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.

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.

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.

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.

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.

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.

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.

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.

Effect of Different Titanium Surface Treatments on the Adhesion Test Result:Dental Application

The aim of the study was to investigate the effect of different surface treatment of titanium(Ti)on the adhesion test results for dental application.Ti substrates roughened by 400 to 1500-grit SiC polish papers and alumina blasting,alkali treated by 5 molar(M)NaOH and KOH solutions and heat treated at the temperature range of 400-800℃were used in this study.The treated samples were subjected to the adhesion test.According to the results of the adhesion test,the adhesive strength showed the highest value for the blasted titanium among all polished and blasted samples.The Ti samples heated at 650℃showed the highest adhesive strength among all heat-treated samples.Further,the adhesion test results indicated the higher adhesive strength of chemically treated samples treated by NaOH rather than that by KOH.The polished and heated Ti samples showed the highest adhesive strength among all samples.

Finite element analysis of stress at implant-bone interface of dental implants with different structures

The effect of structure,elastic modulus and thickness of lower modulus layer in porous titanium implants on the stress distribution at the implant-bone interface was investigated.Three-dimensional finite element models of different titanium implants were constructed.The structures of the implants included the whole lower modulus style （No.1）,bio-mimetic style （No.2）,the whole lower modulus style in cancellous bone （No.3） and the whole dense style No.4.The stress distributions at bone-implant interface under static loading were analyzed using Ansys Workbench 10.0 software.The results indicated that the distribution of interface stress is strongly depended on the structure of the implants.The maximum stresses in cancellous bone and root region of implant No.2 are lower than those in the other three implants.A decrease in the modulus of the low modulus layer facilitates the interface stress transferring.Increasing the thickness of the low modulus layer can reduce the stress and induce a more uniform stress distribution at the interface.Among the four implants,biomimetic style implant No.2 is superior in transferring implant-bone interface stress to surrounding bones.

Early Osseointegration of Implants with Cortex-like TiO2 Coatings Formed by Micro-arc Oxidation:A Histomorphometric Study in Rabbits

In our previous studies, a novel cortex-like Ti O2 coating was prepared on Ti surface through micro-arc oxidation（MAO） by using sodium tetraborate as electrolyte, and the effects of the coating on cell attachment were testified. This study aimed to investigate the effects of this cortex-like MAO coating on osseointegration. A sand-blasting and acid-etching（SLA） coating that has been widely used in clinical practice served as control. Topographical and chemical characterizations were conducted by scanning electron microscopy, energy dispersive X-ray spectrometer, X-ray diffraction, contact angle meter, and step profiler. Results showed that the cortex-like coating had microslots and nanopores and it was superhydrophilic, whereas the SLA surface was hydrophobic. The roughness of MAO was similar to that of SLA. The MAO and SLA implants were implanted into the femoral condyles of New Zealand rabbits to evaluate their in-vivo performance through micro-CT, histological analysis, and fluorescent labeling at the bone-implant interface four weeks after surgery. The micro-CT showed that the bone volume ratio and mean trabecular thickness were similar between MAO and SLA groups four weeks after implantation. Histological analysis and fluorescent labeling showed no significant differences in the bone-implant contact between the MAO and SLA surfaces. It was suggested that with micro/nanostructure and superhydrophilicity, the cortex-like MAO coating causes excellent osseointegration, holding a promise of an application to implant modification.

Hydroxyapatite/β-tricalcium Phosphate Composite for Guiding Bone Tissue Growth into a Titanium Tube in 8 mm Dog Tibia Cavity Defects

We developed a fixation method and evaluate bone regrowth in the cavities of a Ф4 mm× 8 mm titanium（Ti）tube through porous hydroxyapatite（HAP）/β-tricalcium phosphate（β-TCP）composite filling（group A）,chitosan/calcium phosphate composite filling（group B）,and HAP particle modification（group C）.After 2 and 5 months of implantation in dog tibia defects,new bone formation in the three groups was studied by histology and histomorphometry.Group A displayed the most bone regenerated area in both 2 and 5 months post-operation.The chitosan/calcium phosphate composite in group B mostly degraded 2 months after implantation,leading to fibrous tissue invasion after 5 months.By contrast,less bone formation was observed in group C.These results indicated that filling the cavities of metalprostheses with a porous HAP/β-TCP composite can be used for stable long-term fixation in clinicalsettings.

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.

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.

Influence of Zr content on microstructure and mechanical properties of implant Ti-35Nb-4Sn-6Mo-xZr alloys

The influence of Zr content on the microstructure and mechanical properties of implant Ti-35Nb-4Sn-6Mo-xZr （x=0, 3, 6, 9, 12, 15; mass fraction） alloys was investigated. It is shown that Ti-35Nb-4Sn-6Mo-xZr alloys appear to have equiaxed single β microstructure after solution treatment at 1023 K. It is found that the grains are refined first and then coarsened with the increase of Zr content. It is also found that Zr element added to titanium alloys has both the solution strengthening and fine-grain strengthening effect, and affects the lattice parameters. With increasing the Zr content of the alloys, the strength increases, the elongation decreases, whereas the elastic modulus firstly increases and then decreases. The mechanical properties of Ti-35Nb-4Sn-6Mo-9Zr alloy are as follows： σb=785 MPa, δ=11%, E=68 GPa, which is more suitable for acting as human implant materials compared to the traditional implant Ti-6Al-4V alloy.

Feasibility of a Chronic Foreign Body Infection Model Studying the Influence of TiO2 Nanotube Layers on Bacterial Contamination

Bacterial infections on the surface of medical devices are a significant problem in therapeutic approach, especially when implants are used in the living. In cardiology, pacemaker generator pocket surfaces, made in titanium alloy can be colonized by pathogen microorganism. This contamination represents a major risk of sepsis, endocarditis and localized infections for patients. A way to limit this bacterial contamination is to modify the surface topography using nano-structuration process of the titanium alloy surface of the implanted devices. The aim of this study is to evaluate the influence of TiO2 nanotube layers on bacterial infection in the living, considering the feasibility of an animal model of chronic foreign body infection. TiO2 nanotube layers prepared by electrochemical anodization of Ti foil in 0.4 wt% hydrofluoric acid solution were implanted subcutaneously in Wistar rats. Three weeks after implantation, TiO2 implants were contaminated by a Staphylococcus epidermilis strain using two different concentrations at 106 and 108 colony forming unit (CFU) in order to induce a sufficient infection level and to avoid unwanted over infection consequences on rats health during the experiments. After 28 days in the living, 75% of nanotube layers initially submitted to the 108 CFU inoculum were contaminated while only 25% nanotube layers initially submitted to the 106 CFU inoculum remained infected. This significant result underlines the influence of TiO2 nanotube layers in decreasing the infection level. Our in vitro experiments showed that the synthesized TiO2 nanotubes indeed decreased the Staphylococcus epidermilis adhesion compared to unanodized Ti foil.

A homogeneous dopamine-silver nanocomposite coating:striking a balance between the antibacterial ability and cytocompatibility of dental implants

Silver has been widely used for surface modification to prevent implant-associated infections.However,the inherent cytotoxicity of silver greatly limited the scope of its clinical applications.The construction of surfaces with both good antibacterial properties and favorable cytocompatibility still remains a challenge.In this study,a structurally homogeneous dopamine-silver(DA/Ag)nanocomposite was fabricated on the implant surface to balance the antibacterial activity and cytocompatibility of the implant.The results show that the DA/Ag nanocomposites prepared under the acidic conditions(pH=4)on the titanium surface are homogeneous with higher Ag^(+)content,while an obvious core(AgNPs)-shell(PDA)structure is formed under neutral(pH=7)and alkaline conditions(pH=10),and the subsequent heat treatment enhanced the stability of PDA-AgNPs nanocomposite coatings on porous titanium.The antibacterial test,cytotoxicity test,hypodermic implantation and osteogenesis test revealed that the homogeneous PDA-AgNPs nanocomposite coating achieved the balance between the antibacterial ability and cytocompatibility,and had the best outcomes for soft tissue healing and bone formation around the implants.This study provides a facile strategy for preparing silver-loaded surfaces with both good antibacterial effect and favorable cytocompatibility,which is expected to further improve the therapeutic efficacy of silver composite-coated dental implants.

Improvement of Systemic Symptoms after Dental Implant Removal

Dental implants have spread worldwide in dentistry. The risks and complications reported are limited to local issues. However, in this case, a patient complained of systemic symptoms after dental implantation. This case report aims to demonstrate that systemic symptoms such as lumbago, shoulder stiffness, neck pain, hip joint pain, and facial pain improved after the removal of well-osseointegrated titanium implants. The results suggest that harmful electromagnetic waves received by implants affect body conditions;however, to date, the underlying mechanisms have not been identified. Therefore, further research is required.