The Role Of Systemically Delivered Bone Marrow-derived Mesenchymal Stem Cells In The Repair And Regeneration Of Periodontal Tissues

Progenitor cells that may participate into the repair of the periodontal tissues have been reported to possess properties of mesenchymal stem cells such as self-renewal and multi-lineage differentiation ability(differentiate to fibroblasts, osteoblasts, and cementoblasts). However, the origin of this population is not well identified. In addition, the previous reports showed that these progenitor cells mainly located in the paravascular region of the periodontal tissues and were able to migrate from alveolar bone into the periodontal ligament. All of these suggest that bone marrow may be the origin of these local progenitor cells.Co-cultured with periodontal fibroblasts in ex-vivo sdudy, bone marrow stem cells (BMSCs) exhibited the characteristics of periodontal fibroblasts, in which the expression of osteopontin and osteocalcin were dramatically improved and lower expression of bone sialoprotein was observed. Previously, in the beagle dog periodontal defect model, the autologous BM-MSCs were shown to be able to improve cementum, bone and periodontal ligament regeneration. And in another in vivo study, when GFP+ rat BM-MSCs expanded ex vivo on microcarrier gelatin beads were transplanted into a surgically created rat periodontal defect, the evidence of the GFP positive cells participating directly into the regeneration of bone, cementum and periodontal ligament was observed. The above reports indicate that BMSCs possess the capability to differentiate into the periodontal progenitor cells and enhance the regeneration of the periodontal tissues.BMSCs are known to migrate or home preferentially to injured sites when transplanted in animal models of injury, which demonstrates that MSCs can migrate into the specific niche, adapt to the local microenvironment and improve the function of the tissue. Chemotaxis assays showed that cultured MSCs migrated to the injured sites due to the existence of growth factor, chemokine and extracellular matrix, and chemokines in a dose-dependent fashion drived the migration. The mechanisms involved in the restoration ability of somatic stem cells docking at sites of injured tissues include transdifferentiating to some available cell type, secreting cytokines and other molecules to enhance the function of the endogenous cell population.The previous study demonstrated that luciferase and GFP double-labeled BMSCs systemically transplanted into irradiated mice could be detected in the bone wound site created in the mandibles and calvaria, which imply transplanted BMSCs can migrate to bone defect and participate in bone regeneration in orocraniofacial region. The above reports encourage our hypothesis that systemically transplanted BMSCs can home or dock at the sites of the periodontal defect, systemic bone marrow-derived MSCs can recruit the endogenous MSCs and exert the ability of regulation and synergetic effects on the repair and regeneration of the periodontal tissues, and periodontal ligament stem cells (PDLSCs) or cementoblasts partly derived from bone marrow.Materials and methods:Part I:Rat bone marrow-derived mesenchymal stem cells were isolated by density gradient centrifugation method with percoll. To assess colony-forming efficiency, CFU-F assay was performed by the limiting dilution method. To assess the tri-lineage differentiation potential, rBM-MSCs were induced to differentiate towards osteoblasts(cells cultured in osteogenic induction medium supplemented with a-MEM containing 10% FCS,0.1μM dexamethasone, 10mMβ-glycerophosphate,50μM ascorbate-2-phosphate and 1% antibiotic/antimycotic.), adipocytes(adipogenic induction medium supplemented with a-MEM containing 10% FCS,1μM dexamethasone,200μM indomethacin,10μM insulin,0.5mM isobutyl-methylxanthine and 1% antibiotic/antimycotic) and chondrocytes(chondrogenic induction medium supplemented with a-MEM containing 1% FCS,50nM ascorbate-2-phosphate,10 ng/ml TGF-β1 and 6.25μg/ml insulin and 1% antibiotic/antimycotic), respectively. Alizarin red staining, oil red O staining, and alcian blue staining were performed to detect the differentiation. To trace directly the distribution and differentiation of rBM-BMSCs in vivo, the lentiviral vector with enhanced green fluorescent protein (pBPLV-EGFP) was used to label rBM-BMSCs. The lentiviral system includes pBPLV, pLP1, pLP2 and pLP/VSVG. Lentiviral expression vector was firstly transfected into the packaging cells 293FT, then the viral supernatant were applied for transfection of rBM-BMSCs. To obtain the positive cells with high expression of EGFP, rBM-BMSCs were then sorted by FACS. Meanwhile, the difference of cell proliferation between transfected and untransfected cells was analyzed by using MTT assay.Part II:To improve the efficiency of cell transplantation, we adopted the preparative regimen with 8.0 Gy (dose rate 1.0 Gy/min) total body irradiation on the SD rats. Then we applied the method of intra bone marrow (IBM) and intravenous (IV) transplantation 4h after irradiation, respectively. The periodontal inflammatory defect model was established 3 weeks after cell transplantation. The location of transplanted EGFP positive cells and the precise numbers of the migration and the difference of migrated positive cells among all of the groups and multi-time dots were determined by direct observation with fluorescence microscope 1w,2w,4w,6w after the surgery. H&E staining and Masson staining histomorphometric were performed to observe the ability to restore and repair periodontal tissues in experimental groups. Immunohistochemical staining for GFP, OPN and type I collagen were performed to verify the results of the above observation.Results:Part I:The pure rBM-MSC cells population with stable morphology was obtained by density gradient centrifugation. By classical CFU-F assay, we confirmed that rBM-MSCs were clonogenic with high colony-forming efficiency (CFU-F>50%). After induction under osteogenic medium for 4 weeks and alizarin red staining, deposition of densely stained extracellular matrix and mineralized nodules were observed. After induction under adipogenic medium for 2 weeks and oil red O staining, aggregation and deposition of lipid-rich vacuoles in the rBM-MSCs were detected. Correspondingly, after chondrogenic induction, the positive staining density was detected. The above results demonstrated that rBM-MSCs owned the potential of tri-lineage differentiation (osteoblasts, adipocytes, and chondrocytes). In addition, high purity of 60.02% EGFP+ rBM-MSCs was achieved using the lentiviral system, and 40% of purified cells by FACS with high level of GFP expression were collected for expansion and further study. MTT assay showed that transfected rBM-MSCs displayed well-spread in appearance and slightly lower proliferation compared with untransfected cells, but no statistically difference was observed (P>0.05).Part II:After irradiation by 8.0Gy gamma ray with total body irradiation, all of the rats kept alive and displayed better mental status in the whole experiment in the IBM group compared with the IV group. After the periodontal surgery for 1 week, the EGFP+ rBM-MSCs were observed homing/docking into the injury sites by fluorescence microscope. Moreover, the numbers of EGFP positive cells in the defect reached the maximum at 2w after surgery. Meanwhile, some positive cells were observed locating in the newly formed bone islands, and majority of positive cells docked surrounded the neovessels. In addition, the positive cells were also existed in the newly formed periodontal ligament and cementum 4 weeks after surgery. Compared with the non-inflammatory defect model, larger numbers of homed positive cells were observed visualized by direct observation of cells using a fluorescence microscope in the inflammatory defect model. Compared with the IV group, the homed positive cells number was larger in the IBM group 1 week and 2 weeks after surgery. Histological analysis demonstrated that the ability of new bone formation, osseous maturation, periodontal ligament reconstruction and new cementum deposition were much more stronger in the IBM inflammatory defect model than that in the other groups. Immunohistochemical staining for OPN showed that cells and cell extracellular matrix in the injured sites were positive staining especially in the region of the newly formed bone, and the immunohistochemical staining for CoL I got the same results. Moreover, the expression of GFP was detected in the same area, suggesting that the above cells with the brown signal were derived from transplanted EGFP+ rBM-MSCs.Conclusions:1. rBM-MSCs can be easily isolated and expanded in vitro and possess classical stem cell properties with single colony forming ability and multipotent differentiation potential. The lentiviral system possesses a high efficiency of packaging cells which improve the transfection efficiency and enhance the procedure of our attempt.2. Systemically transplanted EGFP+ rBM-MSCs can home or dock at the periodontal defect sites, and in addition, these positive cells maily locate in the new bone, new periodontal ligament and new cementum. Compared with intravenous transplantation model, the intra-bone marrow transplantation is an efficient method for cell transplantation. H&E staining, Masson staining and immunohistochemical staining combined with the visualization of fluorescence microscope demonstrate that acute inflammatory defect can recruit more EGFP+ rBM-MSCs which participate in the regeneration of the periodontal tissue and directly differentiate into the osteoblasts and osteocytes. Despite lack of cementum-specific markers, the morphology and position in correlation with the new cementum indicated that EGFP positive cells underwent the differentiation to cementoblasts. Some EGFP positive cells in the periodontal ligament region located in the paravascular site indicating that periodontal ligment stem cell partially come from bone marrow and transplanted cells not only participate directly in bone repair but promote new vessel formation by paracrine effects. However, not all fibroblast cells in the new bone, periodontal ligament and new cementum were positive for GFP. This suggests host endogenous progenitor cells may have been recruited by BM-MSCs through secretion of trophic factors.