Strontium-loaded Titania Nanotube Arrays Repress Osteoclast Differentiation And The Related Molecular Mechanisms:In Vitro And In Vivo Studies

Objective1.To produce Tio2-NTs by anodisation at 60 V,and Ti02-NTs were subjected to hydrothermal treatment to form Sr-containing TiO2-NTs(NT-Sr).Then,to analyse the samples characterisation and biological property.2.To detect the effects of NT-Sr on osteoclast formation and activity in RAW264.7 cells and BMMCs.3.To detect the effects of NT-Sr on the molecular mechanisms of osteoclast differentiation in RAW264.7 cells and BMMCs.4.To elucidate the effects of NT-Sr on bone loss and observe osteoclast formation and function in vivo,using an ovariectomised rat model.Methods1.TiO2-NTs were fabricated on Ti surfaces by electrochemical anodisation,and then subjected to hydrothermal treatment for 1 h or 3 h to generate different Sr amount samples(samples NT-Srlh and NT-Sr3h,respectively)in a Sr(OH)2 solution.The samples were characterised by FE-SEM.We used XRD and XPS to evaluate the samples' surface morphology and chemical composition.The samples biological property were observed by protein adsorption assay,CCK-8 assay and cell adhesion assay.2.We estimated the effect of NT-Sr on osteoclast differentiation by TRAP staining and TRAP enzyme activity assy.We tested the effect of NT-Sr on F-actin rings formation by Immunofluorescence staining.The influence of NT-Sr on osteoclast function was explored used Corning Osteo Assay Surface plates.3.We explored the effect of NT-Sr on activation of NF-?B by Western blot and EMSA.The ERK and Akt/NFATcl signaling pathways were also detected by Western blot.What more,we also measured the expression levels of osteoclast marker genes by RT-PCR and Western blot.4.The osteoporosis model were established by ovariectomized method.After treatment with implants for eight weeks,we elucidated the effects of NT-Sr on bone loss by ?-CT scan.We analysied the effects of NT-Sr on osteoclast formation by TRAP staining in vivo.We also measured serum CTX-I levels by ELISA analysis.Results1.The average diameter of TiO2-NTs produced at 60 V was approximately 100 nm,and the diameter between TiO2-NTs and NT-Sr was no significant difference.We found that TiO2-NTs and NT-Sr increased protein adsorption compared with Ti.NT-Sr did not inhibited cell proliferation,but inhibited cell extension.2.NT-Sr suppressed osteoclast differentiation,osteoclast activity,and osteoclast function in vitro.NT-Sr also inhibited F-actin rings formation.3.NT-Sr suppressed RANKL-induced phosphorylation of I?B?,NF-?B p65,and Akt.NT-Sr significantly impaired the DNA-binding activity of the NF-?B.NT-Sr promoted RANKL-induced phosphorylation of ERK and NT-Sr inhibited NFATc1 protein expression.What more,NT-Sr suppressed mRNA expression and protein expression of osteoclast-specific genes such as TRAP,cathepsin K(CK),MMP-9,and NFATc1.4.NT-Sr prevent OVX-induced bone loss and suppressed osteoclast formation in vivo.NT-Sr also decreased the serum levels of CTX-I.ConclusionIn summary,NT-Sr may effectively inhibit osteoclast differentiation by repressing the NF-?B and Akt/NFATc1 pathways and by negatively regulating the ERK pathway in vitro and in vivo.Our results demonstrated that NT-Sr may become an ideal implant material for patients with osteoporosis.