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.

Different Impact of Suspended Al2O3 Nanoparticles on Microbial Communities: Formation of 2D-Networks (Without Humic Acids) or 3D-Colonies (With Humic Acids).

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.

Environmentally Induced Changes of Commercial Carbon Nanotubes in Aqueous Suspensions. Adaptive Behavior of Bacteria in Biofilms.

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.

Staphylococcus aureus biofilm eradication by the synergistic effect exerted by PEG-coated silicon dots immobilized in silica films and light irradiation.

Immobilization of PEG-covered silicon dots, PEGSiDs, on glass substrates was performed following a simple strategy involving particle embedding by a sol-gel process forming a silica film on glass slides. The obtained films, denoted as fSiO x -PEGSiD, constitute a water-wettable, strongly supported, photoluminescent glass coating. The films showed high capacity for photosensitizing singlet oxygen (1O2) in the UVA when immersed in water. Staphylococcus aureus colonies formed on fSiO x -PEGSiDs modified glasses revealed the inhibition of bacterial adhesion and bacterial growth leading to the formation of loosely-packed and smaller S. aureus colonies. Upon 350 nm light irradiation of the biofilmed fSiO x -PEGSiDs -modified glasses, S. aureus growth was inhibited and bacteria killed reducing the number of living bacteria by three orders of magnitude. Eradication of attached bacteria was achieved by the synergistic effect exerted by a less adherent fSiO x -PEGSiDs surface that inhibits biofilm formation and the ability of the surface to photosensitize 1O2 to kill bacteria.

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.

Characterization and antimicrobial effect of a bioinspired thymol coating formed on titanium surface by one-step immersion treatment.

OBJECTIVE: To develop an antimicrobial and anti-adherent thymol (TOH)-containing coating on titanium (Ti) by a bioinspired one-step biocompatible method. METHODS: A nanolayer of adsorbed TOH (TOH-NL-Ti) was formed by an easy deep coating method on Ti surface. The treatment consists in a simple one-step immersion process in a TOH-containing solution. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), potentiodynamic electrochemical technique, open circuit potential records, Atomic Force Microscopy (AFM) and measurements of TOH release were used to characterize TOH-NL-Ti. Live/Dead staining and plate counting were employed to quantify attached and living adhered bacteria, respectively. Biocompatibility and cytotoxicity in fibroblastic and pre-osteoblastic cell lines were evaluated by acridine orange staining and MTT assay, respectively. RESULTS: TOH adsorbed on TOH-NL-Ti was detected by ATR-FTIR and electrochemical techniques. ATR-FTIR results showed that TOH nanofilms development involves spontaneous production of ketonic structures on Ti surface. AFM analysis revealed that the thickness of the TOH-NL was below 80 nm. Finally, microbiological assays confirmed that TOH-NL-Ti can inhibit the adhesion and kill attached bacteria leading to the eradication of leaving cells on its surface. After 24 h of biocidal release, the antimicrobial effect is also significant (a decrease of 3 orders in the number of attached bacteria). SIGNIFICANCE: The formation of TOH-NL-Ti nanolayer is a simple strategy able to be applied by not specially trained personnel, to reduce implant infection risks, ensure highly effective antimicrobial action and inhibition of bacterial adhesion on Ti surfaces without showing toxic effects for pre-osteoblastic and fibroblastic cells.

Comparative study of transdermal drug delivery systems of resveratrol: High efficiency of deformable liposomes.

Trans-resveratrol (3, 5, 4' trihydroxystilbene, RSV) is a natural compound that shows antioxidant, cardioprotective, anti-inflammatory and anticancer properties. The transdermal, painless application of RSV is an attractive option to other administration routes owing to its several advantages like avoiding gastrointestinal problems and first pass metabolism. However, its therapeutic potential is limited by its low solubility and low stability in water and the reduced permeability of stratum corneum. To overcome these inconveniences the encapsulation of this compound in a drug delivery system is proposed here. In order to find the best carrier for transdermal application of RSV various liposomal nanoparticulate carriers like conventional liposomes (L-RSV), deformable liposomes (LD-RSV), ultradeformable liposomes (LUD-RSV) and ethosomes (Etho-RSV) were assayed. Transmission electron microscopic (TEM) and dynamic light scattering (DLS) studies were performed to analyze the surface morphology of these carriers. Structural characterization for these formulations was performed by confocal Raman spectroscopy. The spectroscopic results were analysed in conjunction with calorimetric data to identify the conformational changes and stability of formulations in the different nanoparticles induced by the presence of RSV. Comparison of the results obtained with the different carrier systems (L-RSV, LD-RSV, LUD-RSV and Etho-RSV) revealed that the best RSV carrier was LD-RSV. The increase in the fluidity of the bilayers in the region of the hydrophobic chains of the phospholipid by ethanol probably facilitates the accommodation of the RSV in the bilayer and contributes to the improved encapsulation of RSV without affecting the mobility of this carrier.

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.

Polyethylene glycol-coated blue-emitting silicon dots with improved properties for uses in aqueous and biological environments.

Grafting of polyethylene glycol (PEG) to ultrasmall photoluminescent silicon dots (SiDs) is expected to improve and expand the applications of these particles to aqueous environments and biological systems. Herein we report a novel one-pot synthesis of robust, highly water compatible PEG-coated SiDs (denoted as PEG-SiDs) of (3.3 ± 0.5) nm size. The nanoparticles' synthesis is based on the liquid phase oxidation of magnesium silicide using PEG as reaction media and leading to high PEG density grafting. PEG-SiDs enhanced photophysical, photosensitising, and solution properties in aqueous environments are described and compared to those of 2 nm size PEG-coated SiDs with low PEG density grafting (denoted as PEG-NHSiDs) obtained from a multistep synthesis strategy. PEG-SiDs form highly dispersed suspensions in water showing stable photoluminescence and quantum yields of Φ = 0.13 ± 0.04 at 370 nm excitation in air-saturated suspensions. These particles exhibited the capacity of photosensitising the formation of singlet molecular oxygen, not observed for PEG-NHSiDs. PEG robust shielding of the silicon core luminescent properties is further demonstrated in bio-imaging experiments stressing the strong interaction between PEG-SiDs and Staphylococcus aureus smears by observing the photoluminescence of particles. PEG-SiDs were found to be nontoxic to S. aureus cells at concentrations of 100 mg ml-1, though a bacteriostatic effect on S. aureus biofilms was observed upon UV-A irradiation under conditions where light alone has no effect.

Delivery of fluorophores by calcium phosphate-coated nanoliposomes and interaction with Staphylococcus aureus biofilms.

The delivery capacity and mechanical stability of calcium phosphate (CaP) coated 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) liposomes free and adsorbed on bacterial surface was investigated introducing either acridine orange (AO) or 5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin (TMP) in the aqueous core of the liposomes. The obtained nanomaterials were thoroughly characterized by electron and optical microscopy and by fluorescence techniques. Distribution of the AO and TMP molecules between the aqueous liposomes core and the outer solution was demonstrated by the band shifts and broadening of the excitation-emission matrices and the modified Stern-Volmer model for fluorescence quenching. In aqueous suspensions, c.a. 40% of AO was released to the outer solution while only a small percentage of TMP was observed to reach the outer liposome surface. The nanoliposomes adhesion capacity and the leaking of fluorophore molecules to Staphylococcus aureus (S. aureus) biofilms were further evaluated. A close interaction between liposomes and S. aureus biofilm was evidenced by TEM and SEM imaging. Epifluorescence experiments demonstrated that CaP-coated liposomes have good biofilm staining capability after two hours incubation of the biofilms with the liposomes, thus supporting an important release of the fluorophores when in contact with the biofilm. Altogether, the obtained results strongly suggest that CaP-coated liposomes are capable of activating drug release when in presence of S. aureus biofilms and smears. The studies herein presented, indicate that CaP-coated liposomes are potential vehicles for the selective delivery of drugs to S. aureus biofilms, as is the case of the singlet oxygen photosensitizer TMP, a well known photodynamic antibacterial agent.

Time-Lapse Evaluation of Interactions Between Biodegradable Mg Particles and Cells.

Mg-based implants have promising applications as biodegradable materials in medicine for orthopedic, dental, and cardiovascular therapies. During wear and degradation microdebris are released. Time-lapse multidimensional microscopy (MM) is proposed here as a suitable tool to follow, in fixed intervals over 24-h periods, the interaction between cells and particles. Results of MM show interactions of macrophages (J774) with the magnesium particles (MgPa) that led to modifications of cell size and morphology, a decrease in duplication rate, and cell damage. Corrosion products were progressively formed on the surface of the particles and turbulence was generated due to hydrogen development. Changes were more significant after treating MgPa with potassium fluoride. In order to complement MM observations, membrane damage as detected by a lactase dehydrogenase (LDH) assay and mitochondrial activity as detected by a WST-1 assay with macrophages and osteoblasts (MC3T3-E1) were compared. A more significant concentration-dependent effect was detected for macrophages exposed to MgPa than for osteoblasts. Accordingly, complementary data showed that viability and cell cycle seem to be more altered in macrophages. In addition, protein profiles and expression of proteins associated with the adhesion process changed in the presence of MgPa. These studies revealed that time-lapse MM is a helpful tool for monitoring changes of biodegradable materials and the biological surrounding in real time and in situ. This information is useful in studies related to biodegradable biomaterials.

Degradation of bioabsorbable Mg-based alloys: Assessment of the effects of insoluble corrosion products and joint effects of alloying components on mammalian cells.

This work is focused on the processes occurring at the bioabsorbable metallic biomaterial/cell interfaces that may lead to toxicity. A critical analysis of the results obtained when degradable metal disks (pure Mg and rare earth-containing alloys (ZEK100 alloys)) are in direct contact with cell culture and those obtained with indirect methods such as the use of metal salts and extracts was made. Viability was assessed by Acridine Orange dye, neutral red and clonogenic assays. The effects of concentration of corrosion products and possible joint effects of the binary and ternary combinations of La, Zn and Mg ions, as constituents of ZEK alloys, were evaluated on a mammalian cell culture. In all cases more detrimental effects were found for pure Mg than for the alloys. Experiments with disks showed that gradual alterations in pH and in the amount of corrosion products were better tolerated by cells and resulted in higher viability than abrupt changes. In addition, viability was dependent on the distance from the source of ions. Experiments with extracts showed that the effect of insoluble degradation products was highly detrimental. Indirect tests with Zn ions revealed that harmful effects may be found at concentrations ≥ 150 µM and at ≥ 100 µM in mixtures with Mg. These mixtures lead to more deleterious effects than single ions. Results highlight the need to develop a battery of tests to evaluate the biocompatibility of bioabsorbable biomaterials.

Photodynamic inactivation induced by carboxypterin: a novel non-toxic bactericidal strategy against planktonic cells and biofilms of Staphylococcus aureus.

Microbial related contamination is of major concern and can cause substantial economic losses. Photodynamic inactivation (PDI) has emerged as a suitable approach to inhibit microorganism proliferation. In this work, PDI induced by 6-carboxypterin (Cap), a biocompatible photosensitizer (PS), was analyzed. The growth inhibition of Staphylococcus aureus exposed to artificial UV-A radiation and sunlight in the presence of Cap was investigated. After UV-A irradiation, 50 µM Cap was able to decrease by three orders (with respect to the initial value) the number of S. aureus cells in early biofilms. However, this concentration was 500 times higher than that needed for eradicating planktonic cells. Importantly, under solar exposure, 100 µM Cap was able to suppress sessile bacterial growth. Thus, this strategy is able to exert a bactericidal effect on sessile bacteria and to eradicate planktonic cells by exposing the Cap-containing sample to sunlight.

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.

Synergistic cytotoxic effects of ions released by zinc-aluminum bronze and the metallic salts on osteoblastic cells.

The use of copper-based alloys for fixed dental crowns and bridges is increasingly widespread in several countries. The aim of this work is to study the dissolution of a zinc-aluminum-bronze and the cytotoxic effects of the ions released on UMR-106 osteoblastic cell line. Two sources of ions were used: (1) ions released by the metal alloy immersed in the cell culture and (2) salts of the metal ions. Conventional electrochemical techniques, atomic absorption spectroscopy [to obtain the average concentration of ions (AC) in solution], and energy dispersive X-ray (EDX) spectroscopy analysis were used to study the corrosion process. Corrosion tests revealed a strong influence of the composition of the electrolyte medium and the immersion time on the electrochemical response. The cytotoxicity was evaluated with (a) individual ions, (b) combinations of two ions, and (c) the mixture of all the ions released by a metal disc of the alloy. Importantly, synergistic cytotoxic effects were found when Al-Zn ion combinations were used at concentration levels lower than the cytotoxic threshold values of the individual ions. Cytotoxic effects in cells in the vicinity of the metal disc were also found. These results were interpreted considering synergistic effects and a diffusion controlled mechanism that yields to concentration levels, in the metal surroundings, several times higher than the measured AC value.

Chlorhexidine delivery system from titanium/polybenzyl acrylate coating: evaluation of cytotoxicity and early bacterial adhesion.

OBJECTIVES: The formation of biofilms on titanium dental implants is one of the main causes of failure of these devices. Streptococci are considered early colonizers that alter local environment favouring growing conditions for other colonizers. Chlorhexidine (CHX) is so far the most effective antimicrobial treatment against a wide variety of Gram-positive and Gram-negative organisms as well as fungi. This study was designed to develop a CHX delivery system appropriate for healing caps and abutments, with suitable drug release rate, effective as antimicrobial agent, and free of cytotoxic effects. METHODS: Polybenzyl acrylate (PBA) coatings with and without CHX (Ti/PBA and Ti/PBA-CHX, respectively) and different drug loads (0.35, 0.70, and 1.40%, w/w) were assayed. The cytotoxic effect of CHX released from the different substrates on UMR106 cells was tested by alkaline phosphatase specific activity (ALP), and microscopic evaluation of the cells. Non-cytotoxic drug load (0.35%, w/w) was selected to evaluate the antimicrobial effectiveness of the system using a microbial consortium of Streptococcus species. RESULTS: The kinetic profile of CHX delivered by Ti/PBA-CHX showed an initial fast release rate followed by a monotonic increase of delivered mass over 48 h. The number of attached bacteria decreased in the following order: Ti>Ti/PBA>Ti/PBA-0.35. CONCLUSIONS: PBA-0.35 coating is effective to inhibit the adhesion of early colonizers on Ti without any cytotoxic effect on UMR-106 cells.

Organization of Pseudomonas fluorescens on chemically different nano/microstructured surfaces.

This paper describes bacterial organization on nano/micropatterned surfaces with different chemical properties, which show different interactions with the biological systems (inert, biocompatible, and bactericide). These surfaces were prepared by molding techniques and exposed to Pseudomonas fluorescens (P. fluorescens) cultures. Results from atomic force microscopy and optical imaging demonstrate that the structure of P. fluorescens aggregates is strongly dependent on the surface topography while there is no clear linking with the physical-chemical surface properties (charge and contact angle) of the substrate immersed in abiotic culture media. We observe that regardless of the material when the surface pattern matches the bacterial size, bacterial assemblages involved in surface colonization are disorganized. The fact there is not a relationship between surface chemistry and bacterial organization can be explained by the coverage of the surfaces by adsorbed organic species coming from the culture medium. Viability assays indicate that copper behaves as a toxic substrate despite the presence of adsorbed molecules. The combination of surface traps and biocidal activity could act synergistically as a suitable strategy to limit bacterial spreading on implant materials.

Submicron trenches reduce the Pseudomonas fluorescens colonization rate on solid surfaces.

Bacterial adhesion and spreading on biomaterials are considered key features of pathogenicity. Roughness and topography of the substrate have been reported to affect bacterial adhesion, but little is known about their effect on spreading. Submicron row and channel tuning with bacterial diameter (S2) were designed to test bacterial motility on these surfaces. Random nanometer-sized structures (S1) were used as controls. Optical microscopy and AFM were employed to detect biological and surface pattern details in the micro- and nanoscale, respectively. Results showed that motility strategies (flagella orientation, elongation, aggregation in rafts, formation of network structures, and development of a bacterial frontier) were affected by the presence of submicropatterns. Importantly, the rate of bacterial spreading on S2 was significantly reduced and influenced by the orientation of the submicropatterns. Consequently, submicroengineered substrates could be employed as a tool to downgrade bacterial colonization. Such patterns could impact on the design of proper engineered structures to control biofilm spreading on solid surfaces.

Cytotoxicity of copper ions released from metal: variation with the exposure period and concentration gradients.

The aim of this work is to contribute to the elucidation of the cytotoxic process caused by the copper ions released from the biomaterials. Clonal cell lines UMR106 were used in the experiments. Copper ions were obtained from two different sources: copper salts and metal dissolution. Experiments carried out with constant ion concentrations (copper salts) were compared with those with concentrations that vary with time and location (dissolution of the metal). Present results and others previously reported could be interpreted through mathematical models that describe: (1) the variation of concentration of copper ions with time and location within a biofilm and (2) the variation of the killing rate with the concentration of the toxic ion and time. The large number of dead cells found near the copper sample with an average ion concentration below the toxic limit could be interpreted bearing in mind that these cells should be exposed to a local concentration higher than this limit. A logarithmic dependence between the number of cells and exposure time was found for nearly constant ion concentrations. Apparent discrepancies, observed when these results and those of different researchers were contrasted, could be explained considering the dissimilar experimental conditions such as the source of the ions and their local concentration at real time.