The Treatment Of Systemically Transplanted Gingival Mesenchymal Stem Cells On Periodontitis In Mice

OBJECT: The gingival mesenchymal stem cells were transplanted into mice periodontitis model via tail vein and the homing and differentiation of GMSC were observed to evaluate the treatment of GMSCs on periodontitis.METHODS: The GMSCs were isolated from gingival tissue using enzyme digestion method.Colony-forming unit-fibroblast assay,adipogenic differentiation,osteogenic differentiation and flow cytometry were used to verify capacity of cell proliferation,multiple differentiation and expression of surface markers.GMSCs were labeled by the lentiviral vector with green fluorescent protein(GFP).Thirty-six 8-week-old C57BL/6J mice were randomly divided into Group A and B.Maxillary second molars of both group mice were tied with 5-0 silk ligature for 4 weeks to induce periodontitis.The GMSCs labeled with GFP were injected via tail vein into Group A and Group B was injected with α-MEM medium.Six of both mice were sacrificed at 1,2 and 4 weeks after the transplantation.Alveolar bones with molars were harvested after fixed with 4% paraformaldehyde.The soft tissues cover left alveolar bones were removed after boil.Photos of left alveolar were taken by stereomicroscope to measure distances from the cementoenamel junction(CEJ)to the alveolar bone crest(ABC).The right alveolar bones were demineralized in 10% EDTA for 4 weeks and embedded in paraffin.Hematoxylin and Eosin(HE)staining and masson’s trichrome(MT)staining were performed to evaluate periodontal tissue regeneration.The migration and differentiation of GMSCs were monitored by a fluorescence microscope after DAPI staining and immunohistochemical analysis.RESULTS: GMSCs were isolated from gingival tissue using enzyme digestion and cells formed clonogenic cell clusters.GMSCs were found to differentiate towards adipocytes and osteoblast under specific condition.And flow cytometry analysis showed that GMSCs were positive for CD73、CD90、CD105 and negative for CD45.The expression rate of GFP in infected GMSCs was over 90%.Mice periodontitis model was established after ligating for 4 weeks.The distances from CEJ to ABC in Group A were smaller than Group B(P<0.05).After the transplantation for 1 and 2 weeks,hematoxylin and eosin staining revealed alveolar bone heights of both groups were extremely low.And most of alveolar bone were blue stained immature bone through masson’s trichrome staining.At the 4 weeks,alveolar bone heights of both groups were significantly improved than 1 and 2 weeks.Moreover,distances from CEJ to ABC in group A were significantly less than group B.Meanwhile,more red stained mature bone formed in group A than B.After the injection for 1 and 2 weeks,GFP-positive fibroblast-like cells detected between alveolar and cement by fluorescence microscope observation and immunohistochemical analysis.In addition,GFP-positive osteoblasts were also found in the newly formed alveolar 4 weeks after injection.CONCLUSION: GMSCs could be easily isolated from gingival tissue by enzyme digestion and cells showed stem cell characterizations.The transfected GMSCs could express GFP stably with the cell passage increase.GMSCs injected via tail vein could homing to periodontal defect and part of cells promoted bone regeneration by differentiate into periodontal ligament cells or osteoblasts.