Elimination of methicillin‑resistant Staphylococcus aureus biofilms on titanium implants via photothermally‑triggered nitric oxide and immunotherapy for enhanced osseointegration

Background:Treatment of methicillin-resistant Staphylococcus aureus(MRSA)biofilm infections in implant placement surgery is limited by the lack of antimicrobial activity of titanium(Ti)implants.There is a need to explore more effective approaches for the treatment of MRSA biofilm infections.Methods:Herein,an interfacial functionalization strategy is proposed by the integration of mesoporous polydopamine nanoparticles(PDA),nitric oxide(NO)release donor sodium nitroprusside(SNP)and osteogenic growth peptide(OGP)onto Ti implants,denoted as Ti-PDA@SNP-OGP.The physical and chemical properties of Ti-PDA@SNP-OGP were assessed by scanning electron microscopy,X-ray photoelectron spectroscope,water contact angle,photothermal property and NO release behavior.The synergistic antibacterial effect and elimination of the MRSA biofilms were evaluated by 2′,7′-dichlorofluorescein diacetate probe,1-N-phenylnaphthylamine assay,adenosine triphosphate intensity,O-nitrophenyl-β-D-galactopyranoside hydrolysis activity,bicinchoninic acid leakage.Fluorescence staining,assays for alkaline phosphatase activity,collagen secretion and extracellular matrix mineralization,quantitative real‑time reverse transcription‑polymerase chain reaction,and enzyme-linked immunosorbent assay(ELISA)were used to evaluate the inflammatory response and osteogenic ability in bone marrow stromal cells(MSCs),RAW264.7 cells and their co-culture system.Giemsa staining,ELISA,micro-CT,hematoxylin and eosin,Masson's trichrome and immunohistochemistry staining were used to evaluate the eradication of MRSA biofilms,inhibition of inflammatory response,and promotion of osseointegration of Ti-PDA@SNP-OGP in vivo.Results:Ti-PDA@SNP-OGP displayed a synergistic photothermal and NO-dependent antibacterial effect against MRSA following near-infrared light(NIR)irradiation,and effectively eliminated the formed MRSA biofilms by inducing reactive oxygen species(ROS)-mediated oxidative stress,destroying bacterial membrane integrity and causing leakage of intracellular components(P<0.01).In vitro experiments revealed that Ti-PDA@SNP-OGP not only facilitated osteogenic differentiation of MSCs,but also promoted the polarization of pro-inflammatory M1 macrophages to the anti-inflammatory M2-phenotype(P<0.05 or P<0.01).The favorable osteo-immune microenvironment further facilitated osteogenesis of MSCs and the anti-inflammation of RAW264.7 cells via multiple paracrine signaling pathways(P<0.01).In vivo evaluation confirmed the aforementioned results and revealed that Ti-PDA@SNP-OGP induced ameliorative osseointegration in an MRSA-infected femoral defect implantation model(P<0.01).Conclusions:Ti-PDA@SNP-OGP is a promising multi-functional material for the high-efficient treatment of MRSA infections in implant replacement surgeries.

Effects of pore size and porosity of surface-modified porous titanium implants on bone tissue ingrowth

The effects of surface-modified porous titanium implants with different porosities and pore sizes on osseointegration were investigated in vivo.Three porous titanium implants(A30,A40 and A50 containing volume fractions of space-holder NaCl being 30%,40%and 50%,respectively)were manufactured by metal injection moulding(MIM).The surface-modified implants were implanted into muscles and femurs of hybrid male dogs.Interface osteogenic activity and histological bone ingrowth of porous titanium implants were evaluated at 28,56 and 84 d.The results showed that when additive space-holder amount of NaCl increased from 30%to 50%(volume fraction),the general porosity and mass fraction of macropores of porous titanium rose from 42.4%to 62.0%and from 8.3%to 69.3%,respectively.Histologic sections and fluorescent labeling showed that the A50 implant demonstrated a significantly higher osteogenic capacity at 28 d than other implants.Bone ingrowth into the A30 implant was lower than that into other implants at 84 d.Therefore,the pore structure of A50 implant was suitable for new bone tissue to grow into porous implant.

Bone integration properties of antibacterial biomimetic porous titanium implants

A novel antibacterial biomimetic porous titanium implant with good osseointegration was prepared by freeze-casting and thermal oxidation.Bone integration properties of the porous titanium implant were evaluated by cell proliferation assay,alkaline phosphatase activity assay,X-ray examination and hard bone tissue biopsy.The in vitro cell proliferation and the level of differentiation of the group with a modified nano-porous implant surface were significantly higher than those in the group without surface modification and the dense titanium control group(P<0.05).In vivo,bone growth and osteogenesis were found in the experimental groups with modified and unmodified porous titanium implants;osteoblasts in the modified group had more mature differentiation in the pores compared to the unmodified group.Such implants can form solid,biologically compatible bone grafts with bone tissues,exhibiting good osseointegration.

Osteogenic composite nanocoating based on nanohydroxyapatite, strontium ranelate and polycaprolactone for titanium implants

Titanium and its alloys are commonly used as dental and bone implant materials.Biomimetic coating of titanium surfaces could improve their osteoinductive properties.In this work,we have developed a novel osteogenic composite nanocoating for titanium surfaces,which provides a natural environment for facilitating adhesion,proliferation,and osteogenic differentiation of bone marrow mesenchymal stem cells(MSCs).Electrospinning was used to produce composite nanofiber coatings based on polycaprolactone(PCL),nano-hydroxyapatite(nHAp)and strontium ranelate(SrRan).Thus,four types of coatings,i.e.,PCL,PCL/nHAp,PCL/SrRan,and PCL/nHAp/SrRan,were applied on titanium surfaces.To assess chemical,morphological and biological properties of the developed coatings,EDS,FTIR,XRD,XRF,SEM,AFM,in-vitro cytotoxicity and in-vitro hemocompatibility analyses were performed.Our findings have revealed that the composite nanocoatings were both cytocompatible and hemocompatible;thus PCL/HAp/SrRan composite nanofiber coating led to the highest cell viability.Osteogenic culture of MSCs on the nanocoatings led to the osteogenic differentiation of stem cells,confirmed by alkaline phosphatase activity and mineralization measurements.The findings support the notion that the proposed composite nanocoatings have the potential to promote new bone formation and enhance bone-implant integration.

Review on the Fabrication of Surface Functional Structures for Enhancing Bioactivity of Titanium and Titanium Alloy Implants

Titanium and its alloys have been widely applied in many biomedical fields because of its excellent mechanical properties,corrosion resistance and good biocompatibility.However,problems such as rejection,shedding and infection will occur after titanium alloy implantation due to the low biological activity of titanium alloy surface.The structures with specific functions,which can enhance osseointegration and antibacterial properties,are fabricated on the surface of titanium implants to improve the biological activity between the titanium implants and human tissues.This paper presents a comprehensive review of recent developments and applications of surface functional structure in titanium and titanium alloy implants.The applications of surface functional structure on different titanium and titanium alloy implants are introduced,and their manufacturing technologies are summarized and compared.Furthermore,the fabrication of various surface functional structures used for titanium and titanium alloy implants is reviewed and analyzed in detail.Finally,the challenges affecting the development of surface functional structures applied in titanium and titanium alloy implants are outlined,and recommendations for future research are presented.

Surface modification of titanium implants with micro–nanotopography and NIR photothermal property for treating bacterial infection and promoting osseointegration

A double acid corrosion and subsequent hydrothermal treatment were used to fabricate a micro–nano-structured Ti substrates(Ti–M–N).Afterward,the mesoporous polydopamine(MPDA)nanoparticles as photothermal agent were prepared and immobilized on the surface of Ti–M–N samples,in order to obtain Ti–M–NMPDA sample.Unique micro–nanostructure properties and the photothermal effect of the modified Ti implant caused physical stress on the bacteria and the bacterial membrane damage,and eventually led to bacteria death.More importantly,based on excellent bioactivity and cytocompatibility of mussel-inspired materials,MPDA promoted adhesion,proliferation and osteogenic differentiation of mesenchymal stem cells in vitro.Furthermore,animal experiments in vivo further confirmed that the modified Ti implants could enhance osseointegration.

Antibiotic-loaded amphora-shaped pores on a titanium implant surface enhance osteointegration and prevent infections

Artificial prostheses for joint replacement are indispensable in orthopedic surgery.Unfortunately,the implanted surface is attractive to not only host cells but also bacteria.To enable better osteointegration,a mechanically stable porous structure was created on a titanium surface using laser treatment and metallic silver particles were embedded in a hydrophilic titanium oxide layer on top.The laser structuring resulted in unique amphora-shaped pores.Due to their hydrophilic surface conditions and capillary forces,the pores can be loaded preoperative with the antibiotic of choice/need,such as gentamicin.Cytotoxicity and differentiation assays with primary human osteoblast-like cells revealed no negative effect of the surface modification with or without gentamicin loading.An in vivo biocompatibility study showed significantly enhanced osteointegration as measured by push-out testing and histomorphometry 56 days after the implantation of the K-wires into rat femora.Using a S.aureus infection model,the porous,silver-coated K-wires slightly reduced the signs of bone destruction,while the wires were still colonized after 28 days.Loading the amphora-shaped pores with gentamicin significantly reduced the histopathological signs of bone destruction and no bacteria were detected on the wires.Taken together,this novel surface modification can be applied to new or established orthopedic implants.It enables preoperative loading with the antibiotic of choice/need without further equipment or post-coating,and supports osteointegration without a negative effect of the released dug,such as gentamicin.

Improvement of antibacterial, anti-inflammatory, and osteogenic properties of OGP loaded Co-MOF coating on titanium implants for advanced osseointegration

The bacterial infection,especially for methicillin-resistant Staphylococcus aureus(MRSA),and the associated severe inflammatory response could extremely limit the crosstalk of RAW264.7 cells and mesenchymal stem cells(MSCs)and lead to the undesirable osseointegration of peri–implants.It is highly demanded to modify the surface of titanium(Ti)implant to improve its anti-bacterial and anti-inflammatory properties and facilitate its disabled osseointegration.Herein,in our study,a multifunctional coating of zeolitic imidazolate frameworks-67 encapsulated osteogenic growth peptide(OGP)(ZO)was fabricated on titanium dioxide nanotubes(TNT)substrates(TNT-ZO)via the electrophoresis deposition(EPD)approach.The TNT-ZO substrates exhibited excellent antibacterial activity indicated by the reactive oxygen species(ROS)generation,outer membrane(OM)and inner membrane(IM)permeabilization change,adenosine triphosphate(ATP)decrease,and intracellular compounds(DNA/RNA)leakage.Importantly,the regulation effects of TNT-ZO coating modified titanium substrates on the RAW264.7-MSCs crosstalk could induce the anti-inflammatory and osteogenic microenvironment via multiple paracrine signaling of Runx2,BMP2,VEGF,and TGF-β1.The promoted effects of coating structure were investigated in vivo,including antibacterial effect,osteogenic differentiation of mesenchymal stem cells,and anti-inflammation of RAW264.7 cells,as well as infected bone regeneration and repair in bone defect transplantation model.The results demonstrated that MRSA was effectively eliminated by the hydrolysis of ZIF-67 nanoparticles on TNT-ZO substrates.Furthermore,the excellent osseointegration of peri–implants was realized simultaneously by modulating the crosstalk of RAW264.7-MSCs.This study could provide a novel approach to designing a multifunctional coating on the Ti implants for infected bone regeneration in orthopedic applications.

A HAase/NIR responsive surface on titanium implants for treating bacterial infection and improving osseointegration

Bacterial infection and insufficient osseointegration are critical factors affecting the long-term success of titanium-based implants.Unfortunately,the direct application of antibiotic on Ti implants easily leads to poor cytocompatibility,as well as the production of drug-resistant bacteria.So,in this work,we designed a prospective antibacterial strategy by combining photothermal and ciprofloxacin(CIP).The synergistic effect of photothermal and antibiotic may provide an effective bacteriostatic efficacy without sacrificing osteogenesis at a mild condition of moderate temperature and less antibiotic.Herein,CIP was loaded into mesoporous polydopamine(MPDA)nanoparticles(MPDA@CIP),which were anchored on the surface of titanium and finally covered with sodium hyaluronate-catechol(HAc)coating.The hydrophilic HAc layer could inhibit the early adhesion of bacteria,and some bacteria could secrete bacterial hyaluronidase to accelerate the degradation of HAc.This enabled smart enzyme-triggered release of antimicrobials at the site of infection on-demand and avoided unwanted side effects on normal tissues.In addition,NIR light irradiation had a positive influence on both CIP release and MPDA nanoparticle’s photothermal effect.Moreover,before anchoring MPDA@CIP,by the construction of hydroxyapatite microstructure on Ti sur-face with micro-arc oxidation and alkali heat treatment,the ability of bone formation of Ti could be promoted also.Both in vitro as well as in vivo assays demonstrated that functional Ti has an excellent antibacterial effect and osteogenic ability.

Screening the Optimal Patterned Surfaces Consisting of Cell Morphology Mimicking Micro-pillars and Nanotube Arrays for the Design of Titanium Implants#br#

Micron/nano scale topographic modification has been a significant focus of interest in current titanium(Ti)surface design.However,the influence of micron/nano structured surface on cell or bacterium behavior on the Ti implant has rarely been systematically evaluated.Moreover,except for popular microgrooves,little work has been carried out on the reaction of cells to the bionic structure.In this study,several micro-pillars mimicking cell morphology were prepared on Ti surfaces by lithography and contact printing(ICP)method,and they were further decorated with nanotube arrays by anodization technology.These surface modifications remarkablly increased the surface roughness of pristine Ti surface from 91.17 nm±5.57 nm to be more than 1000 nm,and reduced their water contact angles from 68.3°±0.7°to be 16.9°±2.4°.Then,the effects of these hierarchical micron/nano scale patterns on the behaviors of MG63 osteoblasts,L929 fibroblasts,SCC epithelial cells and P.gingivalis were studied,aiming to evaluate their performance in osseointegration,gingival epithelial sealing and antibacterial ability.Through an innovative scoring strategy,our findings showed that square micro-pillars with 6μm width and 2μm height combined with 85 nm diameter nanotubes was suitable for implant neck design,while square micro-pillars with 3μm width and 3.6μm height combined with 55 nm diameter nanotubes was the best for implant body design.Our study reveals the synergistic effect of the hierarchical micron/nano scale patterns on MG63 osteoblasts,L929 fibroblasts,SCC epithelial cells and P.gingivalis functions.It provides insight into the design of biomedical implant surfaces.

Surface treatment of titanium dental implant with H2O2 solution

The surface treatment is important for titanium and its alloys as promising candidates for dental implantation due to their bioinert surface.Titanium surface samples were modified using H2O2 solution at different times up to 72 h to boost their bioactivity.According to the results of the field emission scanning electron microscopy test,some nanostructures are formed on the surface of treated titanium samples and increased in size by increasing the time of treatment up to 24 h.After 24 h of application,the sharpness of nanostructures decreased and the micro-cracks and discontinuity in the coating surface increased.The results of the X-ray diffraction study and Raman spectroscopy revealed that anatase(TiO2)was formed on the surface of treated titanium samples.The peak intensity of Raman spectroscopy increased with an improvement in treatment time of up to 24 h and then decreased due to the discontinuity of the coating.Full wettability and ability to form apatite were reached at 6 h of treatment.It is clear that the treatment time has a significant effect on the surface treatment of titanium using the H2O2 solution.

Inducing Macrophages M2 Polarization by Dexamethasone Laden Mesoporous Silica Nanoparticles from Titanium Implant Surface for Enhanced Osteogenesis

The study conveys an idea to enhance the osseointegration of titanium implant (Ti) through modulating macrophages M2 polarization. The ?100 nm spherical mesoporous silica nanoparticles (MSN) that compromised of ~4-nm-diameter nano? tunnels were synthesized by the conventional "sol-gel" method, into which the dexamethasone (DEX) was loaded (DEX@ MSN). The DEX@MSN could consistently release DEX and showed favorable cytocompatibility in RAW264.7 cells. The arginase-1 expression, a specific marker for macrophages M2 polarization, was also enhanced by DEX @ MSN treatment. Then, the Ti was pre-treated with anodization under 5 V to generate the titania nanotubes with ?30 nm diameter (NT-30) and the DEX @ MSN was introduced onto NT-30 surface via electrophoretic deposition, with the aid of chitosan. After optimizing the deposition parameters, the supernatants of RAW264.7 from the decorated implant surface could significantly promote the osteogenic differentiation of murine primary bone marrow mesenchymal stem cells. These findings demonstrate that delivery of DEX from implant surface can modulate the macrophages M2 polarization and result in favorable osteogenesis.

Animal Modelling of Lumbar Corpectomy and Fusion and in vivo Growth of Spine Supporting Bone by Titanium Cage Implants: An Experimental Study

In this study a lumbar spinal fusion animal model is established to assess the effect of spinal fusion cage,and explore theminimum area ratio of titanium cage section to vertebral section that ensures bone healing and biomechanical property.Lumbarcorpectomy was conducted by posterolateral approach with titanium cage implantation combined with plate fixation.Titaniumcages with the same length but different diameters were used.After implantation of titanium cages,the progress of bone healingwas observed and the bone biomechanical properties were measured,including deformation and displacement in axial compression,flexion,extension,and lateral bending motion.The factors affecting the in vivo growth of spine supporting body wereanalyzed.The results show that the area ratio of titanium cage section to vertebral section should reach 1/2 to ensure the bonehealing,sufficient bone intensity and biomechanical properties.Some bone healing indicators,such as BMP,suggest that there isa relationship between the peak time and the peak value of bone formation and metabolism markers and the bone healing strength.

Surface modification of titanium using steel slag ball and shot blasting treatment for biomedical implant applications

Surface modification is often performed using grit or shot blasting treatment for improving the performances of biomedical implants. The effects of blasting treatments using steel slag balls and spherical shots on the surface and subsurface of titanium were studied in this paper. The treatments were conducted for 60-300 s using 2-5 mm steel slag bails and 3.18 mm spherical shots. The surface morphology, roughness, and elemental composition of titanium specimens were examined prior to and after the treatments. Irregular and rough titanium surfaces were formed after the treatment with the steel slag balls instead of the spherical shots. The former treatment also introduced some bioactive elements on the titanium surface, but the latter one yielded a harder surface layer. In conclusion, both steel slag ball and shot blasting treatment have their own specialization in modifying the surface of metallic biomaterials. Steel slag ball blasting is potential for improving the osseointegration quality of implants; but the shot blasting is more appropriate for improving the mechanical properties of temporary and load bearing implants, such as osteosynthesis plates.

COMPARISON OF SURFACE PROPERTIES OF Ti-6Al-4V COATED WITH TITANIUM NITRIDE, TiN+TiC+Ti(C,N)/DLC, TiN/DLC AND TiC/DLC FILMS BY PLASMA-BASED ION IMPLANTATION

The surface properties of Ti-6Al-4V alloy coated with titanium nitride, TiN+TiC+Ti(C,N)/DLC (diamond like carbon), TiN/DLC and TiC/DLC films by plasma-based ion implantation (PBII) with nitrogen, PBII with nitrogen then acetylene, PBII with nitrogen then glow discharge deposition with acetylene plus hydrogen and PBII with acetylene then glow discharge deposition with acetylene plus hydrogen respectively were studied. The corresponding films are found getting dimmer, showing light gold or gold, smoky color (uneven), light red in black (uneven), and graphite black separately. The corresponding film resistivities are given. Antioxidation ability of the titanium nitride film is poor, while the existence of carbon (or carbide) improves the antioxidation ability of the films. Having undergone excellent intermediate transitional region of nitrogen and carbon implantation, the top DLC layer of the TiN+TiC+Ti(C,N)/DLC multilayer are formed after the carbon implantation has the best adhesion with the substrate among all the multilayers. Although microhardness of the samples increases in the order of coatings of titanium nitride, TiN/DLC, TiN+TiC+Ti(C,N)/DLC and TiC/DLC, the TiN/DLC and TiC/DLC multilayers have greater brittleness as compared with other films.

Microstructures and mechanical properties of a new titanium alloy for surgical implant application

A new titanium alloy Ti12.5Zr2.5Nb2.5Ta（TZNT） for surgical implant application was synthesized and fully annealed at 700℃for 45 min.The microstructure and the mechanical properties such as tensile properties and fatigue properties were investigated.The results show that TZNT mainly consists of a lot of lamellaα-phase clusters with different orientations distributed in the originalβ-phase grain boundaries and a small amount ofβphases between the lamella a phases.The alloy exhibits better ductility,lower modulus of elasticity,and lower admission strain in comparison with Ti6A14V and Ti6A17Nb,indicating that it has better biomechanical compatibility with human bones.The fatigue limit of TZNT is 333 MPa,at which the specimen has not failed at 10^7 cycles.A large number of striations present in the stable fatigue crack propagation area,and many dimples in the fast fatigue crack propagation area are observed,indicating the ductile fracture of the new alloy.

Corrosion behaviors of a new titanium alloy TZNT for surgical implant application in Ringer’s solution

A new near α-titanium alloy Ti12.5Zr2.5Nb2.5Ta （TZNT） for surgical implants was designed. The potentiodynamic technique was performed to investigate the corrosion behaviors of TZNT in Ringer＇s solution, and Ti6A14V, Ti6Al7Nb, and TA2 were taken as comparison. The structure of the passive film was analyzed using an X-ray photoelectron spectrometer （XPS）. The results indicate that TZNT possesses better corrosion resistance, when compared with Ti6A14V, Ti6A17Nb, and TA2. The passive film formed on the TZNT surface is composed of oxides, such as TiO2, ZrO2, Nb2O5, and Ta2O5. The elements Zr and Ta are rich, whereas Ti and Nb are poor in the passive film. The addition of Zr, Nb, and Ta with relatively low electrochemical reaction potentials can reduce the anode activity and improve passive properties. Other than that, oxides such as ZrO2, Nb2O5, and Ta2O5 with the nobler equilibrium constants make the passive film more stable.

Imaging and Dosimetric Consideration for Titanium Prosthesis Implanted within the Irradiated Region by Cobalt-60 Teletherapy Unit

The aim of this research is to observe dose distributions in the vicinity of titanium prosthetic implants during radiotherapy procedures on 60Co teletherapy machine, Prowess Panther treatment planning system (TPS). Data were obtained using a locally fabricated tissue equivalent phantom CT images with titanium prosthesis which was irradiated with 60Co gamma radiation. Prowess TPS (1.25 MeV) estimated less variations. Proximal ends of the metal recorded slight increase in doses as a result of backscatter with dose increment below acceptable tolerance of ±3%. Doses measured decreases on the distal side of the prosthesis at a distance less than dmax from the plate on each beam energy. The depth dose increases marginally after a certain depth level which generally originated from the unperturbed dose due to increase in the electron fluence. The percentage of depth doses decrease with the increase in plate thickness. A reduction in the above trend was also noticed with an increase in beam energy primarily because scattered photons are more forwardly directed. Prowess TPS (convolution superposition algorithm) was found to be better at reducing dose variation when correction for artifact. Manual calculations on blue phantom data agree with results from Prowess. This treatment system is capable of simulating dose around titanium prosthesis as its range of densities, 0.00121 to 2.83, excludes titanium density (rED for titanium is 3.74).

Effects of Laser Pulse Numbers on Surface Biocompatibility of Titanium for Implant Fabrication

Generally, materials with high biocompatibility are more appropriate for bone and tissue transplant applications, due to their higher effectiveness in the healing process and infection problems. This study presents the effects of laser surface texturing on the surface topography properties, roughness, and wettability of thin titanium sheets, which consequently enhance the biocompatibility of this material. Creating line patterns across the surfaces, the titanium samples are prepared using variety of laser parameters. The apatite inducing ability of each sample is tested through the use of simulated body fluid (SBF). The final biocompatibility level of titanium samples is analyzed through wettability, surface angle measurements, and average surface temperature profile. Overall, the effects of laser parameter, pulse numbers, upon the biocompatibility of titanium are thoroughly examined, with results indicating that a scanning speed of 100 μm/ms results in desirable bone type apatite inducing abilities across the surface of treated titanium sheets.

Fabrication of Micro/Nano-textured Titanium Alloy Implant Surface and Its Infl uence on Hydroxyapatite Coatings

We put forward a protocolcombining laser treatment and acid etching to obtain multiscale micro/nano-texture surfaces of titanium alloy implant.Firstly,the operationalparameters of the laser were optimized to obtain an optimum current.Secondly,the laser with the optimum operationalparameters was used to fabricate micro pits.Thirdly,multiple acid etching was used to clean the clinkers of micro pits and generate submicron and nanoscale structures.Finally,the bioactivity of the samples was measured in a simulated body fluid.The results showed that the micropits with a diameter of 150 μm and depth of 50 μm were built successfully with the optimized working current of 13 A.In addition,submicron and nanoscale structures,with 0.5-2 μm microgrooves and 10-20 nm nanopits,were superimposed on micro pits surface by multiple acid etching.There was thick and dense HA coating only observed on the multiscale micro/nano-textured surface compared with polished and micro-textured surface.This indicated that the multiscale micro/nano-texture surface showed better ability toward HA formation,which increased the bioactivity of implants.