| Background and ObjectivesTitanium implant has a high success rate,however,poor osseointegration and peri-implant-related infection are still the main factors for its failure.Therefore,it is very necessary to improve the surface and antibacterial modification of titanium.In the previous study,we prepared double-layered TiO2 nanotube(NT)structure using titanium nanobowl(TNB)template by twice electrochemical anodization.The results show that this structure can significantly improve the biocompatibility compared to pure titanium,and this structure further increases the specific surface area,adsorption capacity and loading space of titanium compared to single-layered TiO2 nanotube(TNT).Silver,a powerful inorganic antibacterial agent,has obvious advantages such as wide antibacterial spectrum,less bacterial resistance,long antibacterial time.Ag nanoparticles(AgNPs)have attracted more and more attention as a potential substitute for traditional antimicrobial agents because of their large specific surface area and excellent biocompatibility and antibacterial activity.Among the many modifications of titanium surface,nest-like nanofiber(NTNF)structure has potential application prospects.At present,it is mainly prepared by hydrothermal method,which needs a long reaction period,high equipment requirements and technical difficulty.NTNF prepared by a hydrothermal method is prepared on titanium substrate in a bottom-to-top way,and the adhesion is weak.Therefore,it is urgent to develop a simple,easy-to-operate,safe and controllable method to prepare NTNF with high adhesion on titanium surface.Inspired by the fact that TNB and AgNPs can significantly improve the reaction efficiency and performance by increasing the specific surface area,in this study we creatively adopted the method of TNB template combined with alkali etching to construct NTNF structure on titanium surface at room temperature and pressure,we also examed the biocompatibility,biological activity of promoting osteogenesis and differentiation in vitro and the effect of osseointegration in vivo of TNT and NTNF titanium implants in a beagle model.Materials and methods1.Pure titanium sheets and titanium rods were anodized in ethylene glycol electrolyte of 88 mmol/L ammonium fluoride for 2.5 h at a voltage of 60 V to form TNT.The nanotubes were removed by ultrasonic shake in deionized water to form TNB structure.2.TNB was anodized to 40 min at 12 V to form NT,and then vacuum ion sputtered with AgNPs for 1 min under 10 mA in a coating sputtering machine to obtain double-layered TiO2 nanotube with Ag nanoparticles(NT-Ag)structure;the surface morphology and chemical composition and distribution of the samples were characterized by scanning electron microscope(SEM),transmission electron microscope(TEM),energy dispersive spectrometer(EDS)and x-ray photoelectron spectrometry(XPS);Ag+release was detected by inductive coupled plasma emission spectrometer(ICP).The biological activity of NT-Ag and NT in terms of the adhesion,viability,proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSCs)was detected by cell adhesion and morphology,cytoskeleton,living dead cells,cell counting kit-8(CCK-8),alkaline phosphatase(ALP)semi-quantitative and staining,real time quantitative polymerase chain reaction(RT-qPCR),and the antibacterial activity of NT-Ag was detected by adhesion and survival of porphyromonas gingivalis(P.gingivalis).3.TNB was immersed in 4 mol/L KOH solution for 2 h at room temperature,then soaked in deionized water for 2 h.NTNF was prepared at room temperature and pressure.The surface morphology,chemical composition and distribution of the samples were characterized by SEM,EDS,x-ray diffractometer(XRD)and XPS.The three-dimensional morphology and roughness of the sample were observed and measured by atomic force microscope(AFM).Cell adhesion,morphology,cytoskeleton,living and dead cells,CCK-8,ALP semi-quantitative and staining,and RT-qPCR were used to detect the biological activity of TNT and NTNF on the adhesion,vitality,proliferation and osteogenic differentiation of rBMSCs.4.The implant model of beagle dog jaw was established.Calcein(CA)and alizarin red S(ARS)sequence fluorescence labeling were performed at 9 weeks and 11 weeks after titanium rod implantation,respectively.Three months after implantation,the samples were collected.Micro computed tomography(Micro-CT)scanning and three-dimensional reconstruction software analysis,fluorescence double labeling detection of hard tissue sections and Van Gieson histological staining were used to evaluate the effect of TNT and NTNF structure on osseointegration of the implants.Results1.AgNPs was uniformly loaded into the inside and on the wall of the large and small nanotubes of NT by sputtering coating.The release rate of AgNPs increased rapidly within 24 h,remained stable after 468 h,and reached a plateau after 468 h.The loading of AgNPs did not affect the adhesion,activity and proliferation of rBMSCs in vitro,but increased the expression of genes related to osteogenic differentiation.NT-Ag has more antibacterial activity than NT.2.The TNB template can be directly immersed in 4 mol/L KOH aqueous solution at room temperature and atmospheric pressure for 2 h to form a top-down NTNF structure with very high adhesion on titanium substrate.rBMSCs protrudes a large number of slender filamentous pseudopodia on the surface of the structure,and most of the ends of the pseudopodia extend into the nest-like NTNF structure;the cytoskeleton has more stretches,and the elongation of the poles is more obvious;the detection of living dead cells,CCK-8 and ALP showed that rBMSCs have good cell viability,proliferation and differentiation on the surface of NTNF,and increased the expression of genes related to osteogenic differentiation.3.Micro-CT scan and 3D reconstruction analysis showed that the percentage of percent new bone volume(BV/TV),trabecular thickness(Tb.Th)and bone mineral density(BMD)around the titanium rod implant in NTNF group were higher than those in TNT group,while the bone surface/bone volume(BS/BV)ratio was lower than that in TNT group.Fluorescence double labeled hard tissue sections showed that the distance between CA and ARS in NTNF group was significantly larger than that in TNT group.The results of Van Gieson staining showed that there was more new bone formation on the surface of titanium rod in NTNF group than that in TNT group.Conclusions1.TNT can be prepared on titanium surface by electrochemical anodization.After removing TNT by ultrasonic shake,a uniform TNB structure can be obtained,which can significantly increase the specific surface area of titanium surface.Using TNB as a template,honeycomb NT structures with larger loading space and different tube diameters can be prepared by re-anodization,and nested NTNF structures with higher adhesion and stability can also be prepared by alkali etching at room temperature and atmospheric pressure.2.The loading of AgNPs in NT increased the osteogenic and differentiated ability of rBMSCs in vitro and improved its antibacterial properties.3.NTNF structure promotes the early adhesion,vitality,proliferation,osteogenesis-related gene expression and osteogenic differentiation of rBMSCs in vitro,and promotes the osseointegration of titanium rod implants in beagles.4.The methods of electrochemical anodization,ultrasonic shake and alkali etching are simple,economical,efficient,safe and reliable,which is expected to provide a more ideal implant surface modification for clinical usage and provide an experimental foundation for clinical treatment in the future. |
PDF Full Text Request