The Research Of Regulatory Mechanism On Stem Cells From Human Exfoliated Deciduous Teeth During Physiological Root Resorption

OBJECTIVE:In this study,we preliminarily investigated the ability of stem cells from human exfoliated deciduous teeth(SHED)to promote the osteoclastic differentiation of osteoclast precursors and the mechanisms underlying this process in vitro using indirect co-culture approach.METHODS:SHED were isolated,purified and cultured in vitro.The indirect co-culture system of SHED and osteoclast precursor cell line peripheral blood mononuclear cells(PBMCs)was established(co-culture 3,7,10,14 days).In order to evaluate the osteoclastic differentiation of PBMCs after co-culture,the morphology of the osteoclast-like cells was observed by TRAP staining and the number of TRAP+ cells was counted;the expression of TRAP,CTSK,c-Fos,TRAF6 in PBMCs was evaluated by real-time RT-PCR and Western blot.After treatment by TNF-?(0,5,10,50,100 ng/mL),the expression of osteoclastic gene from PBMCs and NF-?B from SHED were determined by real-time RT-PCR and Western blot.We utilized the inflammatory cytokine TNF-a(10ng/mL)to simulate an inflammatory microenvironment in vitro.The addition of BAY11-7082 was used to inhibit the NF-?B signaling pathway.The expression of RANKL/OPG and the NF-?B related genes were determined by real-time RT-PCR and Western blot after SHED were influenced by inflammatory microenvironment.RESULTS:SHED promoted the differentiation of PBMCs into TRAP+ osteoclast-like cells and the expression of TRAP,CTSK,c-Fos,TRAF6 in PBMCs,compared with PBMCs individual culture group.The expression of CTSK,TRAP,and RANKL,the RANKL/OPG ratio,and the expression of cytoplasmic p-I?B? and nuclear p65 were markedly upregulated in SHED after TNF-a treatment but decreased following NF-?B inhibition.CONCLUSION:SHED can promote the differentiation of osteoclast precursors into osteoclast-like cells.SHED promoted osteoclastogenesis under the influence of an inflammatory microenvironment.Additionally,the inflammatory cytokine TNF-a appeared to activate the NF-?B pathway to upregulate the expression of NF-?B,enhancing the ability of SHED to promote osteoclastogenesis via the regulation of RANKL/OPG expression,which likely promotes root resorption and results in the physiological exfoliation of deciduous teeth.