSCs) were injected via thetail vein. RFP-BMSC migration and fracture healing were evaluated byfluorescence imaging, frozen section, Pathology, immunofluorescence and Western-blotmethods. AMD3100(inhibitor of stromal cell-derived factor1[SDF-1]) was alsoinjected before RFP-BMSCs in one group for comparison to verify the mobilizationmechanism of homing of BMSCs by SDF-1/CXCR4passway. 5、RFP-BMSCs were injected via the tail vein on defferent day post-fracture. Toinvestigate the optimal time to inject BMSCs in a C57murine unilateral, transverse,femur fracture model, which were evaluated by in vivo fluorescence assay, Micro-CTand mechanical test.Results1、Develop the equipment to establish closed femoral fracture in murine model.2、 Chemokines (SDF-1and MCP-3) that induce BMSC migration were highlyexpressed at7days after fracture. TGF-β1mRNA expression begin to improving at7days after fracture.3、Callus tissue homogenate can improve migration of BMSCs by using transwell cellinvasion model in vitro. The chemotaxis can be blocked by AMD3100, the specificblocker of SDF-1/CXCR4axis.4、RFP-BMSCs migrated to the fracture site, mostly at endosteum, can be investigatedby fluorescence imaging at42d post-fracture, express osteogenesis related factors OPN,OCN and chemokine SDF-1, and promote the expression of osteogenesis BMP2, TGF-β1and VEGF. RFP-BMSCs improve fracture healing obviously through the HEstaining and Masson staining pathology method at14d and42d.5、RFP-BMSCs were injected via the tail vein on7d should be superior to on1d or14dpost-fracture, which were evaluated by optical density tested by in vivo fluorescenceassay, BMD,BV, BVF tested by Micro-CT and maximum load, maximum radialdegrees, elastic radial degrees and rigidity by mechanical testing.Conclusion1、The equipment designed independently succeed to be used to establish closedfemoral fracture in murine model. Whole process of fracture healing could be observedand be conformed as endochondral ossification process. 2、Most of chemokines that induce BMSC migration were highly expressed at7daysafter fracture, which supported the7d post-fracture is the optimal time to inject bonemesenchymal stem cells for fracture healing in a murine model.3、Transwell cell invasive test shows that the SDF-1/CXCR4pathway played animportant role in promoting cell migration.4、RFP-BMSCs migrated to the fracture site, mostly at endosteum, for long time bysysterm delivery, which improved fracture healing by promoted osteogenesis relatedfactors by autocrine and paracrine.5、The7d post-fracture is the optimal time to inject bone mesenchymal stem cells forfracture healing in a murine model than1d or14d post-fracture. PART TWO Study on migration of BMSCs in murine cartilage injury model.ObjectiveTo research the phenomenon and its possible mechanism of homing of BMSCs to thesubchondral bone after cartilage injury.Materials and Methods1、Establish cartilage defect with2mm in diameter,1mm deep at femur condyle inrat model.2、RFP-BMSCs(106cells/0.2ml) were injected via the articular cavity post-fracture.3、Distribution of BMSCs were observed by frozen section and fluorescence at1、2、3、7d after injury.4、At3、7、14、21d after surgery, protein microarray detection were used for articularsynovial membrane from joint which injected RFP-BMSCs(106cells/0.2ml) orsaline(0.2ml), Chemokines mRNA were detected by RT-PCR for tissue range4 ×4×2mm aroud the defect at femoral condyle of joint which injected RFP-BMSCs(106cells/0.2ml) or saline(0.2ml).5、 Three months later, the defects of cartilage were repaired by fibrocartilage,RFP-BMSCs(106cells/0.2ml) were injected via the articular cavity again. Distributionof BMSCs were observed by frozen section and fluorescence at1、2、3、7d after injury.Results1、RFP-BMSCs were observed in cartilage defect area at1d、2d after injury andRFP-BMSCs injection, while, were observed in bone marrow under the subchondralbone, more than in cartilage defect area at7d later.2、 Three months later, the defects of cartilage were repaired by fibrocartilage,RFP-BMSCs were not observed in cartilage defect area or bone marrow within7d afterinjury and RFP-BMSCs injection.3、Expression of inflammatory cytokines secretion of synovial tissue in articular cavityand chemokines expression of the subchondral bone of with cartilage defect andRFP-BMSCs injection were higher than control group.ConclusionAfter the full-thickness defects of rat femoral condyle cartilage were built, BMSCsinjection to joint may increase the inflammatory cytokines secretion of synovial tissuein articular cavity, and improve the migration of BMSCs to bone marrow under thesubchondral bone by promoted the chemokine expression of the subchondral bone. |
PDF Full Text Request