Biocompatibility and inflammatory response of silver tungstate, silver molybdate, and silver vanadate microcrystals.

Silver tungstate (α-Ag2WO4), silver molybdate (ß-Ag2MoO4), and silver vanadate (α-AgVO3) microcrystals have shown interesting antimicrobial properties. However, their biocompatibility is not yet fully understood. Cytotoxicity and the inflammatory response of silver-containing microcrystals were analyzed in THP-1 and THP-1 differentiated as macrophage-like cells, with the alamarBlue™ assay, flow cytometry, confocal microscopy, and ELISA. The present investigation also evaluated redox signaling and the production of cytokines (TNFα, IL-1ß, IL-6, and IL-8) and matrix metalloproteinases (MMP-8 and -9). The results showed that α-AgVO3 (3.9 µg/mL) did not affect cell viability (p > 0.05). α-Ag2WO4 (7.81 µg/mL), ß-Ag2MoO4 (15.62 µg/mL), and α-AgVO3 (15.62 µg/mL) slightly decreased cell viability (p ≤ 0.003). All silver-containing microcrystals induced the production of O2 - and this effect was mitigated by Reactive Oxygen Species (ROS) scavenger and N-acetylcysteine (NAC). TNFα, IL-6 and IL-1ß were not detected in THP-1 cells, while their production was either lower (p ≤ 0.0321) or similar to the control group (p ≥ 0.1048) for macrophage-like cells. The production of IL-8 by both cellular phenotypes was similar to the control group (p ≥ 0.3570). The release of MMP-8 was not detected in any condition in THP-1 cells. Although MMP-9 was released by THP-1 cells exposed to α-AgVO3 (3.9 µg/mL), no significant difference was found with control (p = 0.7). Regarding macrophage-like cells, the release of MMP-8 and -9 decreased in the presence of all microcrystals (p ≤ 0.010). Overall, the present work shows a promising biocompatibility profile of, α-Ag2WO4, ß-Ag2MoO4, and α-AgVO3 microcrystals.

Extracellular lipids of Candida albicans biofilm induce lipid droplet formation and decreased response to a topoisomerase I inhibitor in dysplastic and neoplastic oral cells.

OBJECTIVE: Some microorganisms, i.e., Candida albicans, have been associated with cancer onset and development, although whether the fungus promotes cancer or whether cancer facilitates the growth of C. albicans is unclear. In this context, microbial-derived molecules can modulate the growth and resistance of cancer cells. This study isolated extracellular lipids (ECL) from a 36-h Candida albicans biofilm incubated with oral dysplastic (DOK) and neoplastic (SCC 25) cells, which were further challenged with the topoisomerase I inhibitor camptothecin (CPT), a lipophilic anti-tumoral molecule. METHODOLOGY: ECL were extracted from a 36-h Candida albicans biofilm with the methanol/chloroform precipitation method and identified with Nuclear Magnetic Resonance (1H-NMR). The MTT tetrazolium assay measured ECL cytotoxicity in DOK and SCC 25 cells, alamarBlue™ assessed cell metabolism, flow cytometry measured cell cycle, and confocal microscopy determined intracellular features. RESULTS: Three major classes of ECL of C. albicans biofilm were found: phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The ECL of C. albicans biofilm had no cytotoxic effect on neither cell after 24 hours, with a tendency to disturb the SCC 25 cell cycle profile (without statistical significance). The ECL-induced intracellular lipid droplet (LD) formation on both cell lines after 72 hours. In this context, ECL enhanced cell metabolism, decreased the response to CPT, and modified intracellular drug distribution. CONCLUSION: The ECL (PI, PC, and PG) of 36-h Candida albicans biofilm directly interacts with dysplastic and neoplastic oral cells, highlighting the relevance of better understanding C. albicans biofilm signaling in the microenvironment of tumor cells.

Disclosing the Biocide Activity of α-Ag2-2xCuxWO4 (0 ≤ x ≤ 0.16) Solid Solutions.

In this work, α-Ag2-2xCuxWO4 (0 ≤ x ≤ 0.16) solid solutions with enhanced antibacterial (against methicillin-resistant Staphylococcus aureus) and antifungal (against Candida albicans) activities are reported. A plethora of techniques (X-ray diffraction with Rietveld refinements, inductively coupled plasma atomic emission spectrometry, micro-Raman spectroscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, field emission scanning electron microscopy, ultraviolet-visible spectroscopy, photoluminescence emissions, and X-ray photoelectron spectroscopy) were employed to characterize the as-synthetized samples and determine the local coordination geometry of Cu2+ cations at the orthorhombic lattice. To find a correlation between morphology and biocide activity, the experimental results were sustained by first-principles calculations at the density functional theory level to decipher the cluster coordinations and electronic properties of the exposed surfaces. Based on the analysis of the under-coordinated Ag and Cu clusters at the (010) and (101) exposed surfaces, we propose a mechanism to explain the biocide activity of these solid solutions.

Inactivation of SARS-CoV-2 by a chitosan/α-Ag2WO4 composite generated by femtosecond laser irradiation.

In the current COVID-19 pandemic, the next generation of innovative materials with enhanced anti-SARS-CoV-2 activity is urgently needed to prevent the spread of this virus within the community. Herein, we report the synthesis of chitosan/α-Ag2WO4 composites synthetized by femtosecond laser irradiation. The antimicrobial activity against Escherichia coli, Methicilin-susceptible Staphylococcus aureus (MSSA), and Candida albicans was determined by estimating the minimum inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC). To assess the biocompatibility of chitosan/α-Ag2WO4 composites in a range involving MIC and MBC/MFC on keratinocytes cells (NOK-si), an alamarBlue™ assay and an MTT assay were carried out. The SARS-CoV-2 virucidal effects was analyzed in Vero E6 cells through viral titer quantified in cell culture supernatant by PFU/mL assay. Our results showed a very similar antimicrobial activity of chitosan/α-Ag2WO4 3.3 and 6.6, with the last one demonstrating a slightly better action against MSSA. The chitosan/α-Ag2WO4 9.9 showed a wide range of antimicrobial activity (0.49-31.25 µg/mL). The cytotoxicity outcomes by alamarBlue™ revealed that the concentrations of interest (MIC and MBC/MFC) were considered non-cytotoxic to all composites after 72 h of exposure. The Chitosan/α-Ag2WO4 (CS6.6/α-Ag2WO4) composite reduced the SARS-CoV-2 viral titer quantification up to 80% of the controls. Then, our results suggest that these composites are highly efficient materials to kill bacteria (Escherichia coli, Methicillin-susceptible Staphylococcus aureus, and the yeast strain Candida albicans), in addition to inactivating SARS-CoV-2 by contact, through ROS production.

Antifungal Activity and Biocompatibility of α-AgVO3, α-Ag2WO4, and ß-Ag2MoO4 Using a Three-Dimensional Coculture Model of the Oral Mucosa.

Fungal infections have become a major concern in the medical community, especially those caused by Candida spp. Within this species, Candida albicans stands out for being an opportunistic commensal fungus that can cause superficial and invasive infections. Current antifungal therapy involves the local and/or systemic use of drugs such as azoles, polyenes, and echinocandins. These antifungals are based on highly specific target sites, and the development of resistance may occur with changes in the enzymatic pathways that serve as the drug targets. Thus, the development of new antifungal drugs is highly recommended to prevent drug resistance. The present investigation evaluated the antifungal activity of silver-containing microcrystals such as silver vanadate (α-AgVO3), silver tungstate (α-Ag2WO4), and silver molybdate (ß-Ag2MoO4). In addition to having antimicrobial activity, such compounds should not cause damage to underlying tissues. Thus, to better assess the biocompatibility of new compounds, a new three-dimensional (3D) coculture model involving three cell lines was developed. The validation of the model was based on fluorescent markers and confocal laser microscopy. The biocompatibility of silver-containing microcrystals was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 3D coculture was infected with C. albicans biofilm and challenged with α-AgVO3, α-Ag2WO4, and ß-Ag2MoO4. The action of microcrystals on C. albicans biofilm was evaluated by colony-forming units (CFU/ml) and LIVE/DEAD staining. In addition, production of proinflammatory cytokines interleukin 6 (IL-6), IL-8, IL-1ß, and tumor necrosis factor α (TNF-α) was measured by cytometric bead array kit using flow cytometry. The 3D coculture model described here proved to be adequate to assess both the biocompatibility of the new materials and the infectious processes. Regarding the biocompatibility of the microcrystals, only α-AgVO3 (15.62 µg/ml) showed a decrease in cell viability. The antibiofilm activity of α-Ag2WO4 was similar to that of the standard drug (fluconazole). Although α-Ag2WO4 was able to induce the production of IL-6, IL-8, and IL-1ß, no differences in cytokine production were observed between noninfected and infected models treated with this microcrystal. ß-Ag2MoO4 inhibits the production of TNF-α in the infected model; however, it showed no antibiofilm activity. Based on the biocompatibility and antifungal findings, α-Ag2WO4 is a promising material for treating C. albicans infection.

Extracellular lipids of Candida albicans biofilm induce lipid droplet formation and decreased response to a topoisomerase I inhibitor in dysplastic and neoplastic oral cells

Abstract Objective Some microorganisms, i.e., Candida albicans, have been associated with cancer onset and development, although whether the fungus promotes cancer or whether cancer facilitates the growth of C. albicans is unclear. In this context, microbial-derived molecules can modulate the growth and resistance of cancer cells. This study isolated extracellular lipids (ECL) from a 36-h Candida albicans biofilm incubated with oral dysplastic (DOK) and neoplastic (SCC 25) cells, which were further challenged with the topoisomerase I inhibitor camptothecin (CPT), a lipophilic anti-tumoral molecule. Methodology ECL were extracted from a 36-h Candida albicans biofilm with the methanol/chloroform precipitation method and identified with Nuclear Magnetic Resonance (1H-NMR). The MTT tetrazolium assay measured ECL cytotoxicity in DOK and SCC 25 cells, alamarBlue™ assessed cell metabolism, flow cytometry measured cell cycle, and confocal microscopy determined intracellular features. Results Three major classes of ECL of C. albicans biofilm were found: phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The ECL of C. albicans biofilm had no cytotoxic effect on neither cell after 24 hours, with a tendency to disturb the SCC 25 cell cycle profile (without statistical significance). The ECL-induced intracellular lipid droplet (LD) formation on both cell lines after 72 hours. In this context, ECL enhanced cell metabolism, decreased the response to CPT, and modified intracellular drug distribution. Conclusion The ECL (PI, PC, and PG) of 36-h Candida albicans biofilm directly interacts with dysplastic and neoplastic oral cells, highlighting the relevance of better understanding C. albicans biofilm signaling in the microenvironment of tumor cells.

Insights into the activation of oral keratinocyte cell death by Candida albicans and Staphylococcus aureus biofilms.

Polymicrobial biofilms comprising Candida albicans and Staphylococcus aureus can increase the frequency and severity of oral diseases. This study assessed oral keratinocyte cell death, apoptosis and/or necrosis, promoted by soluble factors from single and dual biofilms of S. aureus and C. albicans. The soluble factors were obtained from the 16-h biofilm growth media. Cell viability was assessed by MTT and cell membrane damage by LDH. SEM was used for morphology changes. Assessment of apoptosis and necrosis was performed using annexin V and propidium iodide and caspases -2, -3, -6, -8 and -9. Statistical analysis was conducted with ANOVA and Tukey tests (α = 5%). Dual biofilms promoted the greatest harmful effect on oral cells, with a viability rate of 31.76%, damage to cell membranes and LDH released. Dual biofilms also induced higher percentages of necrotic cells (24.95%). Apoptosis was associated with caspases -2, -3, -6 and -8 activation.

Proto-Oncogenes and Cell Cycle Gene Expression in Normal and Neoplastic Oral Epithelial Cells Stimulated With Soluble Factors From Single and Dual Biofilms of Candida albicans and Staphylococcus aureus.

This study was aimed at analyzing proto-oncogenic signaling pathway activation in normal oral keratinocytes (NOK-si) and neoplastic cell lines (SCC 25 and Detroit 562) stimulated with metabolites (soluble factors) from single and dual biofilms of Candida albicans and Staphylococcus aureus. Soluble factors (SF) from early (16-h) and mature (36-h) biofilms of C. albicans and S. aureus were collected and incubated with cell cultures, which were subsequently evaluated using gene expression via RT-qPCR, cell viability via AlamarBlueTM, and flow cytometry cell cycle analysis. In general, exposure to the SF of early and mature biofilms from C. albicans and dual species caused a major reduction in NOK-si cell viability and enhanced the sub G0 phase. This led to a decrease in gene expression. However, in this cell line, SF of S. aureus biofilms upregulated the CDKN1A gene followed by the maintenance of cell viability and a significant increase in the G2/M population. For tumor cells, SCC 25 and Detroit 562, the stimuli of SF biofilms upregulated oncogenes such as hRAS and mTOR, as well as Bcl-2 and CDKN1A. SCC 25 and Detroit 562 cells could survive even after 24 h of stimuli from both SF (early and mature). This occurred without significant changes taking place in the cell cycle progression for SCC 25, and with a significant tendency to increase the G2/M phase for Detroit 562. These results point to the fact that metabolites from prevalent clinical fungal and bacterial biofilms, C. albicans and S. aureus, can disrupt the homeostasis of normal and neoplastic oral epithelial cells. This changes proto-oncogenes' expression, specifically PI3KCA, hRAS, mTOR, BRAF, and cell cycle genes CDKN1A and Bcl-2, thus causing a disturbance in cell viability, survival, and the cell cycle profile.

A long-term controlled drug-delivery with anionic beta cyclodextrin complex in layer-by-layer coating for percutaneous implants devices.

This study demonstrated a drug-delivery system with anionic beta cyclodextrin (ß-CD) complexes to retain tetracycline (TC) and control its release from multilayers of poly(acrylic acid) (PAA) and poly(l-lysine) (PLL) in a ten double layers ([PAA/PLL]10) coating onto titanium. The drug-delivery capacity of the multilayer system was proven by controlled drug release over 15 days and sustained released over 30 days. Qualitative images confirmed TC retention within the layer-by-layer (LbL) over 30 days of incubation. Antibacterial activity of TC/anionic ß-CD released from the LbL was established against Staphylococcus aureus species. Remarkably, [PAA/PLL]10/TC/anionic ß-CD antibacterial effect was sustained even after 30 days of incubation. The non-cytotoxic effect of the multilayer system revealed normal human gingival fibroblast growth. It is expected that this novel approach and the chemical concept to improve drug incorporation into the multilayer system will open up possibilities to make the drug release system more applicable to implantable percutaneous devices.

Selective Synthesis of α-, ß-, and γ-Ag2WO4 Polymorphs: Promising Platforms for Photocatalytic and Antibacterial Materials.

Silver tungstate (Ag2WO4) shows structural polymorphism with different crystalline phases, namely, orthorhombic, hexagonal, and cubic structures that are commonly known as α, ß, and γ, respectively. In this work, these Ag2WO4 polymorphs were selectively and successfully synthesized through a simple precipitation route at ambient temperature. The polymorph-controlled synthesis was conducted by means of the volumetric ratios of the silver nitrate/tungstate sodium dehydrate precursors in solution. The structural and electronic properties of the as-synthesized Ag2WO4 polymorphs were investigated by using a combination of X-ray diffraction and Rietveld refinements, X-ray absorption spectroscopy, X-ray absorption near-edge structure spectroscopy, field-emission scanning electron microscopy images, and photoluminescence. To complement and rationalize the experimental results, first-principles calculations, at the density functional theory level, were carried out, leading to an unprecedented glimpse into the atomic-level properties of the morphology and the exposed surfaces of Ag2WO4 polymorphs. Following the analysis of the local coordination of Ag and W cations (clusters) at each exposed surface of the three polymorphs, the structure-property relationship between the morphology and the photocatalytic and antibacterial activities against amiloride degradation under ultraviolet light irradiation and methicillin-resistant Staphylococcus aureus, respectively, was investigated. A possible mechanism of the photocatalytic and antibacterial activity as well the formation process and growth of the polymorphs is also explored and proposed.

Long-Term Effect of Daily Chemical Disinfection on Surface Topography and Candida Albicans Biofilm Formation on Denture Base and Reline Acrylic Resins.

PURPOSE: This study investigated the effect of long-term daily chemical disinfection on the topographic and Candida albicans biofilm formation on a denture base resin and a reline acrylic resin. MATERIAL AND METHODS: Circular samples (14 × 1.2 mm) were fabricated from a denture base (Vipi Wave) and reline acrylic resins (Tokuyama Rebase Fast II). Samples were kept in 50 ml of distilled water (48 h at 37°C). Subsequently, the samples were immersed in five different solutions: 0.5% sodium hypochlorite; 3.8% sodium perborate; 2% chlorhexidine gluconate; apple vinegar containing 4% maleic acid; and distilled water (control group). The specimen was immersed in the solutions for 8 h daily and transferred to distilled water at 37°C for more 16 h. The surface topographic and Candida albicans (ATCC 90028) biofilm formation were evaluated at baseline (before chemical disinfection) and after 1, 3 and 6 months of immersion. The surface topographic was evaluated by arithmetical roughness average (Ra) and scanning electron microscope (SEM), while the biofilm formation was evaluated by colony-forming units (CFU/ml) method and Alamar Blue assay (cell metabolism). The results were evaluated by three-way analysis of variance (ANOVAs) and post-hoc tests (α = 0.05). RESULTS: The results showed statistically significant effects from the type of acrylic resin (p = 0.029) and time (p <0.001) on the roughness of the specimen. In general, the reline resin had higher roughness than the denture base resin. In addition, the roughness of the samples after 1, 3 and 6 months of immersion in the cleaning solutions was higher than at baseline. In relation to the microbiological assays, there were no statistically significant differences (p >0.055) in the CFU/ml values of the biofilms among the different resins, periods of time and cleaning solutions. Considering the metabolism of the cells within the biofilms, the results showed that, at baseline, it was statistically significantly higher (p <0.05) than after 1, 3 and 6 months of storage. The SEM images showed that all disinfectant solutions provided surface changes of both acrylic resins (base and reline) after 1, 3 and 6 months of immersion. CONCLUSIONS: The roughness of both acrylic resins was affected by the disinfection in all cleaning agents, increasing over time, and this effect was more evident in the reline acrylic resin group. This surface change was also observed in the SEM images. While the number of cells within the biofilms was not affected by immersion in the cleaning agents, their metabolism was lower after 1, 3 and 6 months of immersion.

Unvealing the role of ß-Ag2MoO4 microcrystals to the improvement of antibacterial activity.

Crystal morphology with different surfaces is important for improving the antibacterial activity of materials. In this experimental and theoretical study, the antibacterial activity of ß-Ag2MoO4 microcrystals against the Gram-positive bacteria, namely, methicillin-resistant Staphylococcus aureus (MRSA), and the Gram-negative bacteria, namely, Escherichia coli (E. coli), was investigated. In this study, ß-Ag2MoO4 crystals with different morphologies were synthetized by a simple co-precipitation method using three different solvents. The antimicrobial efficacy of the obtained microcrystals against both bacteria increased according to the solvent used in the following order: water < ammonia < ethanol. Supported by experimental evidence, a correlation between morphology, surface energy, and antibacterial performance was established. By using the theoretical Wulff construction, which was obtained by means of density functional calculations, the morphologies with large exposition of the (001) surface exhibited superior antibacterial activity. This study provides a low cost route for synthesizing ß-Ag2MoO4 crystals and a guideline for enhancing the biological effect of biocides on pathogenic bacteria by the morphological modulation.

Antifungal activity and biocompatibility of α-AgVO3 microcrystals: A promising material against oral Candida disease.

The number of studies on microcrystals containing silver has increased in recent decades. Among the silver-containing microcrystals, α-AgVO3 has gained prominence owing to its polymorphism that allows it to exert interesting antimicrobial activity against pathogenic microorganisms. The aim of this study was to evaluate the antifungal activity and cytotoxicity of three different α-AgVO3 microcrystals when in solution. α-AgVO3 microcrystals were synthesized using the co-precipitation method at three different temperatures (10 °C, 20 °C, and 30 °C), and then characterized by X-ray diffraction and scanning electron microscopy. The antifungal activity of α-AgVO3 microcrystals against Candida albicans was determined by estimating the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Fluorescence images were obtained to confirm antifungal concentrations. To assess the biocompatibility of microcrystals applied at MIC and MFC on keratinocytes cells (NOK-si), an Alamar Blue assay, scanning electron microscopy, and a DNA gel integrity test were carried out. The quantitative and qualitative results showed that, regardless of the co-precipitation method used to synthetize α-AgVO3 microcrystals, C. albicans growth was visibly inhibited at 3.9 µg/mL (MIC) and completely inhibited at 15.62 µg/mL (MFC). The cytotoxic and genotoxic outcomes revealed that the MIC and MFC concentrations did not affect NOK-si cell morphology, proliferation, or DNA integrity. The search for new antimicrobial materials has been the focus of the research community recently because of increases in microbial resistance. The findings reported herein demonstrate a novel antifungal and non-cytotoxic material that could be used in biomedical and dental applications.

Low-temperature plasma on peri-implant-related biofilm and gingival tissue.

BACKGROUND: Evaluate the effect of low-temperature plasma (LTP) on an anaerobic biofilm and on the biological response of an in vitro reconstituted gingival epithelium tissue. METHODS: Porphyromonas gingivalis W83 biofilm was cultured on titanium discs and reconstituted gingival tissues were submitted to similar treatment conditions. TREATMENTS: LTP1-plasma treatment for 1 minute, LTP3-plasma treatment for 3 minute, CHX-0.2% chlorhexidine for 1 minute, GAS-gas only (no plasma) for 3 minute, and NEGATIVE-no treatment. TRITON group was included as a positive control for tissue analysis. Counting of viable colony forming units (CFU/mL) and confocal laser scanning microscopy were performed to evaluate LTP's antimicrobial effect. EpiGingival tissue was evaluated through cytotoxocity, viability, histology, and imunnohistochemistry (Ki67, vascular endothelial growth factor-A vascular endothelial growth factor A [VEGF-A], and terminal deoxynucleotidyl transferase dUTP nick end labeling terminal deoxynucleotidyl transferase dutp nick end labeling [TUNEL] expression). RESULTS: LTP1 and LTP3 presented significantly different reduced CFU/mL reduction in comparison to the negative control (Ρ < 0.001), but it was not as effective as the positive control (CHX). Low cytotoxicity and high viability were observed in gingival epithelium of NEGATIVE, GAS, CHX, and both LTP groups. The morphologic analysis of gingival epithelium revealed minor cell damage in the plasma groups (score 1). LTP1, LTP3, GAS, and NEGATIVE groups exhibited less than 5% of basal layer positive cells. LTP1, LTP3, GAS, and CHX groups were not positive for TUNEL assay. LTP1 and LTP3 showed the most positivity for VEGF. CONCLUSIONS: LTP treatment can be considered as an effective method for reducing P. gingivalis biofilm on implant surfaces, while being safe for the gingival epithelium. Furthermore, plasma treatment may be associated with cell repair.

Promising effects of silver tungstate microcrystals on fibroblast human cells and three dimensional collagen matrix models: A novel non-cytotoxic material to fight oral disease.

Silver tungstate (α-Ag2WO4) microcrystals have shown encouraging results regarding their antimicrobial activity. However, in addition to the promising outcomes in fighting oral disease, cytotoxic tests are mandatory for screening new materials for biological applications. Here, we developed a better understanding of the effects of microcrystals on the behavior of both human gingival fibroblast (HGF) cells and three-dimensional (3D) collagen matrices. To perform these experiments, the lowest concentration of α-Ag2WO4 capable of preventing the visible growth of Candida albicans (C. albicans) planktonic cells was defined as the test concentration, and it ranged from 0.781 (C1) to 7.81 (C2) to 78.1 (C3) µg/mL. Complete medium and lysis buffer (LB) served as negative (C-) and positive (C+) controls, respectively. The effect of the microcrystal concentration on the morphology, remodeling and proliferation of HGF cells was evaluated by different approaches. Quantitative and qualitative assessments demonstrated that α-Ag2WO4 did not affect the mitochondrial enzymatic activity of HGF cells cultured in a monolayer or the cell viability within 3D collagen matrices. These experiments showed that α-Ag2WO4 at the C2 concentration did not damage the genomic DNA. The development of new materials is attractive for the possible treatment of diseases and for avoiding indiscriminate prescribing of antibiotics. These findings provide information on the effect of α-Ag2WO4 on cell behavior and reveal that these microcrystals are non-cytotoxic against human gingival cells over a sufficient period to measure the hazard potential.

Development and characterization of a 3D oral mucosa model as a tool for host-pathogen interactions.

The aim of this study was to (i) design, develop and validate a practical and physiologically relevant reconstituted in vitro oral mucosa tissue model and (ii) to assess its applicability in in vitro host-pathogen interactions with C. albicans and S. aureus. Co-culture organotypic constructions were created by incorporating specific numbers of keratinocytes (NOK-si) onto cellularised, collagen gel scaffolds containing human gingival fibroblasts incubated in KGM media and cultured for 14 days. The detection of the appropriate oral mucosa/epithelial structure was evaluated by histology (hematoxylin and eosin (HE), periodic acid-Schiff (P.A.S.) and Picrosirius red), and immunocytochemistry (cytokeratin 13, cytokeratin 14, Ki-67 and collagen IV) compared to a normal human gingiva. The morphology of the reconstituted tissue was analyzed by Transmission Electron Microscopy. To further quantitate tissue damage, lactate dehydrogenase (LDH) was measured in the tissue supernatant. NOK-si grown upon a gingival scaffold provided an organotypic model in an in vitro setting and exhibited structural characteristics typically associated with normal oral mucosa. Immunocytochemistry revealed the detection of epithelial cytokeratins 13 and 14, Col IV and Ki-67 in the reconstituted oral mucosa model. Infection was detected after 8 h and 16 h. This study presents an in vitro cellularised, organotypic model of reconstituted oral mucosa, which enables close control and characterization of its structure and differentiation over a mid-length period of time in culture.

Soluble factors from biofilm of Candida albicans and Staphylococcus aureus promote cell death and inflammatory response.

BACKGROUND: The objective of this study was to better understand the effects of soluble factors from biofilm of single- and mixed-species Candida albicans (C. albicans) and methicillin-sensitive Staphylococcus aureus (MSSA) cultures after 36 h in culture on keratinocytes (NOK-si and HaCaT) and macrophages (J774A.1). Soluble factors from biofilms of C. albicans and MSSA were collected and incubated with keratinocytes and macrophages, which were subsequently evaluated by cell viability assays (MTT). Lactate dehydrogenase (LDH) enzyme release was measured to assess cell membrane damage to keratinocytes. Cells were analysed by brightfield microscopy after 2 and 24 h of exposure to the soluble factors from biofilm. Cell death was detected by labelling apoptotic cells with annexin V and necrotic cells with propidium iodide (PI) and was visualized via fluorescence microscopy. Soluble factors from biofilm were incubated with J774A.1 cells for 24 h; the subsequent production of NO and the cytokines IL-6 and TNF-α was measured by ELISA. RESULTS: The cell viability assays showed that the soluble factors of single-species C. albicans cultures were as toxic as the soluble factors from biofilm of mixed cultures, whereas the soluble factors of MSSA cultures were less toxic than those of C. albicans or mixed cultures. The soluble factors from biofilm of mixed cultures were the most toxic to the NOK-si and HaCaT cells, as confirmed by analyses of PI labelling and cell morphology. Soluble factors from biofilm of single-species MSSA and mixed-species cultures induced the production of IL-6, NO and TNF-α by J744A.1 macrophages. The production of IL-6 and NO induced by the soluble factors from biofilm of mixed cultures was lower than that induced by the soluble factors from biofilm of single-species MSSA cultures, whereas the soluble factors from biofilm of C. albicans cultures induced only low levels of NO. CONCLUSIONS: Soluble factors from 36-h-old biofilm of C. albicans and MSSA cultures promoted cell death and inflammatory responses.

Mechanism of Antibacterial Activity via Morphology Change of α-AgVO3: Theoretical and Experimental Insights.

The electronic configuration, morphology, optical features, and antibacterial activity of metastable α-AgVO3 crystals have been discussed by a conciliation and association of the results acquired by experimental procedures and first-principles calculations. The α-AgVO3 powders were synthesized using a coprecipitation method at 10, 20, and 30 °C. By using a Wulff construction for all relevant low-index surfaces [(100), (010), (001), (110), (011), (101), and (111)], the fine-tuning of the desired morphologies can be achieved by controlling the values of the surface energies, thereby lending a microscopic understanding to the experimental results. The as-synthesized α-AgVO3 crystals display a high antibacterial activity against methicillin-resistant Staphylococcus aureus. The results obtained from the experimental and theoretical techniques allow us to propose a mechanism for understanding the relationship between the morphological changes and antimicrobial performance of α-AgVO3.

Effect of titanium and zirconia dental implant abutments on a cultivable polymicrobial saliva community.

STATEMENT OF PROBLEM: Peri-implantitis is considered the most important biological complication responsible for late implant failure. The physical chemical properties intrinsic to each material can affect the first step to biofilm development and is an important precursor to the adaptive behavior of pathogenic bacteria species. PURPOSE: The purpose of this in vitro study was to evaluate the effect of 2 commercially available implant abutment materials on the adhesion phase and biofilm formation. MATERIAL AND METHODS: Disks (8 mm in diameter, 2 mm thick) of machined pure titanium (Ti) and yttrium-stabilized zirconia (ZrO2) materials were used to mimic implant abutments. The physical chemical surface properties were investigated using different approaches. Initial adherent bacteria and biofilm formation were evaluated after 16 and 48 hours by incubating the disks in a rich medium containing representative saliva-derived oral microbial community. Unpaired t test, 2 tailed, was used to compare the groups. RESULTS: Ti presented lower hydrophobicity and surface free energy values than the ZrO2, and 6.1-fold fewer bacteria adhered to the Ti. After 48 hours, detailed quantitative analysis showed that biofilm biomass and biofilm density were lower on the Ti disks than on ZrO2. The quantity of phylotypes on the Ti and ZrO2 surfaces was relatively similar during the attachment and early biofilm formation periods. CONCLUSIONS: Although no difference in the bacteria profile was observed between both materials independent of the time point, the highest level of colonization was on ZrO2.

Occlusal Pressure Analysis of Complete Dentures after Microwave Disinfection: A Clinical Study.

PURPOSE: This clinical study evaluated the effect of microwave disinfection protocols on the occlusal pressure pattern of dentures. MATERIALS AND METHODS: Dentures were constructed for 40 patients and evaluated as follows (n = 20). Group 1: Patients had the maxillary dentures submitted to microwave disinfection, once a week, for 4 weeks. Group 2: Patients had the maxillary dentures submitted to microwave disinfection, three times a week, for 4 weeks. Occlusal contacts were recorded on five occasions: 30 days after denture insertion and before first disinfection (baseline or control group); 1 week after disinfection; 2 weeks after disinfection; 3 weeks after disinfection; 4 weeks after disinfection. Occlusal contacts were analyzed by T-Scan III. Intergroup analysis was performed using the Mann-Whitney test and intragroup analysis using the Friedman test with significance of 5%. RESULTS: The results showed no significant difference between groups during the periods. The data on parameters loss of denture adaptation or complaints showed that patients used their dentures regularly for eating and expressed comfort and satisfaction in all experimental periods. The evaluation of functional occlusion revealed that the distribution of the occlusal contacts remained unaltered after disinfection. CONCLUSION: Microwave disinfection protocols as studied in this report did not influence occlusal contacts of the complete dentures.