| Periapical periodontitis of deciduous tooth is a common clinical disease,prevalent disease.The inflammatory injury of the periapical tissue may disturb the root development and can aggravate the immature teeth looseness,or even premature shedding.Current treatments cannot solve the lack of root development,root canal thick,crown and root ratio of uncoordinated.Therefore,we need to explore new ways of treatment.In recent years,tissue engineering technology has attracted increased interests and could provide new ideas for the treatment of apical tissue defects.Stem Cells from the Apical Papilla(SCAP)is one of the five types of dental stem cells,which plays an important role in dentin and root development and is an seed cell for repairing apical tissue defects.In this study,immortalized Stem Cells from the Apical Papilla(iSCAP)were constructed,and maintained the multidirectional differentiation of the original cells and solved the problem of insufficient number of cells.BMP9 is one of the most potent factors that can induce osteogenic differentiation of MSCs.It has been proved that BMP9 can effectively induce osteogenic differentiation of iSCAP cells.In recent years,with the development of tissue engineering technology,the effect of microenvironment on the differentiation of stem cells has been paid more and more attention by researchers.In the periapical period of inflammatory microenvironment,there will be a large number of inflammatory factor released,in which tumor necrosis factor alpha(TNF-?)as one of the most important inflammatory factors of apical periodontitis,The effect of TNF-? on BMP9-induced iSCAP cells osteogenesis differentiation was still unknown.Therefore,the aim of this study is to solve the following problems:(1)To detect the expression of TNF-? and the relationship between TNF-? and the bone defect in the process of periapical periodontitis;(2)To investigate the effect of TNF-? on the BMP9-induced osteogenic differentiation of iSCAP cells(P <0.05).(3)Whether BMP9 can induce osteogenic differentiation of iSCAP cells in the environment of high concentration of TNF-?(10ng / ml).In order to explore the above problems,the first part of this study established a model of periapical periodontitis of deciduous tooth in SD rats.Micro-CT was scanned at different time points and calculating the area of mesial root apical lesion.We found that the root development was disturbed,lesions area was gradually increased from day 3 to day 14,and then sharply increased until 28 days.The lesion area kept rather stably from day 28 to day 35.H & E staining was used to observe the morphology of the apical defect area.We found that the bone defect area was filled with connective tissue and filled with a lot of inflammatory cell.The expression of TNF-? at different time points was detected by immunohistochemistry,and found that TNF-? may be positive correlated with the area of periapical bone defects.The effects of different concentrations of recombinant TNF-? protein(0.1 ng / ml,1.0 ng / ml,10 ng / ml,100 ng / ml)on osteogenic differentiation of BMP9-induced iSCAP cells were investigated.The expression of ALP was significantly decreased in the TNF-?(10 ng / ml,100 ng / ml)group,and the expression of OCN and OPN was also significantly decreased by q PCR and the late stage mineralization was inhibited.All these results indicating that TNF-?(10 ng / ml,100 ng / ml)inhibited BMP9-induced osteogenic differentiation of iSCAP cells.The ectopic osteogenesis in nude mice showed that the volume of ectopic bone mass in BMP9+ TNF-?(10 ng / ml)group was significantly lower than that in positive control group(P <0.05),and the bone trabecula was low using bone Masson staining,and the maturity and mineralization were significantly diminished.In order to investigate whether overexpression of BMP9 can induce the osteogenic differentiation of iSCAP cells in the high concentration of TNF-?(10 ng / ml)environment,we gradually increased Ad-BMP9 expression and found that ALP expression increased gradually,The expression of OCN and OPN was improved by qPCR,and the late stage mineralization was increased,which indicated that BMP9 could induce osteogenic differentiation of iSCAP cells in the high concentration of TNF-?(10 ng / ml)condition.In summary,TNF-? is released during the development of periapical periodontitis and is positively correlated with the area of periapical bone defects.High concentration of TNF-?(10 ng / ml)significantly inhibited BMP9-induced osteogenic differentiation of iSCAP cells.BMP9 is still able to induce osteogenic differentiation of iSCAP cells in a high concentration of TNF-?(10 ng / ml)environment. |
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