Effect Of FGF2 On The Odontoblast Differentiation Of Stem Cells From Human Exfoliated Deciduous Teeth

Background:Clinically,pulpitis or apical periodontitis caused by dental pulp trauma or infection is usually treated by removing the inflammatory pulp and filling with inorganic materials.However,traditional root canal treatment has certain disadvantages.Therefore,based on the principle of tissue engineering,people proposed regenerative endodontic treatment(RET),that is,the use of bioactive factors,stem cells and scaffolds to achieve the regeneration of dental pulp-dentin-like tissue.Under certain conditions,dental-derived stem cells can differentiate into odontoblast-like cells,secrete mineralized matrix,and form tubular dentin,which can thicken the dentin wall,keep the roots growing and reduce the risk of root fracture.We look forward to finding a "seed cell" with obvious advantages for dental pulp regeneration.Stem cells from human exfoliated deciduous teeth(SHED)and human permanent tooth dental pulp stem cells(DPSCs)originate from neural crest ectodermal mesenchyme,with self-renewal ability and multidirectional differentiation potential.They can differentiate into chondrocytes,odontoblasts,osteoblasts,muscle cells,etc.Fibroblast growth factors(FGFs)are a series of growth factors that play an important role in the development,repair and regeneration of damaged bone tissue,including cartilage,bone and teeth.Among them,FGF2 is involved in the proliferation,homing,and migration of normal and inflammatory pulp cells.At the same time,FGF2 plays a vital role in the repair of dentin and the proliferation,migration,differentiation and self-renewal of pulp cells.The combination of FGF2 and FGFR can cause the phosphorylation of ephrinB1.Phosphorylated ephrinB1 can promote the phosphorylation and entry into the nucleus of signal transducer and activator of transcription 3(STAT3)that can enhance the transcription of downstream genes.However,there is no report that FGF2 activates STAT3 and participates in odontoblast differentiation.Purpose:The purpose of this experiment is to find the ideal cell source for dental pulp regeneration by comparing proliferation ability and odontoblast differentiation ability of SHED and DPSCs;to study the effect of FGF2 on odontoblast differentiation of SHED,and to preliminary explore the relevant mechanism,which provides theoretical basis for the use of FGF2 in dental pulp regeneration and tissue engineering technology to achieve clinical dental pulp regeneration.Methods:Part Ⅰ: SHED and DPSCs were separated and cultured by tissue enzymolysis and limiting dilution assays.Differentiation of SHED and DPSCs was induced by odontogenic induction medium.Alkaline phosphatase(ALP)staining and alizarin Red staining were used to detect calcium salt deposition.RT-qPCR was used to detect dentin matrix protein-1(DMP-1),dentin sialophosphoprotein(DSPP),and fibroblast growth factor receptor FGFR1 and FGFR2,ephrinB1 and STAT3 mRNA expression levels.Western Blot assay was used to detect protein expression levels of DMP-1,DSPP and ephrinB1.Part Ⅱ: RT-qPCR was used to detect the expression levels of DMP-1 and DSPP after different concentrations of FGF2 induced SHED,and the optimal concentration of FGF2 was selected.FGF2 was used to induce SHED,and samples were collected at different time points.RT-qPCR and Western Blot were used to detect DMP-1,DSPP and STAT3 expression levels;CCK-8 method was used to detect the cytotoxicity of Stattic to SHED,Western Blot method was used to detect the expression levels of STAT3 and P-STAT3,etc.,and the optimal concentration of Stattic was selected.By group: blank group,FGF2 group,FGF2 + Stattic group and FGF2 + DMSO group treated cells,Western Blot method was used to detect DSPP expression.Results:Part Ⅰ: The SHED and DPSCs were obtained through separation and culture with relatively uniform morphology.Both cells grew rapidly,but the proliferation rate of SHED was faster than that of DPSCs.The results of ALP staining and alizarin red staining in the SHED group were significantly deeper than those in the DPSCs group,which means calcium salt deposition ability of SHED is stronger than DPSCs.Except for the protein expression of DMP-1 in the 3d group,the mRNA and protein expression of DMP-1 and DSPP in the SHED group are higher than those in the DPSCs group.The expression patterns of FGFR1,FGFR2,ephrinB1 and STAT3 are consistent with DMP-1 and DSPP.Part Ⅱ: FGF2(5ng/mL)could effectively promote the expression of odontogenetic markers DMP-1 and DSPP of SHED.Compared with the blank group,the FGF2 group promoted the expression of DMP-1 and DSPP and phosphorylation of STAT3 in a time-dependent manner.Stattic at 6μmol/mL and 10μmol/mL would cause cell death.When the concentration of Stattic was 3μmol/mL,the cells grew well,and the phosphorylation of STAT3 and the expression of DMP-1 and DSPP were inhibited;Stattic can inhibit STAT3 phosphorylation,thereby inhibiting the promotion of DSPP expression by FGF2.Conclusion:SHED has stronger proliferation and odontoblast differentiation ability than DPSCs,and is an ideal “seed cell” source for dental pulp regeneration;FGF2 promotes odontoblast differentiation of SHED by activating STAT3,and the inhibition of STAT3 phosphorylation can inhibit the promotion of odontoblast differentiation of SHED by FGF2,indicating that STAT3 is involved in the regulation of odontoblast differentiation by FGF2,but other mechanisms need further study.