Oxytocin Facilitates The Proliferation,migration And Osteogenic Differentiation Of Human Periodontal Stem Cells

Objective:Periodontitis causes the destruction of gingiva,periodontal ligament,cementum and alveolar bone,eventually resulting in tooth loss.Regeneration of lost periodontium is the main goal of periodontal therapy and periodontal tissue engineering.As an ideal cellular source for periodontal tissue repair and regeneration,periodontal ligament stem cells(PDLSCs)have the capability of differentiating into osteogenic,adipogenic,and chondrogenic cells in vitro when exposing to differentiation inducers.However,the inflammatory environment caused by periodontitis leads to a reduction in the number of PDLSCs as well as their abilities to undergo proliferation and differentiation.Therefore,increasing the number of PDLSCs and improving their biological activities are expected to facilitate periodontal regeneration.Cytokines,chemokines and adhesion molecules have been used to promote periodontal regeneration.However,these growth-supporting factors are expensive and lack good stability in the oral environment.Therefore,the selection of growth factors should be further explored.Hormones such as estrogen,acting through estrogen receptors,has an important role in bone dynamics.In addition,oxytocin(OT)has been found to regulate the anabolic action of estrogen on the skeleton via acting with OT receptor(OTR),which has been found expressed on many different types of cells,such as osteoblasts and adipocytes.In addition to its well-known roles,OT plays an important role in controlling the differentiation of human mesenchymal stem cells(MSCs),regulating the balance of osteoblasts and adipocytes in vivo and reversing bone loss in ovariectomized rat models.Moreover,OT has been shown to promote osteoblast differentiation into the mineralized phenotype by up-regulating the expression of bone morphogenetic protein 2(BMP-2).The loss of periodontal tissue,especially alveolar bone,is closely related to hormone level.A prior study showed that the reduction of estrogen after menopause was associated with increasing resorption of alveolar bone.Moreover,down-regulation of serum OT may lead to osteoporosis in postmenopausal women.Hence,we speculate that OT may play a role in periodontal regeneration.Therefore,in the present study,PDLSCs were stimulated with OT to evaluate the effects of OT on the proliferation,migration and osteogenic differentiation of PDLSCs,as well as to identify the underlying mechanism.Methods:(1)Isolation,cultivation and identification of PDLSCs and detection of OTR:the primary cells were obtained by method of tissue block and passage 3-5 were used for the subsequent experiments.PDLSCs with stem cell characteristics were obtained by finite dilution method and cultured.The expression of MSCs and HSCs surface markers was detected by flow cytometry.The expression of OTR on PDLSCs was detected by immunofluorescence staining(IF).(2)Cell viability:the effects of 0?10?50?100 nM concentrations of OT on the proliferation of PDLSCs were detected according to cell-counting kit-8(CCK8)instructions.(3)Cell migration:Transwell assay was used to detect the effect of50 nM OT?100 nM OT?50 nM OT + 200 nM OT receptor antagonist(OTA)?100 nM OT+200 nM OTA on migration of PDLSCs.(4)Osteogenic differentiation:the effect of 0?10?50?100 nM concentrations of OT on the formation of mineralized nodules was detected byAlizarin Red S staining and calcium deposition for 21 days.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the effects of 0?10?50?100 nMconcentrations of OT on the expression of related osteogenic genes,alkaline phosphatase(ALP),Collagen ?(Col ?),runt related transcription factor 2(Runx2),osteopontin(OPN)and osteocalcin(OCN)at 7,14 and 21 days.Western blot was used to detect the effects of 0?10?50?100 nM concentrations of OT on the expression of related osteogenic proteins ALP,Col ?and Runx2 at 7 and 14 days.(5)Mechanism of osteogenic differentiation:the activation of extracellular signal-regulated kinase(ERK),protein kinase B(AKT)and phosphatidylinositol 3-kinase(PI3K)pathwaywas detected by western blot after simulating with 50 nM OT for 0,2,5,10,15,30 and 60 minutes.Results:(1)Isolation and characteristics of PDLSCs and detection of OTR:PDLSCs were successfully isolated and cultivated by limited dilution method.Flow cytometry results showed that PDLSCs expressed MSCs surface markers CD29 and CD73 positively and negatively expressed HSCs surface markers CD45.IF result showed that OTR expressed on PDLSCs,and the expression of OTR was enhanced with increasing concentration of OT.(2)CCK 8 showed that OT had no obvious cytotoxic on PDLSCs and 50 nM OT promoted the proliferation of PDLSCs(P<0.05).(3)The results of Transwell assay showed that 50 and 100 nM OT could promote the migration of PDLSCs(P<0.005)and the effect of 100 nM OT was significantly better than that of 50 nM OT.The effect on cell migration was significantly inhibited after adding OT receptor antagonist(OTA),which indicated that the effect of OT on PDLSCs migration was induced by binding to OTR.(4)Detection of osteogenic differentiation:Alizarin Red S staining and calcium deposition showed that 10 and 50 nM OT significantly promoted the formation of mineralized nodules at 21 days(P<0.01).qRT-PCR and Western blot indicated that OT could promote the expression of relevant osteogenic genes ALP,Col ?,Runx2,OPN and OCN(P<0.05).Not surprisingly,OT enhanced with the expression of related osteogenic proteins ALP,Col ? and Runx2(P<0.01),and 50 nM OT exhibited the maximal effect.(5)50 nM OT could activate ERK and AKT pathway after acting on PDLSCs for 2 min and inhibit PI3K pathway,demonstrating that OT promoted osteogenic differentiation of PDLSCs via ERK and AKT signaling pathways.Conclusions:OT can promote PDLSCs proliferation,migration and osteogenic differentiation in vitro.This study provides a new idea for periodontal regeneration therapy.