| Complicated biological behaviors were involved in the process of orthodontic tooth movement(OTM), including biomechanics and biology, the former means that mechanical stimulus acting on tooth, while the latter means that tooth transfer the stress to periodontal and trigger a series of cell-biological reaction. Some researches confirm that during the process of OTM, osteoblasts are active in the tension side and mainly behave as bone modeling basing on bone deposition, while the pressure side performing as dynamic balance between osteogenesis and osteoclasia, giving priority to bone remolding. Knowing more about the biological foundation of OTM, contribute to carry out clinical work more safely and effectively.It is generally agreed that periodontal is the key point during the process of OTM. As the stem cells of periodontal, periodontal ligament stem cells(PDLSCs) are surmised to involve in maintaining periodontal homeostasis and play an important role in bone remolding, which make them become ideal cells for periodontal regeneration. It has been studied that the proliferation and differentiation of PDLSCs cultured in vitro may change under stimulation, which involve in complicated mechanism. Wnt signaling pathway exists in organism widely and participates in bone formation and remolding. It has been concerned that Wnt signaling pathway regulates the differentiation of PDLSCs.So we designed experiments to provide evidences for the involvement of PDLSCs in OTM and found whether PDLSCs under mechanical stimulation are functional to the periodontal reconstruct via canonical Wnt pathway.Part I Study on the animal model of OTMObjectiveBuild the model of OTM in rats to verify the involvement of PDLSCs.Methods(1)A total of 24 male SD rats were divided into four groups randomly, received orthodontic treatment for 0,1,3 and 7 days respectively. A nickel-titanium closed-coil spring exerting an orthodontic force of 50 g was link from the central incisors to the maxillary right first molar to induce the molar movement. The other side was used as the control group.(2)HE staining was used to detect the morphological changes of periodontal and TRAP staining was used to detect the the number of osteoclast.(3)The expression of nestin and PDGFRα was detected by immunohistochemistry.Result(1)The results of HE indicated that, with the extension of time, the periodontal space narrowing in the pressure side.In addition, the number of osteoclast in the pressure side was increased.(2)Immunocytochemistry assay showed that the expression of nestin and PDGFRα in experimental group was significantly different from control group.ConclusionThe number of PDLSCs was significantly increased and the distribution was wider, prompts the involvement of PDLSCs during the process of OTM.Part II Static pressure regulated the osteogenetic differentiation of h PDLSCsObjective(1) Isolate and identificate of h PDLSCs.(2) Detect the expression of osteogenesis related indicators of h PDLSCs to explore the effect of pressure on them.methods(1)HPDLSCs were isolated by limiting dilution technique. crystal violet staining was used to detect the cloning efficiency. MTT assay was carried out to detect the proliferation analyses. The CD markers were analyzed by flow cytometry. To assess osteogenic differentiation, ALP staining and alizarin red staining were performed. Lipid accumulation was detected by oil red O staining. In order to further verify the the multi-directional differentiation potential of h PDLSCs, western blot and RT-PCR were utilized to detect the relative index.(2)In order to detect the osteogenic differentiation of h PDLSCs under static pressure, they were subjected to the pressure incubators at 100KPa/1h and 100KPa/12 h.Results(1)HPDLSCs have a cloning formation rate of 28% and a proliferation ability. Identification of cell surface markers via flow cytometry confirmed that they were positive for CD44,CD29,CD105 and STRO-1, but negative for CD45 and CD34. ALP active assess, ALP staining and alizarin red staining revealed that PDLSCs could form mineralized nodules. Oil red O staining showed that the cells had formed lipid droplets. In addition, the protein and gene levels of ALP,Runx2,COL-I,LPL and PPAR-γ were significantly increased after induction.(2)The results of ALP staining and ALP activity assess showed that static pressure of100KPa up-regulated osteogenic differentiation of h PDLSCs at 1h, while down-regulated it at 12 h.The gene and protein levels of ALP and RUNX2 were increased after 1h exposure and decreased at 12 h under 100 KPa.Conclusion(1) HPDLSCs has self-renewal and multi-directional differentiation ability.(2)Short-term pressure increased the osteogenesis differertation of h PDLSCs, while under the long-term treatment condition, the osteogenesis differertation of them was inhibited.Part III Influence of the canonical Wnt signaling pathway in osteogenic differentiation of h PDLSCsObjectiveBy measuring the related indicators of Wnt/β-catenin signaling pathways under pressure, and the change of osteogenetic differentiation of h PDLSCs after regulated the pathway, to confirm the effect of the canonical Wnt signaling pathway on osteogenic differentiation of h PDLSCsmethods(1)The expression of active-β-catenin in the pressure side of periodontal in rats was detected by immunohistochemitry.(2)HPDLSCs were placed in the pressure incubators at 100KPa/1h and 100KPa/12 h, then the expression of GSK-3β, p-GSK-3β, total β-catenin and active-β-catenin was tested by western blot and RT-PCR.(3)HPDLSCs were treated with wnt3 a and DKK-1, then ALP staining and ALP activity assay was utilized as above. In addition, the samples were harvested and subjected to western blot and RT-PCR analyses as described earlier.Results(1)The result of immunohistochemistry showed that the expression of active-β-catenin increased with the extension of experiment time.(2)Western blot showed that the pressure of 100 KPa increased the expression of GSK-3β, while decreased the expression of p-GSK-3β, total-β-catenin and active-β-catenin at 1h. By contrast, when the pressure continues to 12 h, h PDLSCs high expressed p-GSK-3β, total-β-catenin and active-β-catenin, but the levels of GSK-3β were low.The results of RT-PCR showed no significant differences.(3)When conditions is 100KPa/1h, ALP staining and ALP activity assay demonstrated that the activate of the canonical Wnt pathway by wnt3 a suppression the osteogenic differentiation of h PDLSCs.When using DKK1 to inhibit the Wnt/β-catenin pathway at 100KPa/12 h, ALP staining and ALP activity assay exposed the osteogenic differentiation of h PDLSCs was increased.Western blot analysis were consistent.Conclusion(1)The analysis of experimental animals revealed the involvement of the canonical Wnt signaling pathway in the process of OTM.(2)Short-time pressure inhibited Wnt/β-catenin signaling pathway, while long-time pressure activated it.(3)The osteogenic differentiation of hPDLSCs could be inhibited by Wnt/β-catenin signaling pathway. |
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