| Malocclusion is a common oral condition of misalignment relation between teeth in different degrees with/without an incorrect relation between the upper and lower arch,leading to both physiological function depression and facial aesthetic problems,even failures of making normal social connections.In recent years,a fast-growing number of adults are seeking orthodontic treatment with chief complaints for functional and aesthetic improvement.However,a high risk of periodontitis(up to 80%-90%)in our country,makes things more challenging.As we all know,periodontitis is a chronic inflammatory disease caused by bacterial infection with a progressive damage to the supporting connective tissue-periodontal ligament,including a deep periodontal pocket,alveolar absorption,tooth loosening.At present,the treatment of periodontitis is mainly concentrated on inflammation control and the tissue regeneration.Since 2004,Seo successfully separated and characterized a kind of mesenchymal stem cells from human periodontal ligament,with a capacity of self-renewal,high proliferation and multi-directional differentiation,PDLSCs has being an important seed cells for periodontal defect repair.On the other hand,the excellent potential of hPDLSCs,to a great extent,can be disturbed by variable factors.For example,studies have continuously found that TNF-?and IL-1?,with a suitable concentration,could make adverse effects on cell mitosis and the potential of multi-directional differentiation.In addition,considering that PDLSCs obtained from inflammatory environment are responding to orthodontic force in a different way,pilot study also confirmed this conclusion,that is,a proper static stretch tension has an obvious inhibitory effect both on the abilities of proliferation and differentiation.But with mechanism of the phenomenon is still maintaining unknown.At the same time,through consulting literature,we find that mechanical stress and inflammatory micro-environment also play important roles of cytoskeletal metabolism,that being related to multiple cellular events such as cell proliferation and differentiation,contraction and migration,as well as intracellular transportation.The Rho family of small GTPases and its downstream are considered as molecular switches in modulating the dynamic changes and rearrangement of cytoskeleton.However,up to now,Whether Rho signaling pathway is participating in mechanoresponsive properties including cytoskeletal reorganization of h PDLSCs derived from inflammatory environmentalism and the correlation between cytoskeletal metabolism and the characteristics of those mesenchymal cells are still remain unknown.In order to have a further study of strain-induced cytoskeletal rearrangement of h PDLSCs in inflammatory environment and to explore the possible accommodation of stem cell properties of PDL by cytoskeletons,we have successfully separated PDLSCs from healthy and inflammatory tissues of PDL applied to the subsequent experiments,which may provide more considerable clinical evidence for orthodontic treatment of patients suffering periodontal problems.This research mainly consists of three parts in different aspects respectively.1.Culture and identification of the biological properties of HPDLSCs and PPDLSCs;2.Detection of the influence of SMS at a magnitude of 12% on the proliferation,osteogenetic and adipogenetic capacities,as well as the cytoskeletal rearrangement.3.Correlations between the cytoskeletal rearrangement via Rho signaling pathway and SMS induced osteogenetic capacity of HPDLSCs and PPDLSCs.MethodsUsing low density incubation to isolate and culture HPDLSCs and PPDLSCs,mesenchymal stem cell makers were detected by flow cytometry,cell self-renewal viability were quantified by colony-forming assay,MTT or cell cycle test by flow cytometry.Osteogenesis was determined by ALP activity,ALP alizarin red staining,and proteins associated with osteogenetic ratio including ALP and Runx2 were tested by western blot assay.For adipogenesis experiments,oil red O staining was applied.With helps of FITC-labeled phalloidine immunofluorescent staining and the laser scanning confocal microscope,the intracellular stress fibers could be observed and via western blot test,the expressions of Rho signaling pathway associated proteins were measured.Results1.hPDLSCs successfully separated from both healthy and inflammatory periodontal ligament respectively and both of them had a relatively high expression level of the mesenchymal stem cell markers including CD146,CD29,CD90,at the same time,hematopoietic markers CD31,CD14 were negative.For morphological observations,both cells were spindle shaped and oriented randomly,comparing with HPDLSCs,the morphology of PPDLSCs was more slender.PPDLSCs possess a higher ability of proliferation but lose some differentiation potential compared with HPDLSCs.2.Once 12% SMS loading,the situation was reversed,the proliferation ratio of PPDLSCs decreased and its mufti-lineage differentiation became more deteriorating.However,12% SMS significantly played an active role of both multiplication and differentiation.ALP activity,ALP staining,alizarin red staining,oil red O staining and western blot all confirmed that.After strain loading for 12 h,cellular morphology showed that HPDLSCs exhibited a more slender shape,arranged parallel to each other,and the long axis of all cells were vertical to the direction of static strain and in the laser confocal microscope,stress fibers were increased and became more massive.As a contrast,the ratio of irregularly arranged PPDLSCs with cell contraction to some extent increased and intracellular stress fibers decreased and fuzzy.Western blot was applied to examined the proteins related to Rho signal transduction pathway,the quantitative analysis demonstrated that whether 12% SMS loading or not,expression of Rho-A,ROCK1,p-cofilin in PPDLSCs were significantly lower and the expressions of cofilin and Rho-GDI? were higher than that in HPDLSCs.With SMS loading,PPDLSCs showed distinct decrease of Rho-A,ROCK1,p-cofilin protein expressions and slight increase of cofilin and Rho-GDI?,on the other hand,12% SMS acted an positive on expression of Rho-A,ROCK1,p-cofilin protein of HPDLSCs.3.With the application of Y27632,before 12% SMS loading,results showed that the PI values of PPDLSCs significantly descended compared with PPDLSCs treated by DMSO as control,and the group of no strain loading made the highest PI value than other two groups above.Referring to the PI value of HPDLSCs went to DMSO> Y27632> group of no strain loading.As for ALP staining,alizarin red staining and the quantitative protein analysis of osteogenesis and fluorescent staining and protein analysis of cytoskeletal rearrangement,there was more capable of HPDLCs than PPDLSCs in terms of osteogenetic differentiation in the same experimental condition.Moreover,Y27632 inhibited the cytoskeletal rearrangement both of HPDLSCs and PPDLSCs,stress fibers in PPDLSCs with Y27632 rarely observed,by contrast,HPDLSCs showed relatively more fibers,although this amount was significantly lower than other two groups of HPDLSCs.Western blot demonstrated the same tend of signal protein variations with the confocal microscopy.Conclusions1.Inflammation of the micro-environment,to a certain degree,can improve proliferative ability of hPDLSCs,weakening its multi-directional differentiation potential and suppressing cytoskeletal rearrangement at the same time.2.Compared with h PDLSCs derived from the healthy tissue,inflammatory micro-environment obviously weakens cells' endurance to mechanical stress and self-renewal and repair capabilities.On the other hand,the high sensitivity and self-adjusting ability of cytoskeleton to mechanical strain can also be affected by the inflammatory environment.3.The inflammatory micro-environment and mechanical strain are making synergetic roles in both cytoskeletal rearrangement via Rho signaling pathway and potentials of proliferation and differentiation in h PDLSCs. |
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