Design and preparation of Ti-xFe antibacterial titanium alloys based on micro-area potential difference.

Fe was selected as an alloying element for the first time to prepare a new antibacterial titanium alloy based on micro-area potential difference (MAPD) antibacterial mechanism. The microstructure, the corrosion resistance, the mechanical properties, the antibacterial properties and the cell biocompatibility have been investigated in detail by optical microscopy, scanning electron microscopy, electrochemical testing, mechanical property test, plate count method and cell toxicity measurement. It was demonstrated that heat treatment had a significant on the compressive mechanical properties and the antibacterial properties. Ti-xFe (x = 3,5 and 9) alloys after 850 °C/3 h + 550 °C/62 h heat treatment exhibited strong antimicrobial properties with an antibacterial rate of more than 90% due to the MAPD caused by the redistribution of Fe element during the aging process. In addition, the Fe content and the heat treatment process had a significant influence on the mechanical properties of Ti-xFe alloy but had nearly no effect on the corrosion resistance. All Ti-xFe alloys showed non-toxicity to the MC3T3 cell line in comparison with cp-Ti, indicating that the microzone potential difference had no adverse effect on the corrosion resistance, cell proliferation, adhesion, and spreading. Strong antibacterial properties, good cell compatibility and good corrosion resistance demonstrated that Ti-xFe alloy might be a candidate titanium alloy for medical applications.

Immunomodulatory effect of Ti-Cu alloy by surface nanostructure synergistic with Cu2+ release.

The inflammatory response induced by implant/macrophage interaction has been considered to be one of the vital factors in determining the success of implantation. In this study, TiCuNxOy coating with an immunomodulatory strategy was proposed for the first time, using nanostructured TiCuNxOy coating synthesized on Ti-Cu alloy by oxygen and nitrogen plasma-based surface modification. It was found that TiCuNxOy coating inhibited macrophage proliferation but stimulated macrophage preferential activation and presented an elongated morphology due to the surface nanostructure. The most encouraging discovery was that TiCuNxOy coating promoted the initial pro-inflammatory response of macrophages and then accelerated the M1-to-M2 transition of macrophages via a synergistic effect of fast-to-slow Cu2+ release and surface nanostructure, which was considered to contribute to initial infection elimination and tissue healing. As expected, TiCuNxOy coating released desirable Cu2+ and generated a favorable immune response that facilitated HUVEC recruitment to the coating, and accelerated proliferation, VEGF secretion and NO production of HUVECs. On the other hand, it is satisfying that TiCuNxOy coating maintained perfect long-term antibacterial activity (≥99.9%), mainly relying on Cu2O/CuO contact sterilization. These results indicated that TiCuNxOy coating might offer novel insights into the creation of a surface with immunomodulatory effects and long-term bactericidal potential for cardiovascular applications.

Titanium alloys with varying surface micro-area potential differences have antibacterial abilities and a favorable cellular response.

OBJECTIVES: Surface micro-area potential difference (MAPD) can achieve bacteriostatic performance independent of metal ion dissolution. To study the influence of MAPD on antibacterial properties and the cellular response, Ti-Ag alloys with different surface potentials were designed and prepared by changing the preparation and heat treatment processes. MATERIALS AND METHODS: Ti-Ag alloys (T4, T6, and S) were prepared by vacuum arc smelting, water quenching, and sintering. Cp-Ti was set as a control group in this work. The microstructures and surface potential distributions of the Ti-Ag alloys were analyzed by SEM and energy dispersive spectrometry. Plate counting and live/dead staining methods were used to evaluate the antibacterial properties of the alloys, and the mitochondrial function, ATP levels, and apoptosis were assessed in MC3T3-E1 cells to analyze the cellular response. RESULTS: Due to the formation of the Ti-Ag intermetallic phase in the Ti-Ag alloys, Ti-Ag (T4) without the Ti-Ag phase had the lowest MAPD, Ti-Ag (T6) with a fine Ti2Ag phase had a moderate MAPD, and Ti-Ag (S) with a Ti-Ag intermetallic phase had the highest MAPD. The primary results demonstrated that the Ti-Ag samples with different MAPDs exhibited different bacteriostatic effects, ROS expression levels, and apoptosis-related protein expression levels in cells. The alloy with a high MAPD exhibited a strong antibacterial effect. A moderate MAPD stimulated cellular antioxidant regulation (GSH/GSSG) and downregulated the expression of intracellular ROS. MAPD could also promote the transformation of the inactive mitochondria to biologically active mitochondria by increasing the ΔΨm and reducing apoptosis. CONCLUSION: The results here indicated that moderate MAPD not only had bacteriostatic effects but also promoted mitochondrial function and inhibited cell apoptosis, which provides a new strategy to improve the surface bioactivity of titanium alloys and a new idea for titanium alloy design. CLINICAL RELEVANCE: There are some limitations of the mechanism of MAPD. However, researchers will become increasingly aware of the advantages and disadvantages of MAPD and MAPD might provide an affordable solution of peri-implantitis.

A high-hydrophilic Cu2O-TiO2/Ti2O3/TiO coating on Ti-5Cu alloy: Perfect antibacterial property and rapid endothelialization potential.

In order to make novel antibacterial Ti-Cu alloy more suitable for cardiovascular implant application, a Cu-containing oxide coating was manufactured on Ti-Cu alloy by plasma-enhanced oxidation deposition in plasma enhanced chemical vapor deposition (PECVD) equipment to further improve the antibacterial ability and the surface bioactivity. The results of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and water contact angle indicated that a sustainably high-hydrophilic Cu2O-TiO2/Ti2O3/TiO coating with nano-morphology on Ti-5Cu was successfully constructed. The corrosion performance results showed that the coating enhanced the corrosion resistance while releasing more Cu2+, compared with Ti-5Cu. Antibacterial tests confirmed the perfect antibacterial property of the coating (R ≥ 99.9 %), superior to Ti-Cu alloy (R > 90 %). More delightfully, it was observed by phalloidin-FITC and DAPI staining that the coating improved the early adhesion of HUVEC cells mainly due to strong hydrophilicity and nano-morphology. It was demonstrated that the extract of the coated sample significantly promoted proliferation (RGR = 112 %-138 % after cultivation for 1 to 3 days) and migration of HUVEC cells due to the appropriate Cu2+ release concentration. Hemolysis assay and platelet adhesion results showed that the coating had excellent blood compatibility. All results suggested that the coating on Ti-Cu alloy might be a promising surface with the perfect antibacterial ability, blood compatibility and evident promoting endothelialization ability for the cardiovascular application.

Feasibility study on Ti-15Mo-7Cu with low elastic modulus and high antibacterial property.

Titanium and titanium alloy with low density, high specific strength, good biological, excellent mechanical compatibility and easy to process have been widely used in the medical materials, but their application in orthopedics and dentistry often face bacterial infection, corrosion failure and stress shielding. In this paper, Ti-15Mo-7Cu (TM-7Cu) alloy was prepared by high vacuum non-consumable electric arc melting furnace and then treated by solution and aging treatment. The microstructure, mechanical properties, antibacterial properties and cytocompatibility were studied by X-ray diffraction, microhardness tester, electrochemical working station, antibacterial test and Live/Dead staining technology. The results have shown that the heat treatment significantly influenced the phase transformation, the precipitation of Ti2Cu phase, the elastic modulus and the antibacterial ability. With the extension of the aging time, the elastic modulus slightly increased and the antibacterial rate obviously increased. TM-7Cu alloy with a low elastic modulus of 83GPa and a high antibacterial rate of > 93% was obtained. TM-7Cu alloy showed no cytotoxicity to MC3T3. It was suggested that TM-7Cu might be a highly competitive medical material.

A novel Ti-Au alloy with strong antibacterial properties and excellent biocompatibility for biomedical application.

In order to avoid the toxic and side effects on human body of long-term dissolution of metal ions from antibacterial titanium alloys, Au element with non-toxicity and non-side effect was selected as the alloying element to prepare a new Ti-Au alloy with strong antibacterial property. We produced Ti-Au(S) sintered alloy by powder metallurgy and Ti-Au ingot alloy by ingot metallurgy, and investigated the influence of the secondary phase on the relative antimicrobial properties and antibacterial mechanism in this work. The results indicated that the aged Ti-Au(T6) alloy and Ti-Au(S) sintered alloy exhibited strong antibacterial rate against S. aureus due to the formation of Ti3Au phases. In vitro cell culture (MC3T3 cells) experiments showed that Ti-Au alloys had good cytocompatibility and osteogenic properties. The following viewpoints of antibacterial mechanism are that the Ti3Au destroyed the ROS homeostasis of bacteria, causing oxidative stress in bacterial cells and preventing from the biofilms formation.

Antibacterial effect of TiAg alloy motivated by Ag-containing phases.

The precipitates in Ti-Ag alloy made an important contribution to antibacterial activity. In order to study this specific effects, Ti-Ag samples with different forms of precipitates were produced by powder metallurgy and ingot metallurgy followed by heat treatment: Ti-Ag(T4) with no precipitate, Ti-Ag(as-cast) and Ti-Ag(T6) with Ti2Ag and Ti-Ag(PM) with Ti2Ag and Ag-rich phase. Microstructure was analyzed by scanning electronic microscope (SEM), and the antibacterial effects, expression of reactive oxygen species (ROS), protein leakage and biocompatibility were investigated by plate count method, staining technology and cell test. The antibacterial ability was in the following order from low to high: Ti-Ag(T4) < Ti-Ag(as-cast) < Ti-Ag(T6) < Ti-Ag(PM). It was elucidated that Ag-containing phase was the major controlling factor of Ti-Ag antibacterial property and Ti-Ag(PM) with micro-size Ti2Ag and Ag-rich phase exhibited high antibacterial activity. It was proposed that the existence of Ag-containing phases induced high expression of ROS in bacteria, which destroyed the homeostasis of the bacteria and eventually leads to the rupture of the bacterial membrane. Cell test indicated that Ti-Ag samples had no adverse effect on cells and had good biocompatibility.

Development of a low elastic modulus and antibacterial Ti-13Nb-13Zr-5Cu titanium alloy by microstructure controlling.

In order to prepare a titanium with a low elastic modulus and good antibacterial property to meet the requirements as a biomedical material, Ti-13Nb-13Zr-5Cu (TNZ-5Cu) alloy was prepared by high vacuum consume electric arc melting furnace and then subjected to a solution treatment at 950 °C followed by a short-term aging treatment at 600 °C, for 15 min, 30 min, 1 h and 2 h, respectively. The microstructure, mechanical property, antibacterial property and biocompatibility of TNZ-5Cu were investigated in detail. The research results have shown that the solid solution treated alloy was mainly composed of ß-phase and α″-phase, while the aged alloys of ß-phase, α″-phase, α-phase and Ti2Cu. Compared with Ti-13Nb-13Zr alloy (65 GPa) and Ti-6Al-4 V alloy (111 GPa), the elastic modulus of TNZ-5Cu alloy after solution treatment was about 72 GPa and increased with the aging treatment up to 85 GPa, and the hardness was maintained at a higher level than that of Ti-13Nb-13Zr alloys (288 HV). The bacteria plate count results showed that the antibacterial ability of TNZ-5Cu alloy increased with the extension of the aging duration from <60% at 15-30 min to >90% at 1-2 h. Cell experiments showed that all TNZ-5Cu alloy had good cell compatibility. The low modulus and the antibacterial property could provide potential to avoid stress shield and device-related inflection in the clinical application.

The osteoimmunomodulatory effect of nanostructured TiFx/TiOxcoating on osteogenesis induction.

Macrophages play a central role in the host response and the integration of implant materials. The nanostructured TiFx/TiOxcoating (FOTi) on titanium surfaces has shown multiple properties, including antibacterial properties and bioactivity. However, little is known about the effects of these coatings on the regulation of macrophage activity and the subsequent immunomodulatory effects on osteogenesis. In this study, the behavior of macrophages on the FOTi samples was evaluated, and conditioned medium was collected and used to stimulate MC3T3-E1 cellsin vitro. The results showed that the FOTi samples stimulated macrophage elongation and promoted the production of proinflammatory cytokines at 24 h, while induced macrophage polarization to the anti-inflammatory M2 phenotype at 72 h. Furthermore, the immune microenvironment generated by macrophage/ FOTi samples interactions effectively promoted the osteogenic differentiation of MC3T3-E1 cells, as evidenced by improved cell adhesion, enhanced alkaline phosphatase activity and extracellular matrix mineralization, and upregulated osteogenesis-related gene expression. In summary, the FOTi samples mediated macrophage phenotype behaviors and induced beneficial immunomodulatory effects on osteogenesis, which could be a potential strategy for the surface modification of bone biomaterials.

Antibacterial metals and alloys for potential biomedical implants.

Metals and alloys, including stainless steel, titanium and its alloys, cobalt alloys, and other metals and alloys have been widely used clinically as implant materials, but implant-related infection or inflammation is still one of the main causes of implantation failure. The bacterial infection or inflammation that seriously threatens human health has already become a worldwide complaint. Antibacterial metals and alloys recently have attracted wide attention for their long-term stable antibacterial ability, good mechanical properties and good biocompatibility in vitro and in vivo. In this review, common antibacterial alloying elements, antibacterial standards and testing methods were introduced. Recent developments in the design and manufacturing of antibacterial metal alloys containing various antibacterial agents were described in detail, including antibacterial stainless steel, antibacterial titanium alloy, antibacterial zinc and alloy, antibacterial magnesium and alloy, antibacterial cobalt alloy, and other antibacterial metals and alloys. Researches on the antibacterial properties, mechanical properties, corrosion resistance and biocompatibility of antibacterial metals and alloys have been summarized in detail for the first time. It is hoped that this review could help researchers understand the development of antibacterial alloys in a timely manner, thereby could promote the development of antibacterial metal alloys and the clinical application.

Co-Cr-Mo-Cu alloys for clinical implants with osteogenic effect by increasing bone induction, formation and development in a rabbit model.

BACKGROUND: Co-Cr-Mo alloy has been widely used in clinical implants because of its excellent mechanical and anti-corrosion properties, but there is an urgent need to address its disadvantages, such as implant-related infections and implant loosening. We synthesized Co-Cr-Mo-Cu (Co-Cu) alloys with different Cu contents to modify implant performance to be suitable as a bone-compatible implant material. METHODS: Microstructure, phase content and mechanical properties of the Co-Cr-Mo alloy were characterized. Histological and immunohistochemical analyses were performed after implantation in rabbits. The experimental alloy was implanted on the lateral side of the lower tibial condyle and the tibial nodule. RESULTS: Phase content and mechanical properties revealed that the crystallographic structure and wear resistance were changed. Experimental implantation results demonstrated that osteogenic capability was markedly enhanced, ascribed to the excellent antibacterial and osseointegration capacities of Cu phases, and with the release of Cu ions. In particular, Co-Cu alloy containing 2 wt% Cu exhibited the best osteogenic performance among all samples. CONCLUSIONS: The results indicated that osteogenic performance of the Co-Cr-Mo alloy could be enhanced by adding Cu. In particular, the Co-2Cu alloy exhibited the best properties according to both immunohistochemical and histological analyses. Our study not only provides deep insight into the osteogenic effect of Cu but presents a new Co-Cu alloy for clinical implants.

Effect of ultrasonic micro-arc oxidation on the antibacterial properties and cell biocompatibility of Ti-Cu alloy for biomedical application.

In order to improve antibacterial properties and cell biocompatibility of Ti-Cu alloy, an ultrasonic micro-arc oxidation (UMAO) has been applied to Ti-Cu alloy. The corrosion resistance, antibacterial activity and cell compatibility of Ti-Cu alloy before and after UMAO were studied in detail by means of electrochemical test, plate count method and CCK-8 test scanning electron microscopy (SEM) technology to evaluate the application possibilities of UMAO as a surface bio-modification method for Ti-Cu alloy. The surface microstructure characterisation showed that a typical porous coating with a pore diameter of 3-8 µm and a thickness of 5-15 µm was formed on the surface of the Ti-Cu alloy, which significantly improved the surface roughness and hydrophilicity. The plate count method demonstrated that UMAO coatings on Ti-Cu alloy showed strong antibacterial activity (≥99%) against Staphylococcus aureus (S. aureus) even after being immersed in a physiological saline for up to 20 days, indicating that UMAO-treated Ti-Cu alloy had very strong long-term antibacterial properties. It is believed that the strong long-term antimicrobial properties of Ti-Cu-UMAO samples were mainly due to the formation of Cu2O and CuO in UMAO coatings. The results of cell compatibility evaluation showed that UMAO treatment did not bring about cytotoxicity but improved the early adhesion of MC3T3 cell.

A potential strategy for in-stent restenosis: Inhibition of migration and proliferation of vascular smooth muscle cells by Cu ion.

The in-stent restenosis (ISR) often happens after the implantation of metal stents, including both bare metal stents (BMSs) and drug-eluting stents (DESs). Drug release from DESs could reduce significantly the occurrence of ISR but also suppress the revascularization and cause thrombosis. In this study, the effect of Cu ion in a range of 0 to 500 µM on the migration and proliferation of rat aortic smooth muscle cells (RASMCs) was investigated by a series of in vitro experiments including wound-healing assay, cell viability assay and flow cytometric analysis. It has been found that the critical concentration of Cu ion should be at least 250 µM in order to significantly inhibit the migration of RASMCs and the proliferation of RASMCs were impeded by every dose of Cu ion used in this study. In addition, the protein level of caspase-3 was upregulated by 250 µM and 500 µM Cu2+ exposure, which might be the main reason for RASMCs apoptosis. Thus, it is proposed that ISR might be prevented by the constant release of Cu ion.

What controls the antibacterial activity of Ti-Ag alloy, Ag ion or Ti2Ag particles?

Antibacterial metal materials, including Cu- and Ag-containing alloy, have attracted much attention worldwide. As for the antibacterial mechanism of the antibacterial alloys, there are two different views: metal ions sterilization and contact sterilization. For the purpose of revealing the key control factor, Titanium-silver (Ti-Ag) alloys with different silver contents were prepared and a surface acid etching was applied to change the silver ion release and the volume fraction of Ti2Ag on the surface. The microstructure, phase composition, elemental composition, surface roughness, hydrophilicity, silver ion release and antibacterial properties of Ti-Ag alloys were studied comprehensively by X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), contact angle and roughness test, silver ion dissolution test and antibacterial test. The results have shown that the increasing of Ag content did not bring about any change to the surface roughness and hydrophilicity but enhanced the Ag ion release while the surface acid etching improved the hydrophilicity, enhanced the Ag ion release and made more Ti2Ag particles appear on the surface. The antibacterial experiments have shown that the antibacterial properties increased with the increasing of Ag content in Ti-Ag alloys and that the surface acid etching improved the antibacterial activity significantly. The calculated results and surface microstructure observation and XPS analysis demonstrated that the antibacterial activity of Ti-Ag alloys was mainly controlled by Ti2Ag particle in a contact sterilization mode. Silver ion release from Ti-Ag alloy also contributed to antibacterial activity of Ti-Ag, but the Ag ion sterilization was not the key antibacterial mechanism. Finally, the CCK-8 results showed that all Ti-Ag alloys exhibited good cell compatibility.

Effect of heat treatment on the bio-corrosion properties and wear resistance of antibacterial Co-29Cr-6Mo-xCu alloys.

Co-Cr-Mo alloys have been widely used in hip implants due to their good corrosion resistance and good wear resistance. However, complaint is still raising due to infection and inflammation. The addition of Cu has been proven to be an effective way to develop a new kind of Co-based alloy with good antibacterial properties. In this paper, the effect of heat treatment on the corrosion property, the tribology property and the antibacterial property of Cu containing Co-based alloys were investigated in detail. The microstructure observation showed that the as-cast alloys mainly consisted of a dendritic matrix with carbide dispersion at grain boundaries and a fine Cu-rich phase in the matrix and at the carbide/matrix interface. The carbide precipitates and the distribution of Cu phases affected significantly the friction coefficient and wear resistance of Co-xCu alloy. Annealing at 1060 °C/24 h promoted the precipitation of carbide and in turn increased the hardness and wear resistance markedly. Heat treatments, including annealing, solid solution and ageing treatment, enhanced the corrosion resistance of Co-xCu alloy without reduction in antibacterial properties. However, the addition of Cu increased the corrosion resistance and antibacterial properties but reduced the wear resistance especially at high Cu content.

In vivo antibacterial property of Ti-Cu sintered alloy implant.

In-vivo antibacterial property of Ti10Cu sintered alloy was investigated in comparison with pure titanium (cp-Ti) by implanting the alloys with S. aureus suspension in the muscles of rabbits. The general appearance, the white blood cell (WBC) number, the bacteria number were checked and the pathological examination were analyzed. It has been shown that serious inflammation at day 4 and fester at day 14 were observed after implantation in cp-Ti group while only mild infection was observed at day 4 in the case of Ti10Cu implants. Bacterial incubation results have also shown that lots of S. aureus were found in cp-Ti group at all intervals while only several bacteria at day 1 and day 4 and no bacteria after 7 days postimplantation can be found in Ti10Cu group. All these results demonstrate the strong in vivo antibacterial property of Ti10Cu alloy. The strong antibacterial property suggests that Ti10Cu alloy might have potential application in orthopedic surgery and dental implant to reduce the implant-related infection or inflammation.

Anti-bacterium influenced corrosion effect of antibacterial Ti-3Cu alloy in Staphylococcus aureus suspension for biomedical application.

Titanium and titanium alloys have been widely used as dental and orthopedic materials. The infection and the bacterium influenced corrosion both are concerned problems. Ti-3Cu alloy exhibits strong antibacterial properties against E. coli and S. aureus. The strong antibacterial properties of Ti-3Cu provides with a potential new method to reduce the bacterium influenced corrosion. S. aureus suspension was selected to simulate a serious bacterial condition. The corrosion behavior of Ti-3Cu alloy in S. aureus suspension was investigated by an electrochemical testing and an immersion test in comparison with pure titanium. Electrochemical results showed that Ti-3Cu exhibited a much better anti-corrosion property than cp-Ti in S. aureus suspension. Surface observation demonstrated that no corrosion pit was observed on Ti-3Cu alloy after 30 days immersion in the suspension while lots of corrosion pits were found on cp-Ti. The biofilm formation on the surface was observed by scanning electronic microscopy (SEM) in different periods. It has been revealed that S. aureus could grow and gather on the surface of cp-Ti to form biofilm after 18 h immersion, but only several bacteria were found on Ti-3Cu alloy even after 24 h immersion, displaying that Ti-3Cu alloy exhibits very strong anti-adhesion properties against S. aureus. It was concluded that Ti-3Cu performs a super anti-corrosion property due to the strong anti-adhesion property, in which Ti2Cu precipitate plays a critical role.

Antibacterial activities and biocompatibilities of Ti-Ag alloys prepared by spark plasma sintering and acid etching.

In this work, Ti-Ag sintered alloys (Ti-Ag) with different Ag contents were prepared by a spark plasma sintering (SPS) technique, which is a new, efficient and convenient method of powder metallurgy. The Ti-Ag samples were then treated with a mixture of 40 wt% hydrofluoric acid (HF) and nitric acid (HNO3). The surface features, ion release, antibacterial activities and biocompatibilities of the acid-etched Ti-Ag (Ti-Ag(AE)) samples were systematically characterized. The surface characterization results revealed the formation of micropits and particles with high Ag contents. Antibacterial experiments demonstrated that the antibacterial ratios of the Ti-Ag samples increased significantly after the acid etching treatment, and the particles with high Ag contents are thought to play a key role in the antibacterial mechanism. Biocompatibility experiments indicated that the cell proliferation, cell morphology, and osteoblastic differentiation did not significantly differ between the pure titanium (cp-Ti) and Ti-Ag(AE) samples. The Ti-Ag(AE) samples with 3 wt% and 5 wt% Ag not only possessed sustained antibacterial activities for at least 30 days but also did not have impaired biocompatibility.