Enhanced BMSCs Homing To Repair Alveolar Bone Defects In Rats With Postmenopausal Osteoporosis

BackgroundIt is a challenge for both researchers and clinicians to repair the alveolar bone defects caused by trauma, periodontal diseases and inflammation. Both local factors and systemic conditions are considered as risk factors adversely affecting the self-regeneration of the periodontal bone tissues. Among them, the postmenopausal osteoporosis(PMO) was found to play an important role in the progression of periodontal diseases especially among aged women. PMO is a systemic skeletal disease characterized by the reduction of bone density and micro-architectural deterioration. In periodontal diseases, PMO may accelerate the destruction of alveolar bone trabecula and reduce the height of residual ridge, thereby affecting the subsequent implantation or denture treatment. Therefore, there is increasing need to find pathomechanism of PMO as well as effective and practical methods to repair the defects of alveolar bone tissues in women suffered with PMO. In order to increase the repair efficiency, it is important to improve the possibility and potentiality of autogeneic or allogeneic BMSCs homing into area of defects, especially when only a limited number of BMSCs are transplanted. Special attention has focused on SDF-1α/CXCR4 axis due to its ability to increase the homing efficiency of BMSCs. In the present study, it is the first time to investigate and compare the SDF-1α/CXCR4 related biological characteristics as well as related molecular mechanisms between BMSCs isolated from PMO rat model(OVX-BMSCs) and normal rat(Sham-BMSCs) in vitro experiments.Currently, special attention has been paid to periodontal tissue engineering, which is considered as a potential effective strategy to achieve a complete, reliable and reproducible periodontal regeneration. Comparing with other cell populations, bone marrow-derived mesenchymal stem cells(BMSCs) have been demonstrated with substantial potential for clinical use to treat alveolar bone degenerative disorders, due to its capability of osteogenic differentiation and feasibility to be isolated and amplified in vitro. In general, two methods of BMSCs transplantation including system transplantation and local transplantation, are used. Most researchers consider the local transplantation of BMSCs is commonly used in tissue engineering. However, our group’s previous research found that local transplantation of BMSCs have disadvantages including the low number of homing cells to the injured sites, etc. The advantage of BMSCs system-transplantation is that under the microenvironment of the host body, the BMSCs are prone to reach the injured sites with the blood circulation. However, the method suffers the drawback that the transplanted cells will be accumulated in the liver and lungs, etc. Currently there are few studies on the use of BMSCs system-transplantation for the treatment of periodontal bone defects. Therefore, the system-transplantation of BMSCs was used in our task in order to evaluate the recruitment effects of SDF-1α/CXCR axis on the system-transplantation of BMSCs to repair the alveolar bone defects. AimTo compare the SDF-1α/CXCR4 related biological characteristics as well as related molecular mechanisms between BMSCs isolated from PMO rat model(OVX-BMSCs) and normal rat(Sham-BMSCs) in vitro experiments.To investigate the homing effectiveness and alveolar bone regeneration result after system implant BMSCs combination of transplantation of SDF-1α-Hydrogel graft or alone in PMO rat models with the maxillary periodontal defects in vivo experiments. Method1. 3 months after underwent ovariectomies in rats, using Micro-CT and mechanics test to verify the animal model. Isolation and culture the BMSCs from Sham-rat and OVX-rat group, and analyse the general characteristics by colony-forming assay and CCK-8 assay. The immunophenotype of the cultured BMSCs(P3) was analyzed by flow cytometry. The multi-differentiation potential was assessed by the ALP staining and Oil Red staining were proceeded after osteoinduction and adipoinduction.Osteogenic and lipoblast landmark gene were detected by q RT-PCR.2. The chemotactic effects of SDF-1α on cell migration were observed using a transwell membrane system. The CXCR4 expression profiles in two types of BMSCs at two passages(P3 and P6) were further assessed by immunofluorescence staining and q RT-PCR and western blot analysis. Both phosphorylated AKT and ERK phosphorylated protein levels were analyzed using Western blot in the two sets of BMSCs.3. Lentiviral vectors were used to over-express CXCR4 in OVX-BMSCs. Using q RT-PCR and western blot analysis to verify the over-expressing of CXCR4. Further identify the migrate ability of OVX-BMSCs by transwell assay.4. In vivo experiments, establish PMO rat models with the maxillary periodontal defects. Analyze the cell homing effectiveness and alveolar bone regeneration after system implant BMSCs labeled by CM-Dil alone or combination of transplantation of SDF-1α-Hydrogel graft. Further detect the expression of protein OCN and Runx-2 in new bone area by immunofluorescence as well as m RNA levels of OCN and Runx-2 and CXCR4 gene. Results1.(1)Compared with the Sham group, BMC,BMD, BV/TV and Max Load in the OVX group were significantly lower, suggesting the postmenopausal osteoporosis(PMO) rat model was successfully established.(2)There was no significant difference in colony-forming ability between the two sets of cells. Likewise, OVX-BMSCs showed stronger proliferation ability than Sham-BMSCs.The results of immunophenotype of the two types of cultured BMSCs exhibited similar patterns on the expression of surface molecules. The results for cell staining and q RT-PCT showed the OVX-BMSCs had weaker osteogenic and chondrogenic ability but higher adipogenic ability than Sham-BMSCs.2.(1)The results in vitro cell chemotactic and chemotactic-blocking assays showed that migration of two groups of cells increasing dependently with the dose of SDF-1α and 100 ng/ml was found as the peak density. The presence of CXCR4 antagonist AMD3100 greatly decrease the migration of both Sham-BMSCs and OVX-BMSCs to nearly half of the SDF-1α-treated groups. In addition, OVX-BMSCs showed significantly impaired chemotactic activity as compared with Sham-BMSCs.(2)At the same cell passage, CXCR4 immunofluorescence, m RNA and protein expression levels in Sham-BMSCs were higher than those in OVX-BMSCs. In addition, it was found that the increasing passage times was likely to adversely affect the the expressions of CXCR4, since both Sham-BMSCs and OVX-BMSCs exhibited higher CXCR4 immunofluorescence, m RNA and protein expression levels at passage 3(P3) than those at P6.(3)To further investigate the signal transduction mechanisms involved in SDF-1α-induced chemotactic activity,both total AKT and ERK phosphorylated protein levels were analyzed using Western blot in the two sets of BMSCs. For AKT pathway, SDF-1α sharply increased the protein levels of p-AKT at 30 min in Sham-BMSCs and at 60 min in OVX-BMSCs group, which then declined at 120 min. For ERK pathway, SDF-1α sharply increased the protein levels of p-AKT at 30 min in Sham-BMSCs and at 60 min in OVX-BMSCs group, but there was no significant defference between 60 and 120 min.At the same time point, the cells pretreat with AMD3100 exhibited the declined protein levels of p-AKT and P-ERK than cells pretreated with no receptor antagonist.3. The number of 80% positive transfection cells were observed after using lentiviral vectors to over-express CXCR4 in OVX-BMSCs. The expression of CXCR4 in OVX-BMSCs were successfully up-regulated according to the results of q RT-PCR and western blot analysis. The migrate ability of OVX-BMSCs was increased markblely via transwell assay data.4.(1)Micro-CT scan and new bone density analysis showed that,at 4 weeks, the im-BMSCs group showed significantly improved new bone formation with more extensive calcification and reduced defects than blank control group. Meanwhile, the local implantation group(im-BMSCs+SDF-1α-Hy) showed significantly higher regeneration of bone defect density compared with both blank control group and im-BMSCs group. These results suggested that local SDF-1α implantation after bone defects were associated with better repair of the alveolar fossa defects in osteoporotic rats.(2)Hematoxylin and eosin staining and Masson staining showed that the defects in the control group and im-BMSCs group were mostly filled with osteoblasts or osteogenetic cells and connective tissue except but sparsely trabecula of bone. In im-BMSCs+SDF-1α-Hy group, the defect sites showed better healing results with compacted trabecular pattern. In the local nascent bone tissue, osteoclasts and bone cells were more than the other groups.(3)More labeled BMSCs in im-BMSCs+SDF-1α-Hy group were found homming to injure area than in the im-BMSCs group. Furthermore, the immunohistochemical results demonstrated larger amounts of both Runx2 and OCN-positive cells in the defect areas from the im-BMSCs+SDF-1α-Hy sections, which verified that more newly formed bone tissues were regenerated by co-implantation of BMSCs and local SDF-1α compared with mono-BMSCs.(4) The results of m RNA levels of Runx2, OCN and CXCR4 genes in newly formed tissue at bone defect revealed that the implantation of BMSCs significantly increased the m RNA levels of Runx2, OCN and CXCR4 genes and co-implantation of BMSCs and SDF-1α further elevated the m RNA levels. Conclusion1. In summary, our findings confirmed that OVX-BMSCs exhibited the impaired osteoblastic ability as well as lower chemotactic activity towards SDF-1α, which was associated with lower expression levels of CXCR4. SDF-1α/CXCR4 axis enhanced BMSCs migration via the phosphorylation of AKT, whereas postmenopausal osteoporosis attenuated the CXCR4 expression and thus restricted the phosphorylation of AKT. The migration ability of OVX-BMSCs increased after the CXCR4 gene level was up-regulated.2. In addition, local transplantation of SDF-1α combing with system transplantation of BMSCs showed better alveolar bone regeneration than BMSCs alone in PMO rat models with the maxillary periodontal defects. Taken together, these findings suggest that locally transplantation of SDF-1α-Hydrogel graft can help to enhance exogenous BMSCs homing to repair alveolar bone defects in rats with postmenopausal osteoporosis.