| Objective For periodontal regeneration tissue engineering,a large number of seed cells is the key and difficult point.With the development of regenerative medicine and tissue engineering technology,the seed cells obtained from the patient’s autologous source are expanded and proliferated in vitro to a certain number,and then compounded with biomaterials,re-implanted into the periodontal defect area,and repairing the periodontal defect is to solve the problem of periodontal bone loss.New development direction.As we all know,propolis is a naturally occurring substance with a variety of pharmacological effects.Previous studies have confirmed that flavonoids are the main active part of propolis.On the one hand,propolis has antibacterial,anti-inflammatory and analgesic effects(detoxification and swelling);on the other hand,it can inhibit the degradation and absorption of periodontal tissue,and promote tissue repair,regeneration and alveolar bone protection(astringent muscle growth).Therefore,this project proposes the following hypothesis: Based on the "convergence and muscle growth" effect of propolis,the effect of apigenin,a flavonoid monomer in propolis on cell proliferation and osteogenic differentiation,is used to explore the effect of apigenin on the proliferation and osteogenic differentiation of dental pulp mesenchymal stromal cells.The effect of the treatment on bone defect is realized,and it provides new ideas for the treatment of clinical periodontitis.Methods Select the P3 generation human dental pulp mesenchymal stromal cells cultured in the laboratory with good growth status,and use 10% FBS DMEM medium containing different concentrations of apigenin(0.1,1,5,10,50 umol/L)to culture the cells,Use cell proliferation and toxicity test kit(cck-8)to detect cell proliferation.The effect of apigenin on the osteogenic differentiation of human dental pulp mesenchymal stromal cells was detected by the expression of alkaline phosphatase,alizarin red staining and real-time fluorescent quantitative PCR.A rabbit skull defect model(4round defects with a diameter of 8 mm)was constructed on 10 New Zealand white rabbit skulls,divided into four groups: group A blank group,group B bone meal group,group C ordinary cultured h DPSCs compound bone meal group,group D It is the h DPSCs composite bone meal group cultured with 5 umol/L apigenin.Specimens were taken at 4 and 8 weeks after operation for Micro-CT three-dimensional reconstruction to analyze the ratio of BV/TV in the defect area.Results The results of the proliferation and toxicity test kit show that apigenin at a concentration of 0.1 ～ 10 μmol/L has no obvious toxic effect on human dental pulp mesenchymal stromal cells,and apigenin at 5 μmol/L can significantly promote the proliferation of h DPSCs.Compared with other control groups,the difference was statistically significant(F=14.640,P<0.001),the promotion effect was more obvious at72 h,and the difference was statistically significant(F=33.843,P<0.001).When the dose reached 50 μmol/L,apigenin had a tendency to inhibit the proliferation of h DPSCs;the ALP activity level after 1,4,and 7 days of culture was detected with the ALP kit.On the 4th day,compared with the control group,the intracellular ALP activity of the 5umol/L apigenin group was significantly increased(F=310.121,P<0.001);on the 7th day,the OD value increased significantly,and the difference was statistically significant(F=404.063,P<0.001);See the 5 umol/L apigenin group compared with the mineralized group for Alizarin Red staining.More calcium nodules were formed,while no nodules were formed in the control group.After 14 days of in vitro osteogenic induction,ALP staining microscope showed that the 5 umol/L apigenin group and the mineralization group had blue-purple formation compared with the control group,and the 5 umol/L apigenin group had a deeper color and a larger range;Real-time PCR results showed that after 7 days After osteogenic induction,the relative expression of h DPSCs osteogenic related gene OCN and the relative expression of RUNX2 m RNA in the 5 umol/L apigenin group increased compared with the control group,and the difference was statistically significant(P<0.05);4 Peripheral bone volume fraction detection showed that compared with group C,group D had a larger bone volume fraction,and there was no significant new bone formation in groups A and B,and the difference was statistically significant(F=94.389,P<0.01);8 weeks after surgery Bone volume fraction detection showed that group D formed more new bone than group C,and the difference was statistically significant(F=38.139,P<0.01).A and B groups formed a small amount of new bone.Conclusions Appropriate concentration of apigenin can promote the proliferation and osteogenic differentiation of human dental pulp mesenchymal stromal cells.And the h DPSCs cultured with apigenin at a concentration of 5umol/L combined with Bio-oss bone meal and then implanted into the rabbit skull defect area has a significant effect on promoting bone formation,providing new ideas for the treatment of later periodontal defects. |
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