Innovative Anodic Treatment to Obtain Stable Metallic Silver Micropatches on TiO2 Nanotubes: Structural, Electrochemical, and Photochemical Properties.

Electrochemical modification of the Ti surface to obtain TiO2 nanotubes (NT-Ti) has been proposed to enhance osseointegration in medical applications. However, susceptibility to microbial adhesion, linked to biomaterial-associated infections, and the high TiO2 band gap energy, which allows light absorption almost exclusively in the ultraviolet (UV) region, limit its applications. Modifying the TiO2 semiconductor with metals such as Ag has been suggested both for antimicrobial purposes and for absorbing light in the visible region. The formation of NT-Ti with Ag micropatches (Ag-NT-Ti) is pursued with the objective of enhancing the stability of the deposits and preventing cytotoxic levels of Ag cellular uptake. The innovative process proposed here involves immersing NT-Ti in a AgNO3 solution as the initial step. Diverging from previously reported electrochemical methods, this process incorporates anodization within the TiO2 oxide formation region instead of cathodic reduction generally employed by other researchers. The final step encompasses an annealing treatment. The treatments result in the in situ Ag1+ reduction and formation of stable and active micropatches of metallic Ag on the NT-Ti surface. Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), Raman, diffuse reflectance spectroscopy (DRS), wettability assessment, and electrochemical characterizations were conducted to evaluate the modified surfaces. The well-known properties of NT-Ti surfaces were enhanced, leading to improved photocatalytic activity across both visible and UV regions, significant stability against detachment, and controlled release of Ag1+ for promising antimicrobial effects.

Antimicrobial-Loaded Polyacrylamide Hydrogels Supported on Titanium as Reservoir for Local Drug Delivery.

Arthroplasty is a highly successful treatment to restore the function of a joint. The contamination of the implant via bacterial adhesion is the first step toward the development of device-associated infections. The emerging concern about antimicrobial resistance resulted in a growing interest to develop alternative therapeutic strategies. Thus, the increment in the incidence of bacterial periprosthetic infections, the complexity of treating infections caused by organisms growing in biofilms, together with the rise in antibiotic resistant bacteria, expose the need to design novel surfaces that provide innovative solutions to these rising problems. The aim of this work is to develop a coating on titanium (Ti) suitable for inhibiting bacterial adhesion and proliferation, and hence, biofilm formation on the surface. We have successfully prepared polyacrylamide hydrogels containing the conventional antibiotic ampicillin (AMP), silver nanoparticles (AgNPs), and both, AMP and AgNPs. The release of the antibacterial agents from the gelled to aqueous media resulted in an excellent antibacterial action of the loaded hydrogels against sessile S. aureus. Moreover, a synergic effect was achieved with the incorporation of both AMP and AgNPs in the hydrogel, which highlights the importance of combining antimicrobial agents having different targets. The polyacrylamide hydrogel coating on the Ti surface was successfully achieved, as it was demonstrated by FTIR, contact angle, and AFM measurements. The modified Ti surfaces having the polyacrylamide hydrogel film containing AgNPs and AMP retained the highest antibacterial effect against S. aureus as it was found for the unsupported hydrogels. The modified surfaces exhibit an excellent cytocompatibility, since healthy, flattened MC3T3-E1 cells spread on the surfaces were observed. In addition, similar macrophage RAW 264.7 adhesion was found on all the surfaces, which could be related to a low macrophage activation. Our results indicate that AMP and AgNP-loaded polyacrylamide hydrogel films on Ti are a good alternative for designing efficient antibacterial surfaces having an excellent cytocompatibility without inducing an exacerbated immune response. The approach emerges as a superior alternative to the widely used direct adsorption of therapeutic agents on surfaces, since the antimicrobial-loaded hydrogel coatings open the possibility of modulating the concentration of the antimicrobial agents to enhance bacterial killing, and then, reducing the risk of infections in implantable materials.

Effect of degradation products of iron-bioresorbable implants on the physiological behavior of macrophages in vitro.

The degradation of bioresorbable metals in vivo changes the physicochemical properties in the environment of an implant, such as a stent in the artery wall, and may induce the alteration of the functions of the surrounding cells. The Fe-degradation, from bioresorbable stents, is a particularly intricate process because it leads to the release of soluble (SDP) and insoluble degradation products (IDP) of varied composition. Macrophages are involved in the resorption of the exogenous agents coming from degradation of these materials. In the present work an Fe0 ring, made with a pure Fe wire, in contact with macrophage cell cultures was used to simulate the behaviour of a biodegradable Fe-based implant in a biological environment. Non-invasive time-lapse optical microscopy was applied to obtain images of macrophages exposed to Fe-degradation products, without using staining to avoid distortions and artefacts. It was noticed that as metal degraded, the IDP formed in situ accumulated close to the Fe0 ring. In this zone, the macrophages showed a dynamic process of uptake of dark Fe-containing products, confirmed by SEM-EDX. These macrophages showed alterations in the morphology and decrease in the motility and viability. The inability of the macrophages to move and to degrade the engulfed products caused a long persistence of IDP in the zone closest to the metal. The deleterious effects of IDP accumulated close to the ring, were significantly worse than those observed in the experiments made with (1) concentrated salt solutions (Fe3+ salt 3 mM), with the same amount of precipitates but uniformly distributed in the well, and (2) diluted salt solutions (Fe3+ salt 1 mM) with mainly soluble species. The results were confirmed by standard staining protocols that revealed dead cells close to the Fe0 ring and oxidative stress in cells exposed to both soluble and insoluble species.

Is there any difference in the biological impact of soluble and insoluble degradation products of iron-containing biomaterials?

The interactions that could be built between the biomaterials and tissue- microenvironments are very complex, especially in case of degradable metals that generate a broad variety of degradation products. The interfacial problems are particularly relevant for Fe-based materials that have been proposed for the development of biodegradable implants. The cell metabolism could be affected by the accumulation of insoluble Fe-containing degradation products that has been observed in vitro and in vivo as a coarse granular brownish material around the implant. However, the relative importance of each Fe-species (soluble and insoluble) on the cellular behavior of the surrounding cells, particularly on the generation of reactive species (RS), is not completely elucidated. The aim of this study is to evaluate the processes occurring at the Fe-biomaterial/cells interfacial region, and to discriminate the effects of soluble and insoluble corrosion products released by the bulk metal (Fe- microparticles (Fe0p) or Fe0 ring) on the adjacent cells, mainly in relation to RS generation. With this purpose Fe0p and Fe0 ring were incubated with fibroblast cells (BALB/c 3T3 line) for 24 and 48h periods. Then different techniques were used, such as the dichlorofluorescein diacetate assay (DCFH2-DA) for detection of RS, acridine orange dye for cell viability, total protein content determinations, Prussian Blue staining and TEM observations. To individualize the effects of soluble and insoluble species, independent experiments with Fe3+-salts were performed. Overall data indicate that RS generation by cells exposed to the degradation products of Fe-based biomaterials is more dependent on the presence of insoluble products than on soluble Fe species.

Cytotoxicity of corrosion products of degradable Fe-based stents: relevance of pH and insoluble products.

Fe-based biodegradable metallic materials (Fe-BMMs) have been proposed for cardiovascular applications and are expected to disappear via corrosion after an appropriate period. However, in vivo studies showed that Fe ions release leads to accumulation of orange and brownish insoluble products at the biomaterial/cell interface. As an additional consequence, sharp changes in pH may affect the biocompatibility of these materials. In the present work, the experimental protocols were designed with the aim of evaluating the relative importance that these factors have on biocompatibility evaluation of BMMs. Mitochondrial activity (MTT assay) and thiobarbituric acid reactive substances (TBARS) assay on mammalian cells, exposed to 1-5 mM of added Fe3+ salt, were assessed and compared with results linked exclusively to pH effects. Soluble Fe concentration in culture medium and intracellular Fe content were also determined. The results showed that: (i) mitochondrial activity was affected by pH changes over the entire range of concentrations of added Fe3+ assayed, (ii) at the highest added Fe3+ concentrations (≥3 mM), precipitation was detected and the cells were able to incorporate the precipitate, that seems to be linked to cell damage, (iii) the extent of precipitation depends on the Fe/protein concentration ratio; and (iv) lipid peroxidation products were detected over the entire range of concentrations of added Fe3+. Hence, a new approach opens in the biocompatibility evaluation of Fe-based BMMs, since the cytotoxicity would not be solely a function of released (and soluble) ions but of the insoluble degradation product amount and the pH falling at the biomaterial/cell interface. The concentration of Fe-containing products at the interface depends on diffusional conditions in a very complex way that should be carefully analyzed in the future.