Pamidronate Disodium Leads To Bone Necrosis Via Suppression Of Wnt/β-catenin Signaling In Human Bone Marrow Mesenchymal Stem Cells In Vitro＆Case Report

Purpose:Bisphosphonate-related osteonecrosis of the jaw is poorly understood at the cellular and molecular levels.In this paper,the molecular mechanism of osteonecrosis caused by bisphosphonate is discussed from the perspective of osteogenesis.Materials and Methods:P5 human BMMSCs were used.Flowcytometric characterization of surface markers expression pattern on BMMSCs.A proliferation assay was performed to determine the experimental concentration of pamidronate disodium(PD).Alkaline phosphatase(ALP)activity,ALP staining and Alizarin red S(ARS)staining were assessed after treatment with PD(0,0.1,0.5,1,5,10 μg/mL).Quantitative polymerase chain reaction(qRT-PCR)and western blotting specific for Wnt/β-catenin signaling genes or proteins were performed after treatment with 0.5μg/mL PD.Wnt3a was used to observe osteogenic differentiation of BMMSCs during treatment with 0.5 μg/mL PD.Results:STRO-1:57.26%,CD146:88.49%,CD105:93.17%,CD90:95.73%,CD34:2.56%,CD 14:1.03%.As expected,1,5 and 10 μg/mL PD were unfavorable for BMMSC growth(P<0.05),while 0.1 and 0.5 μg/mL PD did not affect BMMSC growth(P ≥ 0.05).BMMSCs treated with 0.5 μg/mL PD had lower ALP activity,ALP staining and ARS staining(P<0.05),and BMMSCs treated with low concentrations of(<0.5 μg/mL)PD had the same level ALP activity,ALP staining and ARS staining as the control(0 μg/mL).PD(0.5 μg/mL)decreased the expression of genes and proteins involved in Wnt/β-catenin signaling.BMMSCs with Wnt3a and 0.5 μg/mL PD treatment had higher ALP activity,ALP staining and ARS staining(P<0.05).Conclusions:Pamidronate disodium inhibited Wnt/β-catenin signaling,which controls osteogenic differentiation in BMMSCs.Wnt3a can reduce the negative effects caused by PD which may rescue the occurence of BRONJ.