Involvement Of PI3K/Akt/mTOR Mediated Autophagy In Tantalum Particle-induced Proliferation In MC3T3-E1 Cells

BackgroundTantalum(Ta)has good physical properties and stable chemical properties for its high melting point,high strength,abrasion resistance and corrosion resistance.The biological tissue is easy to grow on the surface of Ta.So,Ta is also known as"biological metal".Porous tantalum has high porosity and surface energy,good biocompatibility and cellular infiltration,and closer elastic modulus to bone.So,porous tantalum is known as "bone trabecular metal." Studies showed that,porous tantalum implant is beneficial to form a three-dimensional bone integration,promote early attachment,proliferation,differentiation,mineralization of osteoblast,and improve early stability of implant.However,the tantalum particles deposited on the surface of implant contacted directly with tissues and cells surrounding implant and caused stress responses that may affect the dynamic balance of bone formation and absorption.So,what's the effect of tantalum particles on the proliferation of osteoblasts?And what is the mechanism behind?Till now,there is no relevant report.ObjectiveTo analyze the effect of tantalum particles on cell proliferation and further explore the role of PI3K/Akt/mTOR mediated autophagy in tantalum particles induced proliferation,mouse MC3T3-E1 osteoblasts were cultured with tantalum micro particles(Ta-MPs)and tantalum nano particles(Ta-NPs)of different concentrations to simulate the microenvironment of the surface of porous tantalum implant.Methods1.First,transmission electron microscope(TEM),scanning electron microscope(SEM)and dynamic light scattering(DLS)were used to analyze the characteristics of tantalum particles.2.Second,cultured MC3T3-E1 cells with Ta-MPs and Ta-NPs of different concentrations for 6,12,24,48 h and chose the groups that promoted proliferation most according to CCK-8 assay.3.Then,Western Blot,laser confocal microscope,TEM were used to detect autophagy and further determine the role of autophagy in cell proliferation combined with autophagy inhibitor.4.Finally,pathway inhibitors were used to interfere with the PI3K/Akt/mTOR signaling pathway.Western Blot and CCK-8 were used to detect autophagy and cell proliferation to clarify the effect of PI3K/Akt/mTOR on the regulation of autophagy and cell proliferation.Results1.Ta-MPs are irregular in shape,ranging from 100 nm to pms,with an average hydrodynamic size of 1281 nm;Ta-NPs are spherical,with regular shape about 8?15 nm,and the average hydrodynamic size is 292 nm.2.The groups that promoted proliferation most were 100 ng/mL Ta-MPs and 20?g/mL Ta-NPs according to the results of CCK-8.3.In the 100 ng/mL Ta-MPs treated group,no obvious autophagy was detected;in the 20 ?g/mL Ta-NPs treated group,there had increased LC3-II expression and decreased p62 expression(P<0.05),as well as obvious autophagy fluorescence and autophagic structures.Autophagy inhibitors could weak the effect of Ta-NPs on cell proliferation through decreasing its autophagy level.4.PBK/Akt/mTOR pathway inhibitors could further increase the autophagy and proliferation of 20 ?g/mL Ta-NPs treated cells.Conclusions1.Tantalum particles could promote the proliferation of MC3T3-E1 osteoblasts.2.Ta-NPs promoted cell proliferation by inducing autophagy,while Ta-MPs through non-autophagy pathways to promote cell proliferation.3.The PI3K/Akt/mTOR signaling pathway could negatively regulate the Ta-NPs induced autophagy and proliferation.