DAPT Inhibits Titanium Particle-induced Osteolysis By Suppressing The RANKL/Notch2 Signaling Pathway

PART ? DAPT INHIBITS OSTEOCLAST DIFFERENTIATIONObjective:To identify the effect of DAPT,a gamma-secretase inhibitor,on bone marrow-derived macrophages(BMMs)induced by receptor activator of NF-?B ligand(RANKL)and macrophage colony-stimulating factor(M-CSF).Methods:In vitro experiments,primary BMMs were extracted from the femurs and tibias bone marrow of three to five week-old C57/BL6 male mice,and the cells were cultured in complete a-MEM medium to achieve the ideal number and state of adherence.The cytotoxic effects of DAPT were assessed by cell counting kit-8(CCK-8)following the manufacturer's instructions.Osteoclasts with more than 3 nuclei were considered as positive for tartrate-resistant acid phosphatase(TRAP)staining and counted under a microscope.The osteoclast bone resorption was determined using a 24-well Osteo Assay Plate.The mRNA levels of osteoclast-related genes such as NFATc1,TRAP,c-Fos,CtsK,CTR,DC-STAMP were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)under different concentrations of DAPT.Results:The results of CCK-8 showed that DAPT exerted cytotoxic effect on BMMs at the concentration higher than 80?M.The IC50 of DAPT was 316.2?M,281.8?M and 288.4?M after incubation for 48h,72h and 96h,which indicated that the maximum concentration(10?M)used in this study did not cause any cytotoxicity.The results of TRAP staining showed that a large number of osteoclasts were formed under the induction of RANKL and M-CSF(191 cells/well).DAPT significantly decreased the number of osteoclasts and nearly no multinucleated osteoclasts formed at 10?M of DAPT(p<0.05).The bone resorption area in DAPT group was observably smaller than that in the control group(76.8%),and the resorption area was dose-dependent.About 39.9%,14.2%and 6.8%resorbed bone area were measured in the DAPT treatment group with 2,5?M,5?M and 10?M,respectively,which is statistically significant(p<0.05).The results of RT-qPCR showed that compared with the control group,the expression levels of NFATc1,TRAP,c-Fos,CtsK,CTR,and DC-STAMP,which were upregulated during RANKL and M-CSF-induced osteoclast differentiation,were dramatically declined after DAPT treatment in a dose-dependent manner.Conclusions:Therapeutic concentration of DAPT was able to effectively inhibit the formation and function of osteoclast induced by RANKL and M-CSF without cytotoxicity in vitro.PART ? STUDY ON THE MECHANISM OF DAPT INHIBITING OSTEOCLAST DIFFERENTIATIONObjective:To demonstrate the underlying molecular mechanism of DAPT in osteoclastogenesis induced by RANKL and M-CSF.Methods:The effects of DAPT on RANKL and M-CSF-induced osteoclast proliferation and differentiation were investigated using BMMs.Western blot was used to detect the expression of proteins such as NFATc1,NICD2,HES-1,I?B?,p65 and phosphorylated p65(p-p65).Computer molecular docking technology was used to simulate the potential targets of DAPT on p65 protein.Results:The results of western blot showed that RANKL and M-CSF induced the high expression of NFATc1,NICD2,HES-1,p65 and p-p65.However,the expression level of I?B? protein was the same with or without DAPT.The expression level of NFATc1,NICD2,HES-1,p65 and p-p65 decreased after administration of DAPT.Computer molecular docking technology showed that the potential target of DAPT on p65 protein could be Arg-201 and Ser-203 sites.Conclusions:DAPT could inhibit the proliferation and differentiation of osteoclast induced by RANKL and M-CSF via NF-?B and Notch2 signaling pathways.The potential targets of DAPT on p65 protein could be Arg-201 and Ser-203 sites.Therefore,these results provided a new idea to prevent and treat aseptic prosthetic loosening.PART ? DAPT INHIBITS TITANIUM PARTICLE-INDUCED CALVARIAL OSTEOLYSISObjective:To investigate the therapeutic effect of DAPT on osteolysis model induced by titanium particles in mice,providing new ideas for the treatment of clinical patients with aseptic prosthesis loosening.Methods:Twenty healthy 8-week-old C57/BL6 male mice were randomly assigned to four groups:sham operation group(Sham),titanium particles group(Vehicle),low treatment concentration of DAPT(0.5mg/kg/day)group(Low)and high treatment concentration of DAPT(2mg/kg/day)group(High).After anesthesia,pretreated titanium particles were then embedded on the surface of the calvaria around the middle suture.During the following 14 days,mice in the experimental group were injected intraperitoneally with two different concentrations of DAPT,respectively.Sham group and Vehicle group were injected with PBS every day.The mice were finally killed and the calvaria were fixed for Micro-CT and H&E staining analysis.Results:Micro-CT showed that the osteolysis of the calvaria in the Vehicle group was more obvious than that in the sham group,while the osteolysis in the treatment group significantly reduced.Compared with the Vehicle group,the BV/TV%in the low-dose treatment group and the high-dose treatment group increased by 15.9%and 29.1%respectively.However,the number of porosity in the low-dose treatment group and the high-dose treatment group reduced by 42.2%and 65.6%respectively.Histological examination showed that substantial inflammatory infiltration such as macrophages and TRAP-positive osteoclasts were observed on the surface of calvaria after induction of titanium particles.In contrast,the osteolysis area reduced in DAPT-treated group,and so was the number of inflammatory cells(p<0.05).Conclusions:DAPT was able to inhibit the osteolysis and inflammatory response in mice calvaria induced by titanium particles.