| Introduction In the development of orthodontics,the research on the mechanism of maxillary deformity and the mechanism of correction has always been the most important research direction.The orthodontic oriented movement of dislocated teeth is the result of the coordinated and orderly action of osteoclast and osteogenesis in the periodontal membrane Under the action of orthodontic force,the osteoclast mediated osteoclast activity in the periodontal membrane of the pressure side of the misaligned tooth is the premise of orthodontic tooth movement.In the tension-side periodontal membrane,osteoblast-mediated new bone formation and deposition are the histological basis for orthodontic teeth to maintain stability after moving.The study of periodontal tissue remodeling can find new targets for the formation of alveolar bone,provide a theoretical basis for optimizing the treatment,shortening and improving the safety of orthodontic treatment.We believed that these research results can also provide help for clinical technological innovation in the periodontal field from the perspective of enhancing the ability of osteogenesis repair.However,osteoblasts barely not exist in the normal periodontal membrane.In the process of stress remodeling,the tissue source of osteoblasts required for the periodontal membrane osteogenesis has long confused the researchers of oral orthodontics.With the deepening of the research on the osteogenesis of the periodontal membrane,Kaku et al.transplanted the bone marrow derived stem cells expressing green fluorescent protein(GFP)into the bone marrow of the femur of immunodeficient rats.Bone marrow derived mesenchymal stem cells also migrated to the periodontal membrane during periodontal bone tissue repair and tooth replantation.Bone marrow mesenchymal stem cells(BMSCs)are somatic stem cell with multidirectional differentiation ability,especially strong osteogenic differentiation ability.According to these results,osteoblasts differentiated from BMSCs in peripheral blood may also participate in the osteogenesis process of periodontal tissue stress modification.Inspired by the osteogenic mechanism of BMSCs,Seo was the first to isolate and extract periodontal ligament stem cells(PDLSCs)from the human periodontal ligament tissue.BMSCs and PDLSCs are both belong to the mesenchymal stem cells and have similar osteogenic differentiation mechanism.However,the proliferation and differentiation of PDLSCs,especially the regulation mechanism of osteogenic differentiation and periodontal stress remodeling,are still lacking in in-depth research.Supported by the National Natural Foundation of China(81600873),our research group successfully conducted an experimental study on the differentiation of PDLSCs into osteoblasts under dynamic tensile stress.We found that human PDLSCs could transform into osteoblasts,suggesting that PDLSCs,like BMSCs,may be one of the important sources of osteoblasts in the periodontal membrane.In studies on the mechanism of osteogenic differentiation of BMSCs,Notch1 is a key signaling pathway that initiates the proliferation and differentiation of BMSCs,and has different regulatory effects on various stages of osteogenic differentiation of BMSCs.Notch1 signals are widely expressed in the process of periodontal tissue remodeling.However,the synergistic regulation of Notch1 on the proliferation and differentiation of PDLSCs and BMSCs and other osteogenic signals still needs further study.Mesenchymal stem cells are in capacity of multidirectional differentiation.After Notch1 initiates the differentiation of mesenchymal stem cells,the expression of osteoblast regulators is required to promote the differentiation of cells towards osteoblast.Runx2 is a classic osteoblast regulator,which is highly expressed in BMSCs and immature osteoblasts,and is also widely expressed in the process of periodontal tissue remodeling.Runx2 is required for the proliferation and osteogenic differentiation of mesenchymal stem cells.Runx2 promotes osteogenesis and osteoblast maturation by regulating classical osteogenic signals such as Wnt.Thus,we speculated that the stress proliferation and differentiation of PDLSCs and BMSCs in periodontal tissues may also be regulated by Notch1 and Runx2 pathways.In order to improve the mechanism of stress differentiation of PDLSCs regulated by Notch1 and Runx2 pathways,chemical agents were applied in this study to regulate the Notch1 signaling pathway.The expression changes of Notch1 Runx2,ALP BMP2 Ocn and other early osteogenic differentiation markers during the stress differentiation of PDLSCs were detected,to explore the specific regulatory mechanisms of Notch1 and Runx2 pathways in the stress osteogenic differentiation of PDLSCsAt present,the mechanism of Runx2 pathway regulating osteogenic differentiation of BMSCs has been discovered,but the specific mechanism of post-transcriptional regulation of Runx2 expression in BMSCs has not been clarified.N6-methyladenosine(m6A)modification is the most extensive post-transcriptional modification in human somatic cells.Methyltransferase 3(Mettl3,methyltransferase-like 3)is commonly seen in the regulation of m6 A modifications associated with mesenchymal stem cell differentiation.Many studies have found that m6 A modification plays a key regulatory role in the differentiation of BMSCs.Another post-transcriptional regulatory mechanism,mi RNAs also play an important role in the osteogenesis of BMSCs.In previous experiments,we found that mi R-141-3p was highly expressed in bone marrow tissue samples of osteoporotic mice.Currently,the regulatory mechanisms of multiple mi RNAs on BMSCs have been discovered,but the regulation of Runx2 by mi R-141-3p in osteogenic differentiation of BMSCs has not been reported.Therefore,in this study,m6 A methylation of Runx2 and specific mechanisms of mi R-141-3p targeted regulation of osteogenic differentiation of BMSCs were investigated by Me RIP-PCR,RIP-PCR,m RNA stability detection luciferase reporting test and other techniques.With the deepening of osteogenic mechanism research,the potential therapeutic targets of targeted drugs are increasing,but the research of drug carrier materials is still in the exploratory stage.In order to find the carrier with application value,more research attention has been paid to iron based nanomaterials,iron oxide nanoparticles(Fe3O4,IONPs)is one of them.IONPs is an ideal material for clinical application because of its colloidal stability,low toxicity,biocompatibility and biodegradation.Recent studies have found that IONPs can not only carry drugs but promote osteogenic differentiation of mesenchymal stem cells.However,whether IONPs can affect the repair of alveolar bone remains to be studied.In this study,we studied the treatment process of IONPs on SD rat model of enhanced periodontitis,and confirmed the effect of internal stress loading on Notch1 signaling pathway.To investigate the role of Notch1 signaling pathway in IONPs repair of periodontal injury under stress conditions.Through these studies,we will improve the theoretical basis of Notch1 and Runx2 as potential targets for promoting tooth reattachment and alveolar bone repair,and lay a theoretical foundation for the clinical application of a new generation of nanoparticle materials IONPs.We believe that these research results can provide theoretical support for improving the safety of orthodontic clinical treatment and increasing the means of periodontal repair.Objective The expression of NICD protein in PDLSCs was regulated by chemical agents through stress loading in vitro,and the mechanism of synergistic regulation of Notch1 pathway and Runx2 pathway on stress osteogenic differentiation of PDLSCs was investigated.To investigate the specific action sites of Runx2 m RNA by RNA methylation modification in BMSCs in vitro.To investigate the specific mechanism of the regulation of mi R-141-3p in osteogenic differentiation of BMSCs,and confirm the regulatory effects of mi R-141-3p on downstream genes.To explore the role of Notch1 channel and IONPs particles in the inflammatory repair process of SD rats with periodontitis under stress conditions.Methods1.PDLSCs were isolated from the primary tissue,cultured in vitro and screened.After passage to the third generation,the epithelial cells were excluded by immunofluorescence detection of keratin,and the tissue source was determined by detection of vimentin.The expression of surface markers was identified by flow cytometry.Multidirectional differentiation ability of PDLSCs was identified and sorted by chemical staining.2.PDLSCs are stressed using a Flexer cell-4000 tension(FX4000T)dynamic stress loading data computer.Jagged1 and DAPT were used to up-regulate and down-regulate NICD,respectively,to explore the regulatory mechanism of Notch1 on the early stage of stress differentiation of PDLSCs.PDLSCs proliferation and early osteogenic markers were detected to show the differentiation of PDLSCs.3.Knockdown and overexpression transfection were performed to confirm the osteogenic promotion effect of mettl3 on BMSCs.Bioinformatics methods were used to predict mettl3 recognition sites in Runx2 m RNA sequences.The effect of methylated Runx2 m RNA stability on osteogenic differentiation of BMSCs was examined by methylated RNA immunoprecipitation(Me RIP)-q PCR and m RNA stability detection.4.In the process of BMSCs differentiation induced by osteogenic induction medium,the effect of mi R-141-3p on the osteogenic differentiation of BMSCs was detected.The m RNA expression of mi R-141-3p,Runx2 and osteoblast-related factors was detected by real-time quantitative PCR,and the protein expression was detected by WB.BMSCs activity was determined by CCK-8 method.The downstream genes of mi R-141-3p were predicted by targetscan.com and confirmed by luciferase reporter assay,and the regulatory effects of mi R-141-3p on the downstream genes were explored.5.The Sprague-Dawley(SD)rat model of periodontitis was established by ligation of rubber chain between mandibular incisor and neck of first molar.HE staining was used to observe the difference between normal periodontal tissue and inflammatory periodontal tissue.Immunohistochemical staining was used to detect NICD expression in inflammatory periodontal tissues under stress.6.Periodontal tissue and gingival crevicular fluid were extracted from the gingival crease after IONPs intervention to detect Notch1 signaling pathway and the expression levels of inflammatory markers,and to observe the tissue changes during osteogenic repair of periodontal inflammatory injuries.To explore the role of Notch1 and related signaling molecules in osteogenic differentiation and repair of periodontal tissue.Results1.Primary PDLSCs were screened and cultured from in vitro orthodontic teeth by an improved limited dilution method.The primary cells were derived from mesenchymal tissue with strong differentiation potential and proliferation ability,and the cell phenotype was consistent with the characteristics of PDLSCs.2.Stress loading successfully induced osteogenic differentiation of PDLSCs,and the isolated PDLSCs are chemically regulated through the Notch1 signaling pathway after continuous dynamic tension is applied.The results showed that inhibition of Notch1 expression in PDLSCs increased the osteogenic ability of PDLSCs in the early stage of stress differentiation,but decreased the osteogenic ability in the long term.After upregulation of Notch1,Runx2 expression increased,proliferation ability of PDLSCs increased,early stress differentiation osteogenic ability decreased,and long-term osteogenic ability increased.3.The expression level of mettl3 in BMSCs increased in the in vitro osteogenesis induction experiment,confirming that upregulation of mettl3 promoted the differentiation of BMSCs.m RNA stability detection proved that mettl3 increased Runx2 m RNA stability and promoted bone formation of BMSCs through m6 A modification mediated by reading protein IGF2BP1.4.The expression of mi R-141-3p was decreased in osteogenic BMSCs.The cell activity of mi R-141-3p mimic group was inhibited,and SIRT1 was predicted to be the downstream gene of mi R-141-3p.This prediction was confirmed by luciferase reporter gene test.SIRT1 reversed the inhibitory effect of mi R-141-3p on the osteogenic differentiation ability of BMSCs.Mi R-141-3p inhibited the osteogenic differentiation of BMSCs regulated by Runx2/Wnt signaling pathway by down-regulating SIRT1.5.The periodontitis model of SD rats under stress was established by afterligation and feeding with high sugar.Typical manifestations of periodontitis were observed in periodontal tissue sections.Immunohistochemical staining showed that NICD was highly expressed in periodontal tissue after stress loading,and Notch1 signaling pathway was activated,which confirmed the involvement of Notch1 signaling pathway in the periodontal tissue repair process.6.The periodontal pockets were deepened and the expression of inflammatory markers increased in SD rats under stress.After IONPs intervention,the repair speed of periodontal injury increased,the expression of Notch1 signaling pathway increased,and the expression of inflammatory markers decreased compared with the non-intervention group.The intervention effect of IONPs was terminated by DAPT,a Notch1 inhibitor.ConclusionsThe results showed that Notch1 signaling pathway was involved in the regulation of PDLSCs stress differentiation through Runx2,and overexpression of NICD signaling pathway in the early stage of PDLSCs differentiation inhibited their differentiation and promoted their proliferation.Notch1 firstly inhibits differentiation and increases the number of stem cells during the early osteogenesis of PDLSCs,and up-regulates Runx2 pathway to promote osteogenesis.The expression of methyltransferase Mettl3 was increased during osteogenic differentiation of BMSCs.Runx2 m RNA was predicted to be the target of Mettl3 by software.On this basis,we determined that Mettl3/IGF2BP1 promoted stable Runx2 m RNA through methylation,and prolonged Runx2 expression time to promote osteogenic differentiation of BMSCs.The expression of Mi R-141-3p decreased during osteogenic differentiation of BMSCs,and the target of Mir-141-3p was verified as SIRT1 by luciferase reporter assay.In the process of osteogenic differentiation,mi R-141-3p can target the expression of SIRT1 and down-regulate the osteogenic ability of BMSCs by inhibiting the expression of Runx2/Wnt pathway.In the stress-induced periodontitis model of SD rats,the IONPs intervention group increased the repair speed of alveolar bone injury and increased the expression of Notch1 signaling pathway during the repair period.Osteogenesis is the physiological basis of orthodontic tooth fixation and alveolar bone defect repair.When clinically required,PDLSCs proliferation can be achieved by promoting Notch1-Runx2 pathway activity first,and then IONPs can promote osteogenesis,which can increase the efficiency of orthodontic tooth fixation and periodontal defect repair.mi R-141-3p and DAPT can be used as osteogenic inhibitors to accelerate orthodontic tooth movement when treated with appropriate dosage forms.This study demonstrated the broad application prospect of Notch1-Runx2 pathway and IONPs in orthodontic and periodontal diseases,which could provide theoretical support for the optimization of orthodontic treatment procedures.It is believed that this study can also provide help for clinical technological innovation in the periodontal field from the perspective of enhancing osteogenic repair ability. |
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