Nano-Modified Titanium Implant Materials: A Way Toward Improved Antibacterial Properties

Titanium and its alloys have superb biocompatibility, low elastic modulus, and favorable corrosion resistance. These exceptional properties lead to its wide use as a medical implant material. Titanium itself does not have antibacterial properties, so bacteria can gather and adhere to its surface resulting in infection issues. The infection is among the main reasons for implant failure in orthopedic surgeries. Nano-modification, as one of the good options, has the potential to induce different degrees of antibacterial effect on the surface of implant materials. At the same time, the nano-modification procedure and the produced nanostructures should not adversely affect the osteogenic activity, and it should simultaneously lead to favorable antibacterial properties on the surface of the implant. This article scrutinizes and deals with the surface nano-modification of titanium implant materials from three aspects: nanostructures formation procedures, nanomaterials loading, and nano-morphology. In this regard, the research progress on the antibacterial properties of various surface nano-modification of titanium implant materials and the related procedures are introduced, and the new trends will be discussed in order to improve the related materials and methods.

Based on the dimension criteria, antibacterial nanomaterials can be divided into four categories:zero-dimensional–nanoparticles, one-dimensional–nanowires, two-dimensional—nanofilms, and three-dimensional–nanoblocks ( Figure 1 ) ( Saleh, 2020 ). Besides, antibacterial nanomaterials can also be classified according to the structural form or antibacterial active ingredients ( Gleiter, 2000 ). FIGURE 1 Schematic illustration of the nanomaterial’s classification based on dimensionality. Classification by the Structural Form Antibacterial nanomaterials based on the structural form can be classified into antibacterial NPs, antibacterial nanosolids, and antibacterial nano-assembled structures. NPs are known as tiny particles in the size range of 1∼100 nm. Their specific structures induce some sort of surface and interface effects such as small size effect, macroscopic quantum tunneling and quantum size effect ( Morris, 2018 ). As a result, nanomaterials with a series of excellent properties enhance the proficiency of nano-antibacterial agents. Compared with ordinary materials, nanomaterials have irreplaceable characteristics, especially in the antibacterial field. Hence it is worthwhile to expand the scope of their applications. Antibacterial nanosolids can be formed by the aggregation of nano-sized antibacterial particles. They can be further divided into bulk, thin-film, and nanofibrous nanomaterials. Antibacterial nano-assembled structures refer to the artificially assembled and synthesized antibacterial nanomaterial systems. These systems are composed of antibacterial nanoparticles, nanofilaments, or tubes as the basic unit. These various nanostructures can be assembled and arranged in one dimensional, two or three-dimensional space to form the desired nanomaterial structures ( Mageswari et al., 2016 ; Khan, 2020 ). Nanomaterials can be synthesized through various methods such as construction and destruction ( Figure 2 ) ( Saleh, 2020 ). On the one hand, NPs can be obtained from the atomic level and then integrated into the desired materials. The methods of this kind of synthesis include self assembly ( Zhang et al., 2019b ; Deng et al., 2020 ), laser pyrolysis ( Laurent et al., 2010 ; Dumitrache et al., 2019 ), condensation ( Sano et al., 2020 ), CVD ( Gutés et al., 2012 ; Tyurikova et al., 2020 ), sol-gel method ( Gonçalves, 2018 ), soft lithography ( Fu et al., 2018 ), hydrothermal methods ( Zhen et al., 2019 ; Moreira et al., 2020 ), microwave methods ( Henam et al., 2019 ), sonochemical ( Gupta and Srivastava, 2019 ; Moreira et al., 2020 ), synthesis using plant extracts ( Ogunyemi et al., 2019 ; Ranoszek-Soliwoda et al., 2019 ), and green synthesis ( Gour and Jain, 2019 ; Irshad et al., 2020 ). On the other hand, macroscopic level materials can be trimmed down to NPs by different methods, including mechanical grinding ( Sviridov et al., 2017 ; Haque et al., 2018 ), ball milling ( Li Y. et al., 2020 ), lithography ( Fu et al., 2018 ), vapor deposition ( Choi et al., 2018 ), arc-plasma deposition ( Ito et al., 2012 ; Takahashi et al., 2015 ), ion beam technique ( Heo and Gwag, 2014 ; Yang J. et al., 2018 ), severe plastic deformation ( Cui et al., 2018 ; Sarkari Khorrami et al., 2019 ), chemical etching ( Wareing et al., 2017 ; Pinna et al., 2020 ), sputtering ( Pišlová et al., 2020 ), and laser ablation ( Pandey et al., 2014 ; Abid et al., 2020 ). FIGURE 2 A schematic representation of preparation procedure for various nanoparticles.

Titanium-based nanostructure (NS) materials have become the focus of current research because of their unique properties in optical, biological, and electrical fields ( Miao et al., 2015 ; Gupta et al., 2018 ; Wei et al., 2020 ). Different preparation processes can be used to construct NS titanium surfaces, common nano-morphologies for titanium surfaces with antibacterial functions are nanotube and nano-coating forms. Titanium surfaces with different physical and chemical properties can influence the biological interactions and the adhesion of cells and bacteria ( Zhou J. et al., 2018 ; Elbourne et al., 2019 ), which in turn affects the ability of early osseointegration and the risk of implant infection. Preparation of Nanotubes Titanium dioxide nanotubes have received extensive attention because of their controllable size and highly ordered surface arrangement. Nanotubes have a larger specific surface area and storage space than other NS forms such as nanorods, nanospikes, and nanowires. These characteristics make it a good candidate among the other NS morphologies for the storage and release of antibacterial agents. The preparation of nanotube structures on the surface of titanium is mainly achieved by three methods: template synthesis ( Jung et al., 2002 ; Lee et al., 2005 ), electrochemical anodization ( Jun et al., 2012 ; Roman et al., 2014 ; Cao S. et al., 2018 ; Shang et al., 2019 ; Zhang et al., 2019d ), and hydrothermal treatment ( Tsai and Teng, 2004 , 2006 ), they have different advantages and disadvantages listed in Table 1 . TABLE 1 Different nanotube formation processes. Preparation technique Shape and size Characteristics References Template synthesis (1) Tubular arrays or loose aggregates (2) Diameter:10∼500 nm (3) Length: Nanometer to micrometer (1) Nanotubes with different diameters can be prepared (2) Removing the template may destroy the morphology of the nanotubes Choi et al., 2017 ; Luo et al., 2018 Electrochemical anodization (1) Highly ordered array of nanotubes (2) Diameter:10∼500 nm (3) Length:100 nm∼100 μm (1) Highly ordered (2) Low degree of aggregation Fathy Fahim et al., 2009 ; Zhao et al., 2014 Hydrothermal treatment (1) Single or loose block tube (2) Diameter:2∼20 nm (3) Length: nanometer to micrometer (1) Simple process (2) It can prepare small diameter nanotubes (3) Difficult to form nanotube arrays Wang L. et al., 2014 ; Aal et al., 2015 Template Synthesis The template synthesis method can be categorized into a hard template and a soft template method according to the nature of templating agent. The hard template method uses columnar single-crystal anodized aluminum oxide (AAO) ( Hoyer, 1996 ; Ma, 2007 ; Newland et al., 2018 ) as a template to prepare nanotube structures by electrochemical deposition. The preparation process for the hard template is complicated, and the shape and size of the nanotubes are dependent on the size and shape of the template hole. Besides, the nanotubes destruction is possible during the separation step from the matrix template so unfortunately, it has poor reproducibility. In the soft template procedure surfactants used as templates ( Bernal et al., 2012 ; Choi et al., 2017 ). Firstly, the surfactant should be mixed with water, titanium alkoxide, and other substances, then the polymerization takes place under certain conditions. After drying and calcination, the nanotube structure is prepared. The soft template method overcomes the shape and size dependence to the template holes, which is one of the big limitations of hard templates. However, due to the necessity of high temperatures in removing step of soft templates, the nanotubes may collapse. Template synthesis can prepare nanomaterials upon design demands and obtain well-formed nanoarrays, but it still has some drawbacks. For example, the AAO template as a commonly used master plate is mechanically weak which makes it difficult to prepare metal templates with large areas ( Masuda and Fukuda, 1995 ). Moreover, when using solvents to remove the organic compound templates, the structure of the nanomaterials may encounter some damages. Therefore, this synthesis method has the potential to be further optimized to synthesize utility materials with the desired functions using a simple operation ( Bera et al., 2004 ).

Hydrothermal treatment is among the momentous methods for nanotube production ( Harsha et al., 2011 ). This method has the potential to produce titanium dioxide nanotubes with suitable crystalline structures ( Swami et al., 2010 ). In this procedure, usually TiO 2 NPs were utilized as a titanium source to carry out a chemical reaction in a concentrated alkaline solution at high temperature. Subsequently, after ion exchange and calcination, the nanotube structure is reached ( Wong et al., 2011 ). The hydrothermal method can synthesize nanotubes with different diameters and lengths by controlling the reaction conditions ( Chi et al., 2007 ; Nakahira et al., 2010 ; Khoshnood et al., 2017 ; Mi et al., 2017 ). Hydrothermal synthesis is performed at high temperatures, and the rate of heating is a critical factor ( Seo et al., 2001 ). Based on the rate of heating, the hydrothermal method can be divided into two categories: conventional and microwave hydrothermal procedures. In the traditional hydrothermal synthesis method, the sample is simply heated in a general water bath. However, its heating rate is slow and the reaction cycle is long. Additionally, the heating of the reaction system is not uniform. The reaction system is exposed to microwave radiation during the microwave hydrothermal synthesis method. Since microwave heating is a rapid method of heating, the temperature of the reaction system very rapidly increases which results in a significantly reduced reaction period and the more uniform heating ( Ribbens et al., 2008 ; Liu et al., 2014 ; Meng et al., 2016 ). Titanium dioxide nanotubes can be synthesized by template synthesis, electrochemical anodization, and hydrothermal treatment. Additionally, there are some studies on the fabrication of nanotube structures by plasma electrolytic oxidation. Among the various possible methods, electrochemical anodization is the most commonly used method. By controlling the variables of the electrochemical anodization method, it is feasible to fabricate nanotube structures that satisfy the research needs. It is of great importance to delicately control the release of antibacterial agents in nanotubes and regulate the optimal size of nanotubes in order to promote cell adhesion, proliferation, and differentiation.

In the PVD technology, a physical phenomenon is used to vaporize the surface of source material (solid or liquid) into gaseous atoms, molecules, or into ions by ionization under vacuum condition. After the vaporization step, a low-pressure gas (or plasma) is implemented and a functional thin film is deposited on the substrate surface ( Chouirfa et al., 2019 ). The PVD technology can deposit not only metallic and alloy films but also it is capable of depositing ceramics and polymers. The main PVD methods are vacuum evaporation, sputtering deposition, and ion plating. Vacuum evaporation uses laser and electron beam heating to evaporate the material source into atoms or ions, and subsequently deposits atoms or ions onto the surface of the substrate to form a coating ( Kumar et al., 2009 ; Garbacz et al., 2010 ). The resultant coating from this method has relatively large pores and poor adhesion to the substrate ( Xingfang et al., 1988 ; Scott et al., 2003 ; Krysina et al., 2020 ). Sputter plating uses the base material as the anode and the target material as the cathode. By using the sputtering deposition effect, the argon ions generated by argon ionization knocks out the target material atoms and deposits them on the surface of the base material. The characteristic of this kind of coating is the presence of a few pores, but it can combine with the substrate more efficiently ( Ratova et al., 2017 ; Makówka et al., 2019 ; Zhang et al., 2019a ). Ion plating is involved with the ionization of gasses or vaporized substances under vacuum conditions, during the bombardment of gas ions or vaporized material ions, evaporates, or other reaction products are deposited on the substrate. The coating prepared by this method is uniform and dense, basically free of pores with a strong binding with the substrate ( Li et al., 2017c ; Zhang et al., 2018a ; Tian et al., 2019 ). With the development of PVD methods, many advanced PVD-based technologies have been derived which facilitate the production of high-quality nano-coatings. These new developed technologies include activated reactive evaporation ( Bulla et al., 2004 ; Biju et al., 2009 ; Yuvaraj et al., 2010 ), activated reactive sputtering ( Alajlani et al., 2016 , 2017 ), activated reactive ion plating ( Xin et al., 2000 ), magnetron sputtering ( Lelis et al., 2019 ; Avino et al., 2020 ; Vuchkov et al., 2020 ), magnetron sputtering pulsed laser deposition (MSPLD) ( Endrino et al., 2002 ; Jones and Voevodin, 2004 ), ionized magnetron sputtering ( Kusano et al., 1999 ; Tranchant et al., 2006 ), pulsed laser deposition (PLD) ( Paneerselvam et al., 2020 ; Wang et al., 2020 ), and etc.

One of the coating preparation methods entitled as the spin coating is able to precisely control the thickness. However, the size of the substrate is limited by the size of the spinning device ( Abu-Thabit and Makhlouf, 2020 ). The thickness of the coating prepared by the spin coating method is in the range of 30 and 2000 nm. The typical spin coating method is mainly divided into three steps: glue dispensing, high-speed rotation, and drying. First, the spin-coating droplets are injected onto the surface of the substrate. Then, the spin coating solution is spread on the surface of the substrate through high-speed rotation to form a uniform film. Finally, the remaining solvent is removed by drying; hence the stable coating is obtained. In the process of preparing the coating by spin coating, high-speed rotation, and drying are the key steps to control the thickness, structure, and performance of the coating. The schematic of the spin coating method is shown in Figure 3 . FIGURE 3 Schematic diagram of spin coating method(the target particles are applied onto the substrate, and then it accelerated to a high angular velocity to simultaneously spread the liquid over the entire surface and evaporate the solvent to achieve the target thickness). Spin coating technology has been successfully applied in the fields of optics ( Chtouki et al., 2017 ; Al-Douri et al., 2018 ) and electricity ( Oytun and Basarir, 2019 ; Yildiz et al., 2019 ). Simultaneously, the spin coating method is also used in the preparation of functional thin films in the fields of biology and medicine. For example, a hydrophilic or hydrophobic film is produced on the surface of the base material to achieve the purpose of antibiosis ( Kaviyarasu et al., 2017 ; Huang Y. et al., 2019 ; Li H. et al., 2020 ) and anti-corrosion ( Kim et al., 2013 ; Akram et al., 2020 ).

Layer-by-layer self-assembly technology is a relatively new technology in recent years and is widely used in the biology, materials, and nanoscience fields. As shown in Figure 5 ( Zhang et al., 2018d ), this technology can assemble a variety of materials (polyelectrolytes, small organic molecules, NPs, etc.) and can precisely control the surface structure and size of the coating. Nanomaterials with ordered structure prepared by self-assembly technology show unique properties. Self-assembly technology is currently a hotspot in the field of nanomaterial research. FIGURE 5 Schematic illustration of LbL assembly technology, which can load different types of materials on different types of substrates ( Zhang et al., 2018d ). Layer-by-layer self-assembly technology can be used to fabricate filtration membranes ( Rajesh et al., 2016 ), sensors ( Fernandes et al., 2011 ), and optoelectronic devices ( Eom et al., 2017 ). Besides, it is able to produce antibacterial coatings ( Wu et al., 2015 ; Huang J. et al., 2019 ; Li D. et al., 2019 ; Xia et al., 2019 ) or drug controlled release coatings ( Cao M. et al., 2018 ; Silva et al., 2018 ; Zhou W. et al., 2018 ; Sun et al., 2020 ; Wu et al., 2020 ). Hence, this technology seems to would have broad application prospects in the biomedicine field.

Nanomaterials can be used as antibacterial agents on the surface of titanium or titanium alloys and they have a potential to effectively improve the bactericidal properties ( Chen et al., 2014 ; Xin et al., 2019 ; Xu J.W. et al., 2019 ). Most of the studies were focused on the metallic nano-antibacterial agents on the surface of titanium and its alloys ( Liu et al., 2015b ; Gunputh et al., 2018 ; Cheng et al., 2019 ). In this regard, the common metal particles are silver, zinc, copper, etc. ( Vimbela et al., 2017 ). The NPs are tiny particles with a particle size in the range of 1–100 nm. The specific properties of NPs like large specific surface area, small size effect, and quantum size effect makes them an ideal option. Therefore, nanomaterials have a series of excellent properties, which improves the bactericidal effects of antibacterial agents in comparison to traditional antibacterial agents ( Mi et al., 2018 ; Guo et al., 2020 ). Metallic Antibacterial Agents Metallic antibacterial agents have exceptional research value because of their strong antibacterial ability, good biocompatibility, and excellent stability ( Ahmed et al., 2016 ). Inorganic antibacterial agents are usually present on the surface of titanium substrates in the form of NPs. They can be fixed on the surface of the titanium substrate by using a carrier or coated on the titanium substrate’s surface to prepare a nano-coating ( Kheiri et al., 2019 ). Ag Silver has many advantages in a broad-spectrum of antibacterial activity ( Yang Z. et al., 2018 ; Wang L. et al., 2019 ). These advantages make it the most studied and widely used metal-based antibacterial agent. Compared with traditional silver, silver NPs have a larger specific surface area, which significantly enhances their antibacterial ability. In the existing reports, the antibacterial mechanism of silver NPs mainly includes: destroying the structure of bacterial membranes, releasing silver ions and generating ROS to destroy enzymes in the oxidation respiratory chain, and regulating the signal transduction pathway of bacteria ( Park et al., 2009 ; Tang and Zheng, 2018 ). Unfortunately, silver NPs can cause significant cytotoxicity above a specific dose range. The silver ions released by the silver NPs are highly mobile, and their entrance into living cells with high concentrations can kill healthy cells ( AshaRani et al., 2009 ). The silver element forms which served as antibacterial are Ag2O NPs ( Chen et al., 2017 ; Sarraf et al., 2018a ; Lv et al., 2019 ) and Ag NPs ( Cheng et al., 2019 ; Pruchova et al., 2019 ; Surmeneva et al., 2019 ). The silver NPs that were loaded on the surface of the titanium substrate to produce NS, showing different antibacterial effects and performance that is listed in Table 3 . TABLE 3 Antibacterial effects and other related information about different silver-containing nanomaterials. Materials Base materials Manufacturing technique Nanostructure and Size Antibacterial effect/Antibacterial rate Cytotoxicity Cells References Ag-CACS-Ti Cp-Ti In situ reduction Nanoparticles E. col i: 99.8% S. aureus : 99.9% No L929 cells Cheng et al., 2019 Ag-TNTs Ti-6Al-4V Anodization+Chemical reduction Nanoparticles: 102 ± 21 nm S. aureus : 99.15% – – Gunputh et al., 2018 Ag-HA Ti-6Al-4V Laser process Nanoparticles: 20∼30 nm S. aureus : 77.59% – – Liu and Man, 2017 Ag-HA-CS Cp-Ti Pulse electrochemical deposition Nanoparticles: 303–321 nm E. coli : 100% S. aureus : 100% C. albicans : 100% P. aeruginosa : 100% No BMSCs Wang X. et al., 2019 Ag-PDA-TNTs Cp-Ti electrochemical anodization + in situ reduction Nanoparticles: 13.5 ± 4.8 nm E. coli : 54 ± 3.7%/14 days – – Xu et al., 2017 Ag-PDA-TNTs Ti-7.5Mo Anodization + polydopamine assisted immobilization technique Nanoparticles C. albicans : 100%/48 h S. aureus : 100%/48 h No ADSCs Rosifini Alves Claro et al., 2018 Ag-GO Ti-67IMP Physical vapor deposition magnetron sputtering + electrochemical anodization + spin coating Nanoparticles E. coli : 97.56%/24 h S. aureus : 98.15%/24 h No hFOB cells Rafieerad et al., 2019 Ag-Ti Cp-Ti Target-ion induced plasma sputtering + Ag sputtering Nanoparticles: 25 ± 5 nm E. coli : 100%/12 h S. aureus : 100%/12 h Yes L929 fibroblast cells Kim et al., 2018 TAN/TAP Ti-Si Vacuum arc remelting + sol-gel method Nanoparticles: ∼2 μm E. coli : 98-100%/24 h S. aureus : 100%/24 h No L929 fibroblast cells; U-2OS human osteosarcoma cells Horkavcová et al., 2017 Ag-Sr-HA coating Cp-Ti Hydrothermal method Nanoparticles: ∼100 nm E. coli : 99%/24 h S. aureus : 95%/24 h No MG63 cells Geng et al., 2016 Ag-HA coating Cp-Ti Electrostatic spraying Nanorods: length: 50 nm, diameter: 20 nm E. coli : 100%/24 h No Osteoblast Gokcekaya et al., 2017 Ag film Ti-6Al-4V Thermal annealing + DC sputtering Thickness: 20 nm E. coli : 100%/24 h S. aureus : 100%/24 h Yes NIH3T3 fibroblast cells Patil et al., 2019 Ag-TiN multilayers Titanium alloy Multi-arc ion plating Thickness: 120 nm E. coli : 99.88%/24h Yes MC3T3-E1 cells

Zinc is one of the important trace elements in the human body and plays a vital role in the growth and development of bones ( Zhu et al., 2018 ). It was known that Zn can enhance the expression of M2 marker genes and proteins in macrophages. The adhesion, proliferation, and expression of osteoblast-related genes are increased by Zn ( Zhang et al., 2018c ; Chen et al., 2020 ). Zinc NPs are non-toxic and have a higher affinity to bacteria than ordinary zinc, which can lead to better antibacterial effect. In the current research on the antibacterial mechanism of zinc NPs, it has been found that zinc ions can destroy bacterial membranes and promote the production of ROS, thereby achieving antibacterial effects. Related research and results are shown in Table 5 . To confirm the antibacterial effects and the feasibility of clinical applications Zinc-containing nanocoatings were studied, the results have shown excellent antibacterial effects both in vitro and in vivo antimicrobial experiments. Li et al. (2017b) prepared hybrid ZnO/poly-dopamine/arginine-glycine-aspartate-cysteine nanorod arrays on the titanium surface using atomic layer deposition and hydrothermal methods. The material was implanted into the femur of the rabbit model infected with S. aureus . After four weeks, femurs from animal models were tested by H&E staining and Giemsa staining, the zinc-containing nanorod arrays were less infected than the control soft tissues and bones, demonstrating that the release of zinc ions can play an effective anti-infective role. TABLE 5 Different antibacterial properties and biocompatibility of zinc-containing coatings. Materials Base materials Manufacturing technique Nanostructure Antibacterial effect/Antibacterial rate Cytotoxicity Cells References ZnO-Sr-OPDA-TNTs Cp-Ti Anodization hydrothermal treatment + atomic layer deposition ZnO film:thickness<2 nm E. coli : 87%/12 h S. aureus : 91%/24 h No MC3T3-E1 cells Zhang K. et al., 2017 ZnO-CS-CNTs-Ti Cp-Ti Electrophoretic deposition atomic layer deposition ZnO film thickness: <10 nm E. coli : 73%/24 h S. aureus : 98%/24 h Yes MC3T3-E1 cells Zhu et al., 2017 ZnO-TiO2 coating Cp-Ti Hydrothermal low temperature liquid phase Nanoparticle diameters: 50 ± 8∼80 ± 9 nm E. coli : 93.2%/12 h S. aureus : 97.5%/24 h No MC3T3-E1 cells Pang et al., 2019 Zn-Ti nanowires Cp-Ti Sol-gel + alkali heat – S. aureus : 66.58%/3 days P. gingivalis : 45.02%/3 days A. actinomycetemcomitans : 53.42%/3 days No MC3T3-E1 cells Shao et al., 2020 Zn-CHI-GEL multilayer films Cp-Ti LBL self-assembly Film thickness: 14.91 ± 0.97∼ 17.72 ± 0.63 nm E. coli : 47.37%/24 h S. aureus : 52.94%/24 h Yes Osteoblasts Karbowniczek et al., 2017 ZnO-TiO2 coating Cp-Ti Micro-arc oxidation Nanoparticles: Average 30 nm S. aureus : 51.4 ± 14.7%/24 h No MC3T3-E1 cells Zhang et al., 2018b ZnO-Ti coating Cp-Ti Micro-arc oxidation – E. coli : 48.08%/24h – Zhang et al., 2019c ZnO-PPy-HA coating Cp-Ti Electrochemical deposition Nanoparticle: Average 243 nm E. coli : 63.5%/12h S. aureus : 72.8%/12h No BMSCs Maimaiti et al., 2020 Zn-HA coating Ti-6Al-4V Co-precipitation + flame spraying 50–200 nm E. coli : 99.9%/3 h No WST-1 cells Yang et al., 2017 Zn-HA coating Ti-6Al-4V Plasma spraying – E. coli : 63.5%/7 days S. aureus : 36.6%/7 days No Saos-2 cells Sergi et al., 2018

Nickel is an indispensable element in the human body, and its content is in minimal range in the human body. Nickel maintains the structural stability and metabolism of biological macromolecules. Lack of nickel can cause diabetes, uremia, kidney failure, and other diseases. Studies have shown that Ni 2+ can effectively kill bacteria ( Yasuyuki et al., 2010 ), but excessive Ni 2+ will cause cytotoxicity ( Lü et al., 2009 ; Hang et al., 2012 ). A recent study showed that Ni 2+ released from NiTi alloys could exhibit antibacterial properties ( Ohtsu et al., 2017 ). However, due to slight Ni 2+ release from the NiTi alloy, its antibacterial ability is relatively weak. Therefore, in nanometric range the specific surface area of the NiTi alloy is increased, and the release amount of Ni 2+ is increased, thereby enhancing its antibacterial ability. Hang et al. (2018) produced Ni-Ti-O NPs with different lengths (0.55–114 μm) on NiTi alloys by anodization. The antibacterial effect of different samples to S. aureus was determined by the plate counting method. It was found that the antibacterial rate increased as the anodizing time increased. When the length of Ni-Ti-O NPs exceeds 11 μm, the antibacterial rate can reach 100% along with its excellent biocompatibility. Liu et al. (2018) also used anodizing method to prepare Ni-Ti-O NPs on NiTi alloy and studied the effects of different annealing temperatures (200, 400, 600°C) on antibacterial properties and biocompatibility. The length of the NPs is 2.05–2.78 μm. The results show that the antibacterial rate of the smooth NiTi alloy surface is only 36%, and the antibacterial rate of the surface after anodizing reaches 84%. After annealing at 200°C, the antibacterial rate of Ni-Ti-O NPs was close to 100%. Ni-Ti-O NPs annealed at 200–400°C all showed good cell compatibility. Therefore, the preparation of NPs with different sizes on the surface of NiTi alloy can effectively enhance the antibacterial effect of the material by increasing the specific surface area. An enormous number of studies on different inorganic nanoparticles (Ag, Cu, Zn, Au, Ni) indicate that Ag NPs as antibacterial agents have very efficient antibacterial performance compared to other antibacterial agents. At present, the mechanism of antibacterial actions of silver and silver ions is still under controversy. As mentioned previously, Ag NPs stimulate the generation of ROS and induce high oxidative stress, which is thought to be the foremost antibacterial mechanism ( Park et al., 2009 ; Siritongsuk et al., 2016 ). Under normal circumstances, ROS generated in the cell receives a restriction that can be eliminated by antioxidants ( Ramalingam et al., 2016 ). The antibacterial effect of Ag NPs stems from the dehydrogenase inactivation in the oxidative respiration chain along with excessive ROS generation. These circumstances inhibit oxidative respiration and the natural growth of the cells ( Su et al., 2009 ; Quinteros et al., 2016 ). In addition, two antibacterial mechanisms, contact killing, and ion-mediated killing are widely accepted. Ag NPs can anchor to the bacterial cell wall and infiltrate it, which can cause physical changes to the bacterial membrane (bacterial membrane damage, leakage of bacterial contents) and ultimately lead to bacterial death ( Khalandi et al., 2017 ; Seong and Lee, 2017 ). The primary antibacterial form of Ag NPs is the silver ion (Ag + ) ( Liu and Hurt, 2010 ; Le Ouay and Stellacci, 2015 ), and the target of Ag + has been identified as a number of molecules [DNA, peptides (membrane-bound or inside the cell) or cofactors] ( Le Ouay and Stellacci, 2015 ). The interaction of Ag NPs with cellular structures or biological molecules will result in impaired bacterial function and ultimately death. Antibiotics are usually used to attack particular molecules of certain bacteria, but silver ions react with all the nearby molecules, thus having a wide-spectrum antibacterial effect. Silver does not react with water, but can easily dissolve in water with the presence of an oxidizing agent (oxygen), which is termed oxidative dissolution. The oxidative dissolution of silver is also an important mechanism for its antibacterial action ( Molleman and Hiemstra, 2015 ), through the above mentioned multiple mechanisms, which directly or indirectly lead to the ability of Ag NPs to exert efficient antibacterial effects. The issue of cytotoxicity of Ag NPs still needs further studies since it depends on lots of factors, including the shape of NPs, dimension, concentration, etc. ( Attarilar et al., 2020 ), since high concentration of Ag NPs may be cytotoxic. Moreover, some other studies demonstrated the influence of NPs’ size on cytotoxicity. Several studies have shown that Ag NPs with the small size range (<20 nm) may cause varying degrees of cytotoxic effects, while large or condensed particles (>100 nm) sometimes do not have any considerable adverse outcomes ( Marambio-Jones and Hoek, 2010 ;

In recent years, the wings of insects, such as dragonflies and cicadas, have attracted much attention as a model biological system because of their excellent antibacterial and antifungal properties ( Ivanova et al., 2012 ; Diu et al., 2014 ; Kelleher et al., 2016 ). Some studies have shown the presence of physical nano-protrusions on the surface of insect wings. Their antibacterial properties may be due to the fact that when microbial cells come in contact with the protrusions, they possibility enhance the stress and deformation of the membrane structure of the microbial cells, leading to their destruction. It eventually leads to the dissolution and death of the cells ( Ivanova et al., 2013 ; Nowlin et al., 2014 ; Bandara et al., 2017 ). By investigating the surface structure of insect wings as a model and preparing bionic structures according to it, new ideas for preparing modern antibacterial titanium alloys have emerged. Antibacterial Nanopatterns and Fabrication Methods It has been shown that modification of the surface morphology of materials can be used to achieve antibacterial effects and inhibition of biofilm formation ( Modaresifar et al., 2019 ; Stratakis et al., 2020 ). By changing the surface morphology without adding other chemical reagents, the antibacterial and antibiofilm formation properties can also be achieved ( Campoccia et al., 2013 ; Hasan et al., 2013 ). Moreover, it has a slight effect on the mechanical strength of the material. Changing the surface morphology to prepare biomimetic structures with micron and nano-scale surface morphologies, and exploring the effect of surface morphology and size of titanium or its alloys that can effectively attack bacteria are the currently urgent problems to be solved. Table 7 lists some of the antibacterial NS formation methods and their properties. TABLE 7 Antibacterial NS formation methods and the related properties. Feature Manufacturing technique Base materials Antibacterial effect and rate Cells Cytotoxicity Wettability Roughness Morphology References Nanoflowers Chemical etching + hydrothermal oxidation Cp-Ti S. aureus : 43.12%/24 h MRSA : 73.15%/24 h hFOB No Hydrophilicity Ra = 829 nm A Vishnu et al., 2019 Nanowires Hydrothermal synthesis Ti-6Al-4V S. aureus : 74%/18 h Osteoblasts No Hydrophilicity – B Jaggessar et al., 2018 Regular nanotubes Acid etching + anodic oxidation Ti-6Al-4V E. coli : 72.6%/2 h S. aureus : 68.2%/2 h MG63 No Hydrophilicity Ra = 120 nm C Rahnamaee et al., 2020 Irregular nanotubes Electrochemical anodization Ti-6Al-4V E. coli : 48.7%/2 h S. aureus : 50.8%/2 h MG63 No Superhydrophilicity Ra = 360 nm D Rahnamaee et al., 2020 Nanotubes Electrochemical anodization Cp-Ti S. aureus : 36.78%/16 h – – Hydrophilicity R rms = 45.60 nm E, F Simi and Rajendran, 2017 Nano-ripples Femtosecond laser direct writing Cp-Ti E. coli : 56%/24 h MSCs No Superhydrophilicity Ra = 274.6 nm G Luo et al., 2020 Nanoparticles Aerosol flame synthesis Aluminum alloy S. aureus : 80% – – Superhydrophilicity H De Falco et al., 2018 Nanopillars Plasma etching Cp-Ti P. aeruginosa : 87 ± 2%/24 h S. aureus : 72.5 ± 13%/24 h – – Hydrophobicity – I Linklater et al., 2019 Different nano-morphologies can be prepared through different preparation processes: nanoflowers, nanowires, nanotubes, nano-ripples, NPs, and nanopillars are possible morphologies, Figure 6 . Among these nano-topographies, nanopillars show good bactericidal effect, which may be related to its high aspect ratio ( Linklater et al., 2018 ). In each nanotopography, cell activity was not significantly inhibited. Furthermore, in some nanotopographies, the cells’ metabolic activity tends to increase ( Jaggessar et al., 2018 ). FIGURE 6 SEM micrograph of different nano-morphologies on the surface of titanium substrate. (A) Nanoflowers ( Vishnu et al., 2019 ). (B) Nanowires ( Jaggessar et al., 2018 ). (C) Regular nanotubes ( Rahnamaee et al., 2020 ). (D) irregular nanotubes ( Rahnamaee et al., 2020 ). (E,F) nanotubes ( Simi and Rajendran, 2017 ). (G) Nano-ripples ( Luo et al., 2020 ). (H) AFM micrograph of NPs ( De Falco et al., 2018 ). (I) nanopillars ( Linklater et al., 2019 ). There are several methods to fabricate nanopatterns, and the commonly used methods are chemical etching ( Heidarpour et al., 2020 ), reactive ion etching ( Ganjian et al., 2019 ), plasma etching ( He et al., 2013 ), hydrothermal synthesis ( Jaggessar and Yarlagadda, 2020 ; Wategaonkar et al., 2020 ), and anodic oxidation ( Mohan et al., 2020 ). The antibacterial nanopatterns with different antibacterial efficiencies have been prepared by these methods, but the antibacterial effect of these patterns is still not very satisfactory, which may be due to the fact that the production of antibacterial surfaces on titanium and its alloys are more difficult than other materials [silicon, polymethylmethacrylate (PMMA), etc.]. The development of nanopatterns with efficient antibacterial properties can enable the better clinical appli

The design parameters (height, diameter, and spacing) of the nanopatterns have a significant influence on the antibacterial properties. Nanopatterns with different parameters (height from 100 nm to 900 nm, diameter from 10 nm to 300 nm, and spacing less than 500 nm) have been reported in the literature to exhibit bactericidal properties ( Modaresifar et al., 2019 ). Velic et al. (2019) studied the effect of varied design parameters of nanopatterns on bacteria behavior by developing a two-dimensional finite element model and demonstrated that reducing the pillar diameter could effectively promote bactericidal efficiency. Additionally, preparing nanopillar structures with similar diameters but different densities and heights showed that extra stretched nanopillars with varying heights leads to rupture in the bacterial cell membranes (the membranes exceeded the threshold value of stretching) ( Wu et al., 2018 ). The occurrence of implant material-related infections often begins with bacterial adhesion to the implant surface subsequently the colonization of bacteria and biofilm formation happens. Biofilms are bacteria produced microbial communities formed by the EPS substrate to inhibit the damage of bacteria. Statistically, about 99% of bacteria could exist with biofilm status ( Zimmerli and Sendi, 2017 ). Biofilms exist as reservoirs for bacteria, often leading to chronic and systemic infections. Therefore, reducing the opportunity of bacteria to adhere to the surface of the implants is essential to prevent infection. The adhesion of bacteria to a material surface depends on the surface properties of the material, such as surface roughness, wettability, and topography. Bacteria are more likely to adhere to rough rather than smooth surfaces ( Sarró et al., 2006 ; Fan et al., 2013 ) and more likely to adhere to hydrophobic rather than hydrophilic surfaces ( Pagedar et al., 2010 ). At the same time, the nanoscale surface has a higher resistance to bacterial adhesion than the micron and macroscale surfaces ( Singh et al., 2011 ; Spengler et al., 2019 ). The severe plastic deformation (SPD) process is a recently developed technique for the fabrication of nanostructures ( Dyakonov et al., 2019 ; Shuitcev et al., 2020 ). It is noteworthy that by reducing the grain size to the sub-micron range (100–500 nm) through SPD processes, the mechanical properties of commercially pure titanium are comparable to those of conventional Ti-6Al-4V alloys ( Singh et al., 2011 ). It was found that the increase in Ra of the pure titanium surface after SPD processing was followed by an increase in the adhesion density of S. aureus , which was independent to P. aeruginosa . The effect of surface hydrophilicity on bacterial adhesion can be explained by thermodynamics ( Bruinsma et al., 2001 ), with a higher affinity of hydrophobic bacteria for hydrophobic surfaces and of hydrophilic bacteria for hydrophilic surfaces ( Sabirov et al., 2015 ).

JQL, JL, and SA contributed equally to the design, reorganize the figures, and writing the manuscript. CW, MT, KX, and CY collated the resource. JY, LW, CL, and YT helped with editing the manuscript. YT conceived and designed the outline of this review. All the authors read and approved the final manuscript.