Monica Fernández Lorenzo de Mele

“Green’’ nanotechnologies have emerged as environmentally friendly alternatives against microbial multidrug-resistant biofilms. In this study, bactericidal “green” nanolayers (NL) were developed on Ti surfaces using two isomeric phytocompounds, carvacrol (Carv-Ti-NL) and thymol (TOH-Ti-NL). These NLs were fabricated using a one-step immersion treatment method based on a simple and spontaneous self-assembly process. Both NLs revealed strong antimicrobial activity, displaying anti-biofilm and biocidal effects. Notably, TOH-Ti-NL exhibited superior osteogenic performance compared to Carv-Ti-NL, as evidenced by enhanced pre-osteoblast cell attachment and growth, and the production of ALP, collagen type I and Ca ²⁺ deposition. In contrast, fibroblastic cells exhibited reduced attachment on TOH-Ti-NL and enhanced proliferation on Carv-Ti-NL. Considering the biological differential effects, the physicochemical properties of these conformational isomers’ NLs were studied to elucidate potential differences that could impact on cell response. Although the ATR-FTIR spectra of the NLs were similar and indicated the spontaneous oxidation of Carv and TOH leading to ketonic structures, distinct contributions were observed after the electrooxidation of each NL. Slight differences in hydrophilicity were found for both nanostructures, but higher roughness was found for TOH-Ti-NL. Furthermore, the release curves of Carv and TOH from the NLs revealed distinct profiles over time. Overall, Carv and TOH formed self-assembled layers on Ti able to eradicate Staphylococcus aureus biofilms. Their different physical and chemical characteristics induced distinct responses from eukaryotic cells attached to the NLs. Given these characteristics one might envisage the use of either Carv-Ti-NL or TOH-Ti-NL in order to fine-tune specific chemical physical properties of Ti-based implants. Graphical

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.

The use of metal-based and, particularly, Al₂O₃ nanoparticles (Al₂O₃-NP) for diverse purposes is exponentially growing. However, the growth of such promissory market is not accompanied by a parallel extensive investigation related to the impact of this pollution on groundwater and biological systems. Pseudomonas species, ubiquitous, environmentally critical microbes, frequently respond to stress conditions with diverse strategies that generally include extracellular polymeric substances (EPS) formation. The aim of this study is to report that changes in the aqueous environment, particularly, the addition of Al₂O₃-NP without and with humic acids, induce different adaptive strategies of Pseudomonas aeruginosa early biofilms. To this purpose, early biofilms were incubated in diluted culture media without (control) and with Al₂O₃-NP, and with humic acids (HA-control, HA-Al₂O₃-NP) for 24 h. 3D colonies with EPS strings and isolated bacteria in their surroundings were detected in the control biofilms. Unlikely, an unusual adaptive behaviour was developed in the presence of Al₂O₃-NP. Bacteria opt to disassemble the 3D arrangements and to implement a 2D network promoting morphological and size changes of bacterial cells (small coccoid shapes). Remarkably, this strategy allows their temporarily non-EPS-depending survival without decreasing the number of cells. This behaviour was not observed with ZnO-NP, HA-Al₂O₃-NP, or HA-ZnO-NP. Physicochemical analysis revealed that HA were adsorbed on Al₂O₃-NP and promoted the Al(III) ions complexation. This supports the hypothesis that the reduction of toxicity of Al ions and the 3D colony formation in the presence of HA-Al₂O₃-NP is promoted by the complexation of the metal ions with HA components. Graphical abstract

The use of metal-based and, particularly, Al₂O₃ nanoparticles (Al₂O₃₋NP) for diverse purposes is exponentially growing. However, the growth of such promissory market is not accompanied by a parallel extensive investigation related to the impact of this pollution on groundwater and biological systems. Pseudomonas species, ubiquitous, environmentally critical microbes, frequently respond to stress conditions with diverse strategies that generally include extracellular polymeric substances (EPS) formation. The aim of this study is to report that changes in the aqueous environment, particularly, the addition of Al₂O₃-NP without and with humic acids, induce different adaptive strategies of P. aeruginosa early biofilms. To this purpose, early biofilms were incubated in diluted culture media without (control) and with Al₂O₃-NP, and with humic acids (HA-control, HA-Al₂O₃-NP) for 24h. 3D colonies with EPS strings and isolated bacteria in their surroundings were detected in the control biofilms. Unlikely, an unusual adaptive behaviour was developed in presence of Al₂O₃-NP. Bacteria opt to disassemble the 3D arrangements, and to implement a 2D network promoting morphological and size changes of bacterial cells (small coccoid shapes). Remarkably, this strategy allows their temporarily non-EPS-depending survival without decreasing the number of cells. This behaviour was not observed with ZnO-NP, HA-Al₂O₃-NP, or HA-ZnO-NP. Physicochemical analysis revealed that HA were adsorbed on Al₂O₃-NP and promoted the Al(III) ions complexation. This supports the hypothesis that the reduction of toxicity of Al ions and the 3D colony formation in presence of HA-Al₂O₃-NP is promoted by the complexation of the metal ions with HA components.

The effects of environmental factors such as sunlight irradiation and the presence of humic acid (HA) on the physicochemical properties of commercial multiwall carbon nanotubes (MWCNT) suspended in a simulated inorganic matrix (SIM) and their impacts on bacteria growing in biofilms were evaluated. Both solar irradiation and the presence of HA lead to the dissolution of adsorbed metals on the MWCNT, which are residues of synthesis catalysts. Also, preferential adsorption of certain HA components on the MWCNT induces important modifications in the aliphatic/aromatic relationship of HA components in solution and the generation and release of new moieties. Results demonstrated that the variation of such physicochemical parameters strongly affects the interactions of MWCNT with Pseudomonas aeruginosa sessile bacteria. Thus, the number of attached bacteria increased, and stress responses such as decrease in bacterial size were found in the presence of sunlight-irradiated MWCNT with a particular distribution of extracellular polymeric substances (EPS) strands. A shielding effect was observed when HA was added. It was concluded that physicochemical alterations caused by environmental conditions (with/without irradiation, presence/absence of HA) on MWCNT-containing SIM trigger distinctive adaptive behavior of bacteria in biofilms. This information must be taken into account in the development of biologically assisted treatments for organic metal co-contamination of MWCNT-containing media since MWCNT discharge alters the physicochemical properties and composition of the aqueous environment and the response of the biofilms that interact with it.

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.

The interactions between macrophages and bioabsorbable metals during their degradation may lead to altered functions of these cells. The evaluation of this impact "in vivo" is tricky and the use of cell cultures is a preliminary valuable model. In the Fe-biodegradation case, the system is complex because Fe-containing materials may lead to corrosion products of different composition and solubility. Thus, a meticulous study about interactions between macrophages and Fe-based materials, without using staining to avoid its non-desired effects, is proposed here. Multidimensional microscopy was applied to obtain images of unstained macrophages (J774A.1) exposed to Feº rings. Images were taken every 15 min between 24-48h of exposure. For the first time for this system software tools for single-cell tracking (TrackMate - FIJI) was applied for the analysis of the spatiotemporal dependence of different parameters. As metal degrades, macrophages capture and accumulate the degradation products as a dark insoluble material. Significant changes in macrophage morphology, motility, synchrony of duplications and in survival of cells, according to the distance to the metal, were noticed. They account for the deep impact of Fe-degradation products on the macrophages close to the Fe-containing implant.