| Periodontal ligament is composed of intricate fibrous tissue,rich in periodontal microcirculation and cells.This causes teeth to be stable in the alveolar bone,with a certain degree of elasticity,which is beneficial to buffer the chewing force on the teeth and keep the teeth stably to perform the masticatory function.As a group of cells with the characteristics of mesenchymal stem cells in the periodontal ligament tissue,periodontal ligament stem cells(PDLSCs)have the potential of multi-directional differentiation and play an important role in the repair and regeneration of periodontal tissues.Especially for the avulsed teeth after dental trauma,the periodontal ligament tissue is completely broken and damaged.PDLSCs play a vital role in whether the periodontal membrane of the avulsed tooth can heal or not after replantation.In our preliminary animal experiment,platelet-rich fibrin(PRF)extracted from autologous blood was used to construct a new and efficient PDLSCs/PRF double-membrane transplant with autologous PDLSCs.PRF could provide a continuously released multiple growth factors environment for the stem cells in a certain period of time and thus can effectively promote the regeneration and repair of the damaged periodontal ligament.At the same time,it has been found in clinical practice that the use of autologous PRF transplantation,combined with the elastic or semi-elastic fixation recommended by the International Association of Dental Trauma(IADT)for the replanted tooth,enables the replanted tooth to bear a suitable bite force during the whole recovery process,which is helpful for periodontal ligament tissue regeneration and repair.Animal experiments have also found that appropriate bite force stimulation helps to promote the reconstruction of regular and strong periodontal fiber structure,and promote the formation of abundant capillaries in the periodontal ligament.Therefore,we believe that in the healing process of avulsed periodontal ligament,a variety of growth factors in natural ratios from PRF and appropriate bite force stimulation play positive roles in periodontal healing.Moreover,this promotion is likely to work by regulating the differentiation of periodontal ligament stem cells to the vascular endothelium,thereby promoting the reconstruction of periodontal microcirculation.However,the specific regulation mechanism keeps unknown.It is also unclear whether the physical factors of mechanical stimulation and the chemical factors of growth factors derived from PRF have a synergistic effect on periodontal microcirculation reconstruction.Therefore,in the present study,we adopted multi-funtional hydrolic cellular pressure unit independently developed by the research group to simulate the occlusal force on PDLSCs.At the same time,the endothelial differentiation induction fluid or PRF was adopted to provide the chemical induction of endothelial differentiation for stem cells.Then,the vascular endothelial differentiation indicators and angiogenesis ability were tested at the tissue level and the cell level to explore the effect of mechanical stimulation and PRF on the differentiation and angiogenesis of PDLSCs.We hope this work could help to clarify the mechanism of periodontal ligament healing in view of periodontal microcirculation,and provide new ideas for periodontal ligament regeneration from the perspective of biomechanics.Objectives(1)Clarify the effects of periodic dynamic pressure on the endothelial differentiation and proliferation activity of PDLSCs,and screen out the appropriate dynamic pressure condition.(2)Compare the effects of endothelial differentiation medium and PRF on the endothelial differentiation of PDLSCs under appropriate pressure.(3)Investigate the role of Notch signaling pathway in the process of mechanical stimulation and PRF promoting the endothelial differentiation of PDLSCs under the synergistic action of mechanical stimulation and PRF.Methods(1)PDLSCs were sorted by immunomagnetic bead sorting method,and CCK-8 were used to determine the cell growth curve.The determination of clone formation rate,identification of surface markers,osteogenic induction,adipogenic induction and endothelial orientation differentiation induction were used to identify the characteristics of stem cells of PDLSCs.(2)Different periodic dynamic pressures were used to load PDLSCs,CCK-8 method was used to detect the proliferation activity of PDLSCs,Real-time PCR was used to detect the m RNA expression of VEGFR-2 and ANG2 in PDLSCs,and Western blotting was used to detect VEGFR-2,PDLSCs.Protein expression of ANG2,VEGFA and IGF-1.(3)Endothelial differentiation medium of different concentrations of VEGF were used to induce PDLSCs.After 7d,14 d,and 21 d of induction,the cell morphology and the lumen formation experiment were used to detect the lumen forming ability of the cells and cellular immunity.Immunofluorescence assay was used to detect the expression of vWF and VEGFR-2,and Western blotting was used to detect the protein expression of Ang2,VEGFR-2,VEGFA,IGF-1 in the cells.(4)Under periodic dynamic pressure,PDLSCs were induced with endothelial differentiation medium and PRF respectively.The lumen forming ability of the cells was detected by the lumen formation experiment,and the expression of vWF and VEGFR-2was detected by cell immunofluorescence.Western blotting was used to detect the protein expression of Ang2,VEGFR-2,VEGFA and IGF-1 in cells.(5)Under the synergistic effect of mechanical stimulation and PRF,the PDLSCs were induced towards vascular endothelial cells.The γ-endocrine enzyme inhibitor DAPT was added to block the Notch signaling pathway.Real-time PCR and Western blotting were used to detect the m RNA and protein levels of Dll4,Notch1 and Notch4 in PDLSCs.After blocking the lumen forming experiment was used to detect the lumen forming ability of the cells,and Western blotting was used to detect the expression of Ang2,VEGFR-2,VEGFA and IGF-1 in PDLSCs.Results(1)We successfully isolated and sorted PDLSCs,which had ablity of self-renewal,colony formation and multidirectional differentiation,which confirmed as mesenchymal stem cells;(2)Periodic dynamic pressure can effectively promote the proliferation activity of PDLSCs,and up-regulate the expression levels of endothelial differentiation marker genes m RNA VEGFR-2,ANG2 and proteins VEGFR-2,ANG2,VEGFA,IGF-1,and regulate PDLSCs to the endothelium cell differentiation;(3)PDLSCs were successfullu induced towards vascular endothelial cells after the induction.The cells showed in a paving stone-like arrangement,and the lumen forming ability is significantly higher than that of the control group.The protein expressions of vWF,Ang2,VEGFR-2,VEGFA and IGF-1 were up-regulated and induced at high concentrations(50 ng/m L VEGF、10 ng/m L b FGF and 2 ng/m L IGF-1),with the most significant induction effects being on 14 d and 21 d.Under dynamic pressure,the angiogenesis ability of PDLSCs induced by the induction medium and PRF increased significantly.The protein expressions of vWF,VEGFR-2,ANG2,VEGFA and IGF-1were significantly up-regulated.Both PRF and endothelial induction medium can well promote the differentiation of PDLSCs into endothelial cells,with basiclly same effect;(4)Under mechanical stimulation and PRF,the protein expressions of Dll4,Notch1 and Notch4 were significantly up-regulated,indicating that the synergistic effect of mechanical stimulation and PRF can activate Notch signaling pathway.After DAPT was added,the angiogenesis ability of PDLSCs and the protein expression of VEGFR-2,ANG2,VEGFA and IGF-1 were significantly reduced,indicating that blocking the Notch signaling pathway would inhibit the endothelial differentiation of PDLSCs.ConclusionAppropriate dynamic pressure can increase the proliferation activity of PDLSCs and promote endothelial differentiation.Under appropriate dynamic pressure and PRF,expression of both endothelial cell markers and angiogenesis of PDLSCs could be effectively promoted,which is partly regulated by the Notch signaling pathway. |
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