Karan Gulati

Jean-Claude Scimeca

Sašo Ivanovski

Elise Verron
email: elise.verron@univ-nantes.fr

Therapeutic efficacy versus toxicity evaluations of doped titanium implants

Keywords: implants, metal doping, metal ions, metal nanoparticles, toxicity, titanium, nanotubes

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Titanium (Ti) is an ideal material choice for orthopaedic and dental implants, owing to its excellent biocompatibility, corrosion resistance, and mechanics. [START_REF] Chopra | Understanding and optimizing the antibacterial functions of anodized nano-engineered titanium implants[END_REF] Various surface modifications of Ti-based bone/dental implants have been suggested to enhance their integration with bone (osseointegration). [START_REF] Chopra | Understanding and optimizing the antibacterial functions of anodized nano-engineered titanium implants[END_REF] These enhancements in terms of physical, chemical, and biological modifications enable improved integration, especially in compromised patient conditions (poor bone quality/quantity and systemic disease). [START_REF] Guo | Orchestrating soft tissue integration at the transmucosal region of titanium implants[END_REF] Additionally, loading and local elution of potent therapeutics, such as bone-forming proteins and antibiotics, have also been applied to Ti-based implants. It is established that topography dictates bioactivity and follows the trend macro < micro < nano. [START_REF] Gulati | Titania nanotubes for orchestrating osteogenesis at the bone-implant interface[END_REF] Nano-engineering of Ti implants and the incorporation of various metallic ions and NPs (with inherent therapeutic properties) is a promising strategy to augment bioactivity and create a favourable healing environment. [START_REF] Gulati | Titania nanotubes for orchestrating osteogenesis at the bone-implant interface[END_REF] Figure 1 shows the various strategies used to incorporate metals [covering the entire A-Z spectrum from silver (Ag) to zinc (Zn)], including physical, chemical, and topographical modifications, as well as loading inside electrochemically anodized Ti with TiO2 nanotubes. However, it is a 'double-edged sword', with the therapeutic efficacy often reported without thorough toxicity evaluation. In this review, we compare and contrast current understanding of metal-doped Ti implants in terms of achieving desirable outcomes, therapeutic effectiveness, parameters contributing to toxicity, and the research gaps that remain.

Metal-doped implants

The development of NaOH and heat treatment on Ti-based implants to augment bioactivity has led to significant advances, including easy metal doping by using metal ion-containing solutions. [START_REF] Kawashita | Surface structure and in vitro apatite-forming ability of titanium doped with various metals[END_REF] As a result, antimicrobial Ag, and osteoconductive calcium (Ca), strontium (Sr), and Zn have been incorporated into Ti implants. [START_REF] Kawashita | Surface structure and in vitro apatite-forming ability of titanium doped with various metals[END_REF][START_REF] Yamaguchi | Controlled release of strontium ions from a bioactive Ti metal with a Ca-enriched surface layer[END_REF][START_REF] Alvarez | Titanium implants after alkali heating treatment with a [Zn(OH)4]2-complex: analysis of interfacial bond strength using push-out tests[END_REF] Indeed, doping of various metals into Ti via NaOH-heat treatment has been achieved by metal ion solution soaking (various concentrations), to load aluminium (Al 3+ ), Ca 2+ , chromium (Cr 3+ ), copper (Cu 2+ ), Zn 2+ , and Ag + . Whereas Al, Ca, Cr, and Zr were incorporated as stable chemical compounds, Ag and Cu were doped as metallic colloids, which determined the ion release rates and apatite-forming abilities. [START_REF] Kawashita | Surface structure and in vitro apatite-forming ability of titanium doped with various metals[END_REF] However, the applicability of such doping can only be confirmed via in-depth in vitro and in vivo investigations. Table 1 summarizes various strategies used to dope different elements onto Ti implants and their proposed functions. [START_REF] Wang | Flame-sprayed strontium-and magnesium-doped hydroxyapatite on titanium implants for osseointegration enhancement[END_REF][START_REF] Cao | Magnesium and fluoride doped hydroxyapatite coatings grown by pulsed laser deposition for promoting titanium implant cytocompatibility[END_REF][START_REF] Ciobanu | Cerium-doped hydroxyapatite/collagen coatings on titanium for bone implants[END_REF][START_REF] Esparza | Antibacterial response of titanium oxide coatings doped by nitrogen plasma immersion ion implantation[END_REF][START_REF] Wu | Cell-selective titanium oxide coatings mediated by coupling hafnium-doping and UV preillumination[END_REF][START_REF] Sedira | Physical deposition of carbon doped titanium nitride film by DC magnetron sputtering for metallic implant coating use[END_REF][START_REF] Zhao | Osteogenic activity of a titanium surface modified with silicon-doped titanium dioxide[END_REF][START_REF] Gulati | In situ hydrothermal transformation of titanium surface into lithiumdoped continuous nanowire network towards augmented bioactivity[END_REF][START_REF] Zhang | Biocompatibility and antibacterial properties of pure titanium surfaces coated with yttrium-doped hydroxyapatite[END_REF][START_REF] Zhao | Surface functionalization of titanium with zinc/strontium-doped titanium dioxide microporous coating via microarc oxidation[END_REF][START_REF] Kizuki | Preparation of bioactive Ti metal surface enriched with calcium ions by chemical treatment[END_REF][START_REF] Kirmanidou | Assessment of cytotoxicity and antibacterial effects of silver nanoparticle-doped titanium alloy surfaces[END_REF][START_REF] Kheur | Nanoscale silver depositions inhibit microbial colonization and improve biocompatibility of titanium abutments[END_REF][START_REF] Ponomarev | Ag(Pt) nanoparticles-decorated bioactive yet antibacterial Ca-and P-doped TiO2 coatings produced by plasma electrolytic oxidation and ion implantation[END_REF][START_REF] Yao | Microstructure and antibacterial properties of Cu-doped TiO2 coating on titanium by microarc oxidation[END_REF][START_REF] Cochis | The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii[END_REF] Therapeutic metal-doped nano-engineered implants More recently, fabrication of controlled nanotopographies, including Ti dioxide (TiO2) nanotubes (TNTs) via electrochemical anodization of Ti implants, with the ability to load and release therapeutics, have gained attention. [START_REF] Li | Bridging the gap: Optimized fabrication of robust titania nanostructures on complex implant geometries towards clinical translation[END_REF] Various potent metal and semi-metal ions and NPs have been incorporated on/inside TNTs to provide enhanced local therapeutic action, including osteogenic, antibacterial, and immunomodulatory functions. [START_REF] Chopra | Understanding and optimizing the antibacterial functions of anodized nano-engineered titanium implants[END_REF][START_REF] Guo | Orchestrating soft tissue integration at the transmucosal region of titanium implants[END_REF] Various strategies have been used, including chemical reduction, photo-irradiation, spin coating, sputtering, micro-arc oxidation, and drop casting, to enable incorporation of metallic ions/NPs on/inside TNTs. Here, we summarize key advances in this domain.

Calcium-phosphorous-zinc

Ca/phosphorous (P) and Zn, as promoters of osseointegration and antibacterial functions, respectively, were doped into TNTs via reverse polarization and anodization treatments. Augmented biocompatibility, antibacterial performance, electrochemical stability, wear resistance, and reduced mechanical degradation were reported, which were attributed to enhanced adhesion strength with the underlying Ti substrate. [START_REF] Alves | Tribo-electrochemical behavior of bio-functionalized TiO2 nanotubes in artificial saliva: understanding of degradation mechanisms[END_REF][START_REF] Alves | A first insight on the bio-functionalization mechanisms of TiO2 nanotubes with calcium, phosphorous and zinc by reverse polarization anodization[END_REF][START_REF] Yu | Enhancing antibacterial performance and biocompatibility of pure titanium by a two-step electrochemical surface coating[END_REF] Copper Varied amounts of Cu NPs were incorporated inside TNTs via micro-arc oxidation (MAO), and excellent antibacterial functions (against Staphylococcus aureus) via both release and contact killing were obtained. Moreover, the Cu-TNTs enhanced proliferation and adhesion of osteoblasts, although higher concentrations showed cytotoxicity. [START_REF] Zhang | Effects of copper nanoparticles in porous TiO2 coatings on bacterial resistance and cytocompatibility of osteoblasts and endothelial cells[END_REF]

Gallium

Gallium (Ga) III) was solvent casted with poly(-DL-lactic acid) film onto TNTs, which demonstrated excellent antibacterial, immunomodulatory, and osteogenic functions in vivo (male Sprague-Dawley rats). [START_REF] Dong | Gallium-doped titania nanotubes elicit anti-bacterial efficacy in vivo against Escherichia coli and Staphylococcus aureus biofilm[END_REF]

Gold

Gelatin-stabilized gold (Au) NPs loaded inside TNTs enhanced attachment of MC3T3-E1 cells in vitro compared with bare TNTs and Ti surfaces. [START_REF] Neupane | Titania nanotubes supported gelatin stabilized gold nanoparticles for medical implants[END_REF] Alternatively, electro-deposited Au NPs inside TNTs improved corrosion resistance, surface energy, and osteoblast attachment/proliferation. [START_REF] Wang | Enhancement of corrosion resistance and bioactivity of titanium by Au nanoparticle-loaded TiO2 nanotube layer[END_REF] Furthermore, Au-decorated TNTs also exhibited photocatalytic activity via visible-light irradiation, which enabled multispecies antibacterial and softtissue integrating functions. [START_REF] Zheng | Engineering nanotubular titania with gold nanoparticles for antibiofilm enhancement and soft tissue healing promotion[END_REF] More recently, Zhao et al. reported the use of Au NP-functionalized TNTs toward near-infrared (NIR)-triggered drug release. [START_REF] Zhao | NIR light-driven photocatalysis on amphiphilic TiO2 nanotubes for controllable drug release[END_REF] Au has also been co-doped with platinum (Pt) NPs to enhance bioactivity and antibiofouling applications. [START_REF] Moon | Visible light-enhanced antibacterial and osteogenic functionality of Au and Pt nanoparticles deposited on TiO2 nanotubes[END_REF]

Lanthanum

The rare earth element lanthanum (La) was used to enhance the osteogenic potential of Sr-doped TNTs (Sr-TiO3 nanotubes). La-Sr co-doped TNTs exhibited augmented hydrophilicity and protein adsorption, and upregulated expression of osteogenic genes, without any cytotoxicity. [START_REF] Zhang | Synergistic effects of lanthanum and strontium to enhance the osteogenic activity of TiO2 nanotube biological interface[END_REF]

Selenium

Spin coating of chitosan over selenium (Se) electrodeposited inside TNTs enabled simultaneous osteogenic, antibacterial, and anticancer functions. [START_REF] Chen | Fabrication of selenium-deposited and chitosan-coated titania nanotubes with anticancer and antibacterial properties[END_REF]

Silver

Most widely used in metal doping, Ag has been loaded inside TNTs using various strategies, including magnetron sputtering (Ag2O NPs) and plasma immersion ion implantation (Ag ions). [START_REF] Gao | The effects of titania nanotubes with embedded silver oxide nanoparticles on bacteria and osteoblasts[END_REF][START_REF] Mei | Antibacterial effects and biocompatibility of titanium surfaces with graded silver incorporation in titania nanotubes[END_REF] Studies have reported that Ag release enhances both antibacterial and osseointegration abilities. [START_REF] Gao | The effects of titania nanotubes with embedded silver oxide nanoparticles on bacteria and osteoblasts[END_REF][START_REF] Mei | Antibacterial effects and biocompatibility of titanium surfaces with graded silver incorporation in titania nanotubes[END_REF][START_REF] Gunputh | Antibacterial properties of silver nanoparticles grown in situ and anchored to titanium dioxide nanotubes on titanium implant against Staphylococcus aureus[END_REF][START_REF] Chen | Improved immunoregulation of ultra-low-dose silver nanoparticle-loaded TiO(2) nanotubes via M2 macrophage polarization by regulating GLUT1 and autophagy[END_REF] Others have used chemical reduction, photoreduction, micro arc oxidation, and spin coating to modify TNTs with Ag NPs. Alternatively, Ag-doped hydroxyapatite (HA) has also been incorporated on TNTs via electrophoresis to achieve high antibacterial efficacy.

Strontium

Sr ions are known to stimulate bone formation and, as a result, various amounts of Sr ions were incorporated inside TNTs (various diameters) for sustained release over 30 days, with augmented cellular adhesion (rat MSCs in vitro), mobility/spreading, and osteogenic differentiation observed. [START_REF] Zhao | The osteogenic activity of strontium loaded titania nanotube arrays on titanium substrates[END_REF] More recently, Sr and Ca co-doped TNTs were produced via modified anodization and hydrothermal treatment for enhanced osteogenic activity. [START_REF] Zhang | Ca substitution of Sr in Sr-doped TiO2 nanotube film on Ti surface for enhanced osteogenic activity[END_REF] Furthermore, Sr 2+ and Ag NPs have been incorporated inside TNTs toward synergistic osteogenic and antibacterial activity. [START_REF] Pan | Incorporation of Sr2+ and Ag nanoparticles into TiO2 nanotubes to synergistically enhance osteogenic and antibacterial activities for bone repair[END_REF]

Samarium

Anodization, hydrothermal synthesis, and dopamine self-assembly were combined to develop dual samarium (Sm)/Sr-doped TNTs. [START_REF] Zhang | A functionalized Sm/Sr doped TiO2 nanotube array on titanium implant enables exceptional bone-implant integration and also self-antibacterial activity[END_REF] Slow release of Sm and Sr ions demonstrated enhanced antibacterial effect and osteogenesis, respectively.

Tantalum

Vacuum-deposited tantalum (Ta; 20-nm films) onto TNTs promoted osteoblast alkaline phosphatase (ALP) activity, cell mineralization, and bone formation in vitro. [START_REF] Frandsen | Tantalum coating on TiO2 nanotubes induces superior rate of matrix mineralization and osteofunctionality in human osteoblasts[END_REF] Furthermore, Ta coating on TNTs was achieved via plasma spraying, which enhanced the adhesion, differentiation, and mineralization of osteoblasts in vitro. [START_REF] Wang | Tantalum coated on titanium dioxide nanotubes by plasma spraying enhances cytocompatibility for dental implants[END_REF]

Zinc

Zn has a crucial role in stimulating osteoblast adhesion/proliferation and inhibition of osteoclasts, making it suitable for modification of Ti-based implants; hence various attempts have been made to decorate TNTs/Ti implants with Zn. [START_REF] Pan | Preparation of chitosan-Zn2+ complex coating on polydopamine-modified TiO2 nanotubes to enhance osteoblast behaviors[END_REF][START_REF] Pan | Improved blood compatibility and endothelialization of titanium oxide nanotube arrays on titanium surface by zinc doping[END_REF][START_REF] Chen | Zn-Incorporated TiO(2) nanotube surface improves osteogenesis ability through influencing immunomodulatory function of macrophages[END_REF] Rat bone mesenchymal stem cells (MSCs) and s. aureus were separately cultured on Zn-TNTs (hydrothermally incorporated), and Zn dose-dependent enhancement of MSC functions (adhesion, mineralization, and osteogenic differentiation) and antibacterial effects were reported. [START_REF] Huo | Osteogenic activity and antibacterial effects on titanium surfaces modified with Znincorporated nanotube arrays[END_REF] Alternatively, folic acid-conjugated ZnO quantum dots and electrodeposited ZnO NPs have also been used to impart antibacterial function on TNT implants. [START_REF] Xiang | Infection-prevention on Ti implants by controlled drug release from folic acid/ZnO quantum dots sealed titania nanotubes[END_REF][START_REF] Elizabeth | ZnO nanoparticle incorporated nanostructured metallic titanium for increased mesenchymal stem cell response and antibacterial activity[END_REF] Silicon Bai et al. reported anodization of magnetron-sputtered Ti-silicon (Si) coatings to fabricate Si-doped TNTs, which enhanced endothelial cell function and a prohealing macrophage phenotype. [START_REF] Bai | Favorable manipulation of macrophage/endothelial cell functionality and their crosstalk on silicon-doped titania nanotube arrays[END_REF]

Understanding cytotoxicity from doped implants

Generation of nano-scale metals

After in situ implantation, the release of surface-doped metals can be (Figure 2): (i) Intentional: metal ions or NPs intended to provide therapeutic functions; or (ii) unintentional: unintentional release (ions and particles) can result from chemical breakdown, degradation, and wear-tear processes.

Ultimately, mechanical failure and/or corrosion results in the generation of NPs or metal ions from either the implant or its coating. Indeed, the most common reason for revision surgery of joint replacements is the presence of wear particles (mostly <1 μm). [START_REF] Bijukumar | Systemic and local toxicity of metal debris released from hip prostheses: a review of experimental approaches[END_REF] Furthermore, dental implants can undergo damage in the oral environment (chemical/physical deterioration, wear/erosion caused by food, chewing, and bacterial activity). Regardless of the scenario, local metal ion exposure at the site of treatment can be high, as reported with Ti NPs released from dental implants into the surrounding tissues or newly regenerated bone. [START_REF] Schmalz | Scientific update on nanoparticles in dentistry[END_REF] Particles (size range 0.5-40 μm) have been found in human jawbone marrow tissues at distances of 60-700 μm. In addition, NP concentration was highest in mandibular bone (550-1590 μm from the implants) and decreased with distance.

In addition to local release, systemic exposure to free metals (ions/particles) can raise concerns because of their distribution into brain, liver, spleen, lymph nodes, and other organs. [START_REF] Saifi | Cytotoxicity of nanomaterials: using nanotoxicology to address the safety concerns of nanoparticles[END_REF] This is also relevant to dental devices, which remain functional in the oral cavity for long periods. Indeed, as described for TiO2 NPs, there is a high probability that nano-scale metallic species leach out into the saliva and are then absorbed by the digestive tract, mainly via intestinal enterocytes and Peyer plates in the small intestine. The rate of absorption and systemic distribution is enhanced by the small size and the positive charge of these nanomaterials. When ingested, NPs are mainly eliminated within 48 h and the degradation products can accumulate in various organs (spleen, liver, kidney, heart, lungs, bone marrow, or central nervous system). [START_REF] Chang | Neurotoxicity of metal-containing nanoparticles and implications in glial cells[END_REF] Enzymes such as proteases or metallothioneins can be involved in the metabolism of these metallic nanomaterials, resulting in adverse effects such as fever, swelling of the spleen and liver, suppression of the biliary circulation, and acute renal failure.

Cell toxicity of metal ions and nanoparticles

Metals (free ions and NPs) chemically/physically interact with biological fluids. The high surface area of NPs enables the adsorption of numerous biomolecules, such as lipids, proteins, and nucleic acids. This binding process leads to the formation of a dynamic protein corona covering the NPs. Its composition depends not only on the environment at a given time (i.e., nature and concentration of proteins present at the site), but also on material characteristics (shape, structure, size, charge, and hydrophobicity). For example, hydrophobic nanomaterials adsorb more plasma proteins compared with hydrophilic ones, thus resulting in fast opsonization processes (i.e., capture by the immune system for phagocytosis). NPs avidly adsorb albumin, complement proteins, immunoglobulins, fibrinogen, and apolipoproteins. Albumin, which is present at a high concentration, is adsorbed very quickly and will cover the surface of NPs. Subsequently, albumin will be progressively replaced by other proteins present in smaller concentrations but with higher affinity. Thus, for in vitro assessment of materials, these dynamic phenomena highlight the importance of pertinently selecting the medium used to mimic the physiological environment. Ultimately, this corona enables the intracellular delivery of toxic metal ions via a 'Trojan horse' mechanism involving endocytosis or phagocytosis processes, followed by fusion with lysosomes/endosomes.

Once inside the cytoplasm, NPs can be exposed to an acidic environment and degrading enzymes, such as Cathepsin L, which can process almost all bioconjugated NPs. [START_REF] Sée | Cathepsin L digestion of nanobioconjugates upon endocytosis[END_REF] Upon removal of surface molecules, endosomes (pH 6.0) or lysosomes (pH 4.5) can lead to an acidic environment, inducing NP release, thus generating free metal ions. These metal ions, including toxic cationic ions (Ag + , Gd 2+ , Fe 2+/3+ , Zn 2+ , and Au +/3+ ), can affect cellular homeostasis and functions by influencing gene/protein expression. [START_REF] Sabella | A general mechanism for intracellular toxicity of metalcontaining nanoparticles[END_REF] By disrupting electron transport in the inner mitochondria membrane, they interrupt energy production and generate endogenous reactive oxygen species (ROS). Low levels of ROS can activate transcription factors, leading to enhanced expression of inflammation-associated genes. Moreover, a high and sustained production of ROS that are not detoxified by endogenous antioxidant defenses affects cellular membranes (lipid peroxidation and structural proteins alterations), as well as enzymes, ion pumps, or nuclear DNA. For example, ZnO NP dissolution in endosomes and lysosomes of RAW 264.7 cells disrupted cellular Zn homeostasis, leading to lysosomal/mitochondrial damage and, ultimately, cell death. [START_REF] Xia | Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties[END_REF] Moos et al. reported that, when dissolved in cell culture medium, ZnO NP toxicity is independent of the amount of soluble Zn 2+ in the cell culture medium. [START_REF] Moos | ZnO particulate matter requires cell contact for toxicity in human colon cancer cells[END_REF] Alternatively, Wang et al. demonstrated that ZnO NP toxicity is mainly caused by Zn 2+ release, leading to an abnormal increase in Zn 2+ intracellular concentration, and thereby an elevation of ROS levels and cell viability impairment. [START_REF] Wang | Toxicity of ZnO nanoparticles to macrophages due to cell uptake and intracellular release of zinc ions[END_REF] Inert and nonbiodegradable Au is widely used in medicine and restorative dentistry. However, Au can be dissolved in biological environments. [START_REF] Soenen | Cellular toxicity of inorganic nanoparticles: Common aspects and guidelines for improved nanotoxicity evaluation[END_REF] Intracellular Au ions can strongly impede thioredoxin reductase functioning, leading to mitochondrial membrane depolarization or mitochondrial enzyme inactivation. This results in several toxic downstream events, including cellular redox balance alterations, increased ROS levels, and finally apoptosis. [START_REF] Saifi | Cytotoxicity of nanomaterials: using nanotoxicology to address the safety concerns of nanoparticles[END_REF] By contrast, Larsen et al. found that, after a focal brain injury, Au was beneficial in reducing microgliosis and neuronal apoptosis, and enhanced neural stem cell responses. [START_REF] Larsen | Gold ions bio-released from metallic gold particles reduce inflammation and apoptosis and increase the regenerative responses in focal brain injury[END_REF] Metals (ions and NPs) and ROS diffuse easily via nuclear pores and translocate into the nucleus. Indeed, AgNPs, Au, and metal oxides, such as TiO2 or ZnO, act as DNA intercalators and generate ROS responsible for severe oxidative DNA damages. [START_REF] Poier | Effects of zinc oxide nanoparticles in HUVEC: cyto-and genotoxicity and functional impairment after long-term and repetitive exposure in vitro[END_REF] Additionally, interactions between nano-scale metals and plasmatic/cellular proteins affect the functions of not only the materials, but also proteins. Protein denaturation results in loss of structure and/or function and induces protein fibrillation/aggregation to form amyloid fibrils, which can lead to unexpected blood clot formation consecutive to fibrinogen binding. Interactions with the cytoskeleton can also induce conformational changes in tubulin and disrupt its polymerization, as shown with TiO2 NPs, which considerably alter intracellular transport, cell division, and cell migration. [START_REF] Hou | Toxicity and mechanisms of action of titanium dioxide nanoparticles in living organisms[END_REF] Moreover, unfolded proteins can expose new antigenic determinants, which can lead to potential hypersensitivity reactions or inflammation in vulnerable patients. Indeed, metals and their oxides can generate proinflammatory effects through interactions with the immune system cells (leukocytes, neutrophils, monocytes, macrophages, dendritic cells, etc.). [START_REF] Saifi | Cytotoxicity of nanomaterials: using nanotoxicology to address the safety concerns of nanoparticles[END_REF] By stimulating major signaling pathways, nuclear factor (NF)-κB, and mitogen-activated protein kinase (MAPK), Ag, Au, Ti, Cu, or Zn NPs enhance proinflammatory cytokines release (IL-1β, IL-6, IL-8, and TNF-α). [START_REF] Saifi | Cytotoxicity of nanomaterials: using nanotoxicology to address the safety concerns of nanoparticles[END_REF][START_REF] Ickrath | Time-dependent toxic and genotoxic effects of zinc oxide nanoparticles after long-term and repetitive exposure to human mesenchymal stem cells[END_REF] For example, Ag NPs accentuate the release of IL-1β through inflammasome formation and caspase-1 activation. [START_REF] Murphy | Silver nanoparticles induce pro-inflammatory gene expression and inflammasome activation in human monocytes[END_REF] Nanoscale metals can also be recognized by cell surface receptors, which control cell type-specific signaling pathways triggering various responses (apoptosis, cell proliferation, stress-related signaling, or calcium-mediated signal transduction events). For example, necrosis can result from a sustained intracellular release of Ca from endoplasmic reticulum stores. Via chemical affinity, Ag (NPs and ions) interacts with thiol groups harboured by proteins localised in the cytoplasm, cell membranes, and inner membrane of mitochondria. [START_REF] Murphy | Silver nanoparticles induce pro-inflammatory gene expression and inflammasome activation in human monocytes[END_REF] As a result of lipid peroxidation, membrane permeability increases and cell necrosis results from leakage of cytoplasmic contents. Furthermore, exposure to metals modifies subcellular organelles and physiological functions through various mechanisms (Figure 3).

Parameters influencing released metal toxicity

Mechanisms involved in nano-scale metal toxicity mainly depend on various physicochemical parameters. [START_REF] Sukhanova | Dependence of nanoparticle toxicity on their physical and chemical properties[END_REF] Among them, we focus on chemical composition, surface charge, and the size and shape of NPs because they strongly influence toxicity.

First, in terms of the toxicity resulting from chemical composition, it appears that some metal ions (Ag and Cd) are cytotoxic, whereas others (Fe and Zn) are essential biological constituents [START_REF] Sukhanova | Dependence of nanoparticle toxicity on their physical and chemical properties[END_REF] . However, at high concentrations, the latter can also damage cellular pathways. These effects can be prevented by coating NP cores with thick polymer shells or silica layers, or by using nontoxic compounds for NP synthesis. In addition, hydrophilic surfaces (i.e., dextran and polyethylene glycol coatings) offer a neutral chain cloud on the surface that repels plasma proteins and prevents rapid clearance from the systemic circulation. Furthermore, mechanisms of toxicity can differ according to the composition of NPs. For instance, 20-nm ZnO NPs cause oxidative stress, whereas 20-nm SiO2 NPs alter the DNA structure. The chemical instability of NPs or the presence of impurities increase cell reactivity (generation of ROS and inflammatory mediators).

Surface charge also affects cellular uptake. Cationic NPs are more reactive because of an electrostatic attraction with negatively charged cell membrane glycoproteins. In addition, they can connect electrostatically with negatively charged DNA, and damage it. Generally, the more cationic the NPs are, the more toxic impact is observed. [START_REF] Sukhanova | Dependence of nanoparticle toxicity on their physical and chemical properties[END_REF] Thus, modifications of NP charge allow for control over NP localisation and toxicity.

NP size strongly influences the ability of nanoscale metals to penetrate biological barriers. [START_REF] Xia | Size-and cell type-dependent cellular uptake, cytotoxicity and in vivo distribution of gold nanoparticles[END_REF] NPs <5 nm usually overcome cell barriers nonspecifically via translocation, whereas larger particles enter cells using phagocytosis, macropinocytosis, and transport mechanisms, which vary depending on cell size and type. Size also conditions NP reactivity toward biomolecules. As the size decreases, the surface area:volume ratio increases, resulting in a higher reactive surface capable of affecting cell organelles. A reduction in particle size from 30 nm to 3 nm increases the number of surface particles from 5% to 50%, which subsequently increases their chemical reactivity and results in enhanced cytotoxicity and genotoxicity. [START_REF] Saifi | Cytotoxicity of nanomaterials: using nanotoxicology to address the safety concerns of nanoparticles[END_REF] It has also been reported that Ag NP degradation into ions and ROS production depends on NP size. [START_REF] Pan | Size-dependent cytotoxicity of gold nanoparticles[END_REF] In terms of fibroblasts, epithelial cells, or macrophages, 15-nm NPs have been found to be 60 times less toxic compared with 1.4-nm NPs. Moreover, 1.4-nm NPs cause cell necrosis, whereas 1.2nm NPs predominantly cause apoptosis. These data suggest not only that NPs enter the nucleus, but also that the geometric size of 1.4-nm NPs allows their interaction with the negatively charged sugar-phosphate DNA backbone, and blocks DNA transcription. [START_REF] Joris | The impact of species and cell type on the nanosafety profile of iron oxide nanoparticles in neural cells[END_REF] NP shape mediates cellular uptake. It appears that, because of a larger surface:volume ratio, rod-shaped Au NPs are more readily taken up by dendritic cells compared with spherical or cubic-shaped ones, thus resulting in higher toxicity. [START_REF] Niikura | Gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo[END_REF] Although NP shape can physically damage cell membranes, this direct toxicity is prevented by protein adsorption on the NP surface, thereby changing their shape. [START_REF] Hu | Protein corona-mediated mitigation of cytotoxicity of graphene oxide[END_REF] Similarly, crystal structures can vary upon interaction with biological fluids and, for the same size, rutile-like crystal structure NPs caused more cell damage compared with NPs with anatase-like crystal structure.

Research gaps and future directions

The safety evaluation of Ti implants doped with metals is discussed in the context of the general framework for biological evaluation of medical devices, as described in the ISO/TR 13014:2012 and ISO 10993-1:2009 standards. Obviously, the risk assessment should be performed taking into consideration the type of device, the type of tissue contact, and the contact duration, thus identifying the specific exposure scenario. First, a complete physicochemical characterization of materials must include at least size and size distribution, chemical composition, particle and mass concentration, charge (ζ-potential), surface properties, pH, and viscosity. Estimation of exposure for risk assessment is based on measuring the release of metal/metal oxide/nanometals from the implant. However, this can pose analytical challenges, especially for the detection of nanomaterials.

In terms of biological evaluation, traditional experimental methodology fails to provide an accurate and comprehensive description of the effects of NPs on cells. Most of the mechanistic studies are based on in vitro cellular toxicity tests using cell lines (instead of primary cells) and relatively high concentration exposure for limited periods of time (24-48 h), which is not clinically relevant. Extrapolation from these studies to chronic, lowdose exposures in vivo remains problematic. In addition, technical limitations compromise the use of conventional toxicology assays, such as the MTT cytotoxic assay.

However, metal doping shows promise toward enhancing conventional ti-based implants. In this domain, various research gaps remain toward bridging the gap between research and clinics: (i) only the therapy and bioactivity of metal doping are reported, whereas the topographical and chemical alterations (because of the metal doping process) of implant surfaces are not explored; (ii) mechanical stability and long-term survivability of coatings of Ti implant surfaces are not researched, especially given the constant load-bearing conditions of such implants; (iii) the 'race to invade' the implant surface between various cells immediately after implantation can dictate the early acceptance and long-term success of implants. For studies using metal-doped Ti implants, early cellular responses (few seconds to minutes) often hold the key to understanding cellular functions, but, to date, these remain underexplored; and (iv) orthopaedic and dental implants experience varied forces during handling, placement, and survival in constant load-bearing conditions. Currently, there is a lack of long-term in vivo investigations exploring the stability/survivability and toxicity of both implants and their metal modifications. This is urgently needed to evaluate toxicity resulting from the release of particulates upon load.

Concluding remarks

Compromised patient conditions, including osteoporosis and diabetes, demand enhanced osseointegration functionality from Ti implants. Furthermore, bacterial colonization represents a major challenge to achieving longterm implant success. Various potent metal ion or NPs (from Ag to Zn) have been incorporated into Ti implants to address these bioactivity and therapeutic requirements. Whereas in vitro and short-term in vivo investigations have shown promising outcomes in terms of therapy, evaluation of the cytotoxicity of such metal species (released intentionally via local release or unintentionally because of loading/wear) remains a significant research gap. There is broad appreciation of the physical and chemical characteristics of metal species that must be modulated to arrive at tailored therapies with minimal cytotoxicity. Clearly, improved understanding of this 'double-edged sword' holds great promise toward the development of the next generation of advanced therapeutic implants; however, detailed toxicity evaluations from long-term in vivo implantations under load-bearing conditions are needed to bridge the gap. Metals and debris of NPs can interact with biomacromolecules at the surface of the cell and after cell internalization, with phospholipids, proteins, and genetic material, resulting in loss of their structure and activity. These alterations can be amplified through the deleterious action of reactive oxygen species (ROS).

  microarc oxidation (MAO) on microporous TiO 2 Improved early stage osseointegration (in vivo) 13 Lithium (Li) Li nanowire network (via ionic exchange and hydrothermal) on alkalitreated Ti Improved biocompatibility in vitro 14 Yttrium (Y)-HA Ti modified with HA doped with Y via layer-by-layer self-assembly method Enhanced soft-tissue integration (gingival fibroblasts) and antibacterial functions in vitro 15 Zr-Sr Zr/Sr-doping on microporous TiO 2 using MAO Antibacterial activity in vitro and enhanced early osseointegration in vivo {containing the [Zn(OH) 4 ] 2-complex} of mechanical-grinded and smooth cylindrical Ti implants Significantly stronger bone fixation in vivo 6 Ag Direct current (DC) sputtering and electrochemical deposition Antibacterial functions 18, 19 Ag/Pt Ag/Pt decoration (ion implantation) on Ca/P-doped (plasma electrolytic oxidation) TiO 2 coating on Ti Bioactivity and antibacterial functions in vitro 20 Cu Micro-arc oxidation Antibacterial functions 21 Ga/Ag Anodic spark deposition (ASD) treatment Antibacterial functions

Figure 1 .

 1 Figure 1. Schematic of strategies to incorporate various metal ions or nanoparticles inside titanium implants.

Figure 2 .

 2 Figure 2. Release of metal ions and nanoparticles (NPs) upon implantation: intentional (therapeutic) and unintentional (chemical breakdown or physical destruction).

Figure 3 .

 3 Figure3. Schematic of cell toxicity of metal ions and nanoparticles (NPs). Metals and debris of NPs can interact with biomacromolecules at the surface of the cell and after cell internalization, with phospholipids, proteins, and genetic material, resulting in loss of their structure and activity. These alterations can be amplified through the deleterious action of reactive oxygen species (ROS).

Université Côte d'Azur, CNRS, Inserm, iBV, France

Acknowledgment K.G. is supported by the National Health and Medical Research Council (NHMRC) Early Career Fellowship (APP1140699).