| Objective: This study aims to evaluate the effects of smooth titanium(ST),micro titanium(MT)and nano titanium(NT)on the morphology,adhesion,proliferation and osteogenic differentiation of MC3T3-E1 cells,and explore the correlation between mitochondrial fission and fusion(MFF)of MC3T3-E1 cells and topography-regulated osteogenesis,and the preliminary mechanism that may be involved in it.Methods: 1.Material science study: ST was prepared by gradient silicon carbide sandpaper polishing,and on the basis of ST,MT was prepared by 1.25 wt.% hydrofluoric acid(HF)etching for 3 mins,and NT was prepared by anodic oxidation for 1 hour at 20 V under inorganic electrolyte system;Stereoscopic microscope was used for general observation of different titanium surfaces,field emission scanning electron microscope(FE-SEM)and atomic force microscope(AFM)were used for observation of the microstructures or nanostructures on different surfaces,and the roughness of different surfaces was also detected.Contact angle measuring test was performed for evaluation of the hydrophilicity of different surfaces.2.Cytological evaluation: To evaluate the phenotype and biological capacity of MC3T3-E1 cells on surfaces with different topographies,morphology of adhered cells on day 3 was observed by FE-SEM.Cytoskeleton of cells stained with phalloidin after culturing for 3 days on different surfaces was observed by confocal laser scanning microscopy(CLSM).4',6-diamidino-2-phenylindole(DAPI)staining was used to detect the capacity of cell adhesion on different surfaces after incubating for 30 mins,60 mins and 120 mins.CCK-8 experiment was performed for the evaluation of cell proliferation on 1 d,3 d,5 d and 7 d;To evaluate the osteogenic differentiation ability of MC3T3-E1 cells on surfaces with different topographies,the expression level of osteogenesis-related molecules of MC3T3-E1 cells on day 7 was measured by quantitative polymerase chain reaction(qPCR).Alkaline phosphatase(ALP)staining on day 7 and alizarin red staining on day 21 were also conducted to detect the osteogenic differentiation and mineralization of MC3T3-E1 cells;To evaluate the MFF status of MC3T3-E1 cells on surfaces with different topographies,qPCR was used to detect the expression level of MFF-related molecules of MC3T3-E1 cells after 1,2 and 3 days of incubation on different surfaces.The morphology and the number of mitochondria on day 7 were observed with transmission electron microscope(TEM),and mitochondria of MC3T3-E1 cells on day 7 were labeled with Mito-tracker Green,and were also observed.qPCR was applied to measure the expression level of MFFrelated genes of MC3T3-E1 cells after a 7-day incubation.Meanwhile,the expression level of dynamin-related protein 1(DRP1)on day 7 was further measured by western blot.Moreover,the spatial localization and expression of DRP1 on day 7 were observed and analyzed by immunofluorescence staining.3.Molecular mechanism exploration: To explore the possibility that surfaces with different topographies can affect the expression of DRP1 through tuning the reactive oxygen species(ROS)level in MC3T3-E1 cells,ROS level of MC3T3-E1 cells in ST group and NT group on day 3 was detected by DCFH-DA chemical fluorescence staining.qPCR and western blot were used to measure the expression level of DRP1 after transfecting with different DRP1-siRNAs for 24 hours.Meanwhile,combined with qPCR results of mitochondrial-fusion-related genes(mitofusin 1,MFN1;mitofusin 2,MFN2;optic atrophy 1,OPA1),DRP1-siRNA with the highest transfection efficiency was selected,and the effect of it was further verified by Mito-tracker Green;Finally,to explore the mechanism that nanotopography can regulate MFF through affecting DRP1 expression and thus affect the osteogenic differentiation of MC3T3-E1 cells,osteogenesis-related genes of MC3T3-E1 cells in NT group with or without a 24-hour transfection of DRP1-siRNA were measured by qPCR after a 3-day and 7-day osteogenic induction.ALP activity of MC3T3-E1 cells was carried out to confirm it.Results: 1.The ST surface was smooth and mirror-like.The MT surface was grey and presented micro-pit-like structures with sharp edges.The NT surface was golden and presented uniformly distributed nanotubes with a diameter of 100 nm;Surface roughness and hydrophilicity of different groups both ranked as the following: ST < MT < NT;qPCR results showed a trend that mitochondrial fission-related gene(DRP1)significantly increased in MT group and NT group,while fusion-related genes(MFN1?MFN2?OPA1)decreased compared to ST group on day 1 and day 2.However,the expression of these genes presented a contrary trend on day 3.2.On day 3,cells in ST group were spread out with thin and dispersed pseudopodia,and the arrangement of cytoskeleton was uniform and consistent.Differently,the cells in MT group and NT group were elongated and spindle-shaped,with large pseudopodia and polar arrangement of cytoskeleton;The cell adhesion amount per scope and cell proliferation both generally presented as: NT > MT > ST;Compared with ST group and MT group,the expression of osteogenesis-related genes(ALP;osteocalcin,OCN;osteopontin,OPN;Runt-related transcription factor 2,Runx2)all significantly increased in NT group after osteogenic induction for 7 days.Also,ALP activity and osteogenic mineralization capacity in NT group were the strongest.TEM results on day 3 showed that the mitochondria in ST group were short and fission-like,while those in MT group and NT group were thick and continuous;On the 7th day,compared with ST group,the mitochondria in MT group and NT group were more syncretic like a network,with significantly decreased expression of DRP1.Immunofluorescence showed that DRP1 accumulated at sites where the mitochondria were going through a fission,and the expression of DRP1 was the highest in ST group.3.DCFH-DA staining results displayed that ROS level in NT group was lower than that in ST group;The screened DRP1-siRNA could effectively reduce the expression of DRP1,and the mitochondria were in a network-like fusion state after transfection;qPCR results showed that the expression of ALP,Runx2 significantly decreased after transfection with DRP1-siRNA on day 3,and ALP significantly decreased on day 7.ALP staining result also showed a decreased osteogenic differentiation ability on NT in DRP1-siRNA transfected group.Conclusions: 1.ST,MT and NT can be successfully prepared by sandpaper polishing,HF etching and anodic oxidation,and the surface roughness and hydrophilicity of NT is the highest.2.MC3T3-E1 cells spread out well on the surface of NT.Compared with ST and MT,cells on the surface of NT showed the best adhesion ability,the highest proliferation activity,and the strongest osteogenic differentiation capacity.3.There is a certain correlation between topography and the MFF of MC3T3-E1 cells.The mitochondria on MT and NT surface were mainly divided and the expression of DRP1 significantly increased at the early stage(1 d,2 d),while were mainly syncretic and the expression of DRP1 significantly decreased at the latter stage(3 d,7d)compared with ST surface.4.The low level of ROS may act as the upstream molecule to participate in the regulation of DRP1.DRP1-siRNA could effectively reduce the expression level of DRP1 and interfere with MFF balance of MC3T3-E1 cells on NT,but the osteogenic differentiation of MC3T3-E1 cells on NT decreased after the intervention of DRP1-siRNA when compared with the untransfected group. |
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