Study Of The Effect Of Gingival Mesenchymal Stem Cells On Healing Of Critical Size Calvarial Defects In Diabetic Rats

ObjectiveThe purpose of this study is to establish the model of T2 DM calvarial critical bone defects,construct the compound of GMSCs and collagen matrix,transplant to the bone defect area of animal model,and explore the effect of GMSCs on the repair of T2 DM rat calvarial critical bone defects,so as to provide a new idea for the clinical repair of T2 DM patients with critical bone defects.Methods1.GMSCs were isolated from human gingival connective tissue by explant culture method combined with limited dilution method,and identified as mesenchymal stem cells by osteogenic,adipogenic,and chondrogenic differentiation.2.T2 DM rats were induced by high-fat diet and low-dose STZ,two 5mm-diameter calvarial critical bone defects were prepared on T2 DM rats.3.Twelve type 2 diabetic rats were divided into two time points,and each time point was divided into three groups: experimental group: pre-osteogenic differentiated GMSCs + rat tail tendon collagen type I;positive control group: Bio-Oss granules;negative control group: rat tail tendon collagen type I without cells.At 4 and 8 weeks after operation,rats were sacrificed and the calvaria containing the defect sites were harvested.The specimens were observed macroscopically,then fixed in 4% paraformaldehyde for 2 days.At the end of micro CT scanning,the specimens were subjected to HE staining to evaluate new osteogenesis.Result1.GMSCs were successfully isolated from gingival connective tissue by explant culture method combined with limited dilution method.The cell morphology was regular,uniform,long fusiform,and the nucleus was clear and in the middle.After 14 days of induction under standard osteogenic,adipogenic and chondrogenic differentiation conditions in vitro,GMSCs could produce calcified nodules,lipid droplets and cartilage matrix components,respectively.The results showed that our isolated cells had differentiation ability to osteoblasts,adipocytes and chondrocytes and were mesenchymal stem cells.2.Taking high-fat feeding for 4 weeks,the normal SD rats developed obesity,but their drinking water and urination were normal.and then treated with low-dose STZ,which showed symptoms of rising blood glucose(fasting blood glucose 21.63±3.52 mmol / L),polydipsia.We successfully induced T2 DM rats,established animal models similar to the pathogenesis of human T2 DM,and made two 5mm-diameter calvarial critical bone defects.3.Micro CT quantitative analysis results showed that: at 4 weeks after operation,the new bone volume ratio of the experimental group was the highest,followed by the positive control group and the negative control group.The difference between the experimental group and the negative control group was significant(P < 0.05),while the difference between the experimental group and the positive control group,the positive control group and the negative control group was not statistically significant(P > 0.05).At 8 weeks after operation,the percentage of new bone volume from high to low was the experimental group,the positive control group and the negative control group,and the differences were statistically significant(P < 0.05).4.The results of HE staining and micro CT scanning were basically the same.In the experimental group,the amount of new bone were significantly higher than in the control group.The results showed that GMSCs could better promote the repair of calvarial critical bone defects in T2 DM rats.ConclusionThe transplantation of pre-differentiated GMSCs into the calvarial critical bone defects in T2 DM rats can promote the formation of new bone at the defect site,which provides a new alternative for the clinical treatment of bone defect in T2 DM patients.