The Research On The Mechanisms Of The Osteogenesis And Pro-angiogenesis Changes In Inflammatory Periodontal Stem Cells

Periodontitis is a common inflammatory disease that may cause teeth loss,alveolar bone loss and vascular system changes in the supporting tissue.And there is no efficient strategy to restore the supporting tissue damaged by periodontitis so far.Thus the study on the pathology of periodontitis has a profound impact on exploring effective treatment.Mesenchymal stem cells(MSCs)are well known for their pluripotency and paracrine secretion.It enables MSCs to differentiate to other cell linage and affect micro environment or biological function of adjacent cells.Recently a new kind of MSCs were found in the periodontal ligament of the tooth,the periodontal ligament mesenchymal stem cells(PDLSCs).It has been reported that osteogenesis and vascularization process are connected on both physiological and cellular level.The interaction between MSCs and ECs may change the MSCs state.In periodontitis,according to some researches,PDLSCs osteogenesis suffered a huge decrease.Thus we hypothesized that not only did this interaction in inflammation may influence the pathological state of periodontitis,buy also would it change the osteogenesis and vasculature-promoting ability of MSCs.There is very few research focuses on this process.There were proves that the osteogenesis and vasculature both underwent certain changes in periodontitis.Vascular endothelial growth factor(VEGF)is a core factor in vascularization process,which is highly expressed in inflammatory gingival tissue and crevicular fluid.These extra VEGF and hyperplastic blood vessels could further aggravate the periodontitis.Some researches proved that the phosphorylation of ERK increased in the wake of VEGF in inflammatory gingival fibroblasts.We hypothesized that this increase may affect the VEGF secretion by PDLSCs,which in turn change the vasculature condition in periodontal tissue along with micro environment.However,many other researches have proved that ERK cascade may regulate the autophagy while the latter played an important role in cell bio-function.Since ERK cascade could regulate the vascularization-promoting ability in PDLSCs,it is reasonable to question the role of autophagy in this process.Therefore,this study aimed at elucidating the variation of PDLSCs osteogenesis and vascularization-promoting ability from the angle of paracrine secretion.It consists of three part.Part1: ECs effect on PDLSCs osteogenic differentiation ability under inflammation[Objective]To investigate the osteogenic ability variation of PDLSCs regulated by ECs paracrine secretion[Method]PDLSCs were obtained from healthy and inflammatory periodontal ligament tissue(HPDLSCs and IPDLSCs).HPDLSCs were then cultured in medium containing TNF-α to mimic inflammation microenvironment.Conditioned medium from ECs was collected to culture the PDLSCs from the different groups to investigate its effects on the PDLSCs osteogenesis through quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot.The osteogenesis of different PDLSCs co-cultured with ECs was checked with the same methods.[Results]1.No significant change was found in the osteogenesis of PDLSCs in inflammation microenvironment after ECCM culture;2.The osteogenesis of PDLSCs in inflammation microenvironment were attenuated when co-cultured with ECs;3.Co-culturing with ECs reduced the apoptosis level of the PDLSCs from both normal and inflammation microenvironment.[Conclusions]The osteogenesis of PDLSCs was compromised by ECs paracrine secretion,which might result from the cross talk between two types of cell.The weakened function of osteogenesis in PDLSCs was not caused by inflammation.ECs reduced the apoptosis of PDLSCs in inflammation environment.Part 2: PDLSCs vascularization-promoting ability change under inflammation microenvironment[Objective]1.To investigate the variation of PDLSCs vascularization-promoting function on ECs under inflammation microenvironment and the role of VEGF in this phenomenon;2.To investigate the function of ERK cascade in PDLSCs promoting process.[Methods]First,in order to explore the VEGF expression in inflammatory periodontal ligament,teeth with ligament which were pulled out from severe periodontitis patients were collected.Immunohistochemisty assays,qRT-PCR and Western blot were employed to detect the expression of VEGF in periodontal ligament tissue.qRT-PCR and enzyme-linked immunosorbent assay(ELISA)were also used to measure the VEGF expression of PDLSCs treated by inflammatory factor TNF-α and IL-1β.Second,PDLSCs were treated with the inflammatory factors above and then co-cultured with normal or inflammatory ECs.After the co-culture Matrigel tube formation tests and qRT-PCR were employed to investigate the change of the vascularization ability in ECs.Third,qRT-PCR and ELISA was used to investigate the VEGF expression in PDLSCs that was treated by U0126 to suppress ERK.Subsequently,Matrigel tube formation tests and qRT-PCR were employed to measure the vascularization ability of ECs.[Results]1.The expression of VEGF was higher in the periodontal ligament of periodontitis patients than that in the healthy ligament;2.TNF-α and IL-1β led to increase of VEGF secretion by PDLSCs;3.Compared to the inflammation environment,inflammatory PDLSCs showed higher ability in promoting vascularization of ECs;4.The promoting ability of inflammatory PDLSCs was time-dependent;5.TNF-α and IL-1β enhanced the phosphorylation of ERK,thus enhancing the VEGF secretion by PDLSCs;6.The inhibition of ERK cascade could diminish the ability of PDLSCs in promoting vascularization.[Conclusions]The inflammatory microenvironment promoted the VEGF secretion of PDLSCs by enhancing the phosphorylation of ERK,which resulted in the higher ability of vascularization in ECs.Part 3: The effect of autophagy on PDLSCs vascularization-promoting ability[Objective]1.To investigate the variation of autophagy in PDLSCs under inflammatory environment;2.To investigate the relationship between ERK and autophagy in PDLSCs under inflammatory environment;3.To investigate the effect of autophagy in PDLSCs vascularization-promoting ability[Methods]PDLSCs were treated by TNF-α and IL-1β for 0,2,7days.Autophagy marker protein LC3 and Beclin-1 were tested by Western blot and qRT-PCR to reflect its variation.Then the relationship between ERK and autophagy were studied by the same methods above after activating or inhibiting ERK cascade with inflammatory factors and U0126 respectively.Rapamycin and Si-Beclin-1 were employed to enhance or suppress the autophagy in PDLSCs treated with TNF-α and IL-1β,which were then co-cultured with ECs.Matrigel tube formation tests and qRT-PCR were used to check vascularization ability of ECs.[Results]1.The expression levels of LC3 and Beclin-1 were firstly enhanced at the 2nd day and then weakened at the 7th day in PDLSCs treated with TNF-α and IL-1β;2.The activation of ERK caused the up-regulation of autophagy,vice versa;3.With the promotion of autophagy in PDLSCs treated with inflammatory factors for 7 days,PDLSCs show an enhanced ability in promoting vascularization of ECs;4.With the decline of autophagy in PDLSCs treated with inflammatory factors for 2 days,the vascularization of ECs was also attenuated.[Conclusions]ERK regulated the autophagy in inflammatory PDLSCs.And the autophagy modulated the vascularization promoting ability of PDLSCs under inflammation microenvironment.