Periodontal clinical status, microbial profile, and expression of interleukin-1ß in men under androgenic anabolic steroids abuse.

OBJECTIVES: Androgenic anabolic steroids (AAS) abuse is a serious health problem associated to several systemic complications. Here, we evaluated the periodontal clinical status, microbial profile, and expression of total protein (TP) and interleukin (IL)-1ß in men using AAS. MATERIALS AND METHODS: Men using AAS were recruited (case group) and matched for age with men who had never used AAS (control group) but also performed physical activities. Plaque index (PI), marginal bleeding (MB), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BoP) were evaluated. Crevicular fluid and subgingival biofilm were collected from healthy and diseased sites (PD ≥ 4 mm with CAL ≥ 1 mm and BoP) and evaluated for TP, IL-1ß, and proportions of 40 bacterial species. RESULTS: Thirty patients were included (n = 15/group). AAS consumers had significantly higher mean PD and higher percentage of diseased sites; sites with PD ≥ 4 mm or with CAL ≥ 1 mm than non-consumers. Also, AAS users showed a more dysbiotic biofilm containing lower proportions of host-compatible species and higher proportions of pathogens. IL-1ß expression was statistically higher in diseased than in healthy sites only in the control group. A statistically positive correlation was detected between periodontal pathogens and IL-1ß expression. The number of AAS cycles was positively associated with higher percentages of periodontal pathogens, but not with IL-1ß or total protein concentrations. CONCLUSIONS: AAS intake can worsen clinical and immunological periodontal conditions and the biofilm composition in healthy sites. CLINICAL RELEVANCE: Dental care professionals should perform full mouth periodontal screening and schedule regular follow-up appointments for patients under AAS use.

Positive influence of simvastatin used as adjuvant agent for cavity lining.

OBJECTIVES: To assess the biological, antimicrobial, and mechanical effects of the treatment of deep dentin with simvastatin (SV) before application of a glass-ionomer cement (GIC). MATERIALS AND METHODS: Dentin discs were adapted to artificial pulp chambers and SV (2.5 or 1.0 mg/mL) was applied to the occlusal surface, either previously conditioned or not with EDTA (±EDTA). The extracts (culture medium + SV that diffused through dentin) was obtained and then applied to cultured odontoblast-like MDPC-23 cells. Cell viability, alkaline phosphatase (ALP) activity, and mineralization nodule (MN) deposition were evaluated. Untreated discs were used as control. The antibacterial activity of SV (2.5 or 1.0 mg/mL) against Streptococcus mutans and Lactobacillus acidophilus, as well as the bond strength of GIC to dentin in the presence of SV 2.5 mg/mL (±EDTA) were also assessed. The data were analyzed by ANOVA/Tukey tests (α = 5%). RESULTS: EDTA + SV 2.5 mg/mL significantly enhanced the ALP activity and MN deposition in comparison with the control, without changing in the cell viability (p < 0.05). The association EDTA + SV 2.5 mg/mL + GIC determined the highest ALP and MN values (p < 0.05). SV presented intense antimicrobial activity, and the EDTA dentin conditioning followed by SV application increased bond strength values compared with SV treatment alone (p < 0.05). CONCLUSION: SV presents antimicrobial activity and diffuses across conditioned dentin to biostimulate odontoblast-like pulp cells. CLINICAL SIGNIFICANCE: The use of SV as adjuvant agent for indirect pulp capping may biostimulate pulp cells thus preserving vitality and function of the pulp-dentin complex.

In vitro antibacterial and cytotoxic activities of carvacrol and terpinen-4-ol against biofilm formation on titanium implant surfaces.

This study evaluated the antibacterial properties of carvacrol and terpinen-4-ol against Porphyromonas gingivalis and Fusobacterium nucleatum and its cytotoxic effects on fibroblast cells. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were examined. The minimum biofilm inhibition concentration (MBIC) was evaluated by XTT assay. Biofilm decontamination on titanium surfaces was quantified (CFU ml-1), evaluated by confocal laser scanning microscopy (CLSM) and cytotoxic activity by MTT. The MIC and MBC for carvacrol were 0.007% and 0.002% for P. gingivalis and F. nucleatum, and 0.06% for terpinen-4-ol for both microorganisms. The MBIC for carvacrol was 0.03% and 0.06% for P. gingivalis and F. nucleatum, and for terpinen-4-ol was 0.06% and 0.24%. The results indicated anti-biofilm activity using carvacrol (0.26%, 0.06%) and terpinen-4-ol (0.95%, 0.24%) and showed cytotoxic activity similar to chlorohexidine (CHX). However, terpinen-4-ol (0.24%) showed higher cell viability than other treatments. Carvacrol and terpinen-4-ol showed antibacterial activity in respect of reducing biofilms. Moreover, CHX-like cytotoxicity was observed.

KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects against oral pathogens.

This study evaluated the cytotoxicity and antimicrobial activity of analogs of cationic peptides against microorganisms associated with endodontic infections. L-929 fibroblasts were exposed to LL-37, KR-12-a5 and hBD-3-1CV and chlorhexidine (CHX, control), and cell metabolism was evaluated with MTT. The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) of the peptides and CHX were determined against oral pathogens associated with endodontic infections. Enterococcus faecalis and Streptococcus mutans biofilms were cultivated in bovine dentin blocks, exposed to different concentrations of the most efficient antimicrobial peptide and analyzed by confocal laser scanning microscopy. CHX and peptides affected the metabolism of L-929 at concentrations > 31.25 and 500 µg ml-1, respectively. Among the peptides, KR-12-a5 inhibited growth of both the microorganisms tested with the lowest MIC/MBC/MFC values. In addition, KR-12-a5 significantly reduced E. faecalis and S. mutans biofilms inside dentin tubules. In conclusion, KR-12-a5 is a non-cytotoxic agent with potent antimicrobial and anti-biofilm activity against oral pathogens associated with endodontic infections.

Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions.

This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity.

Long-term evaluation of oral gavage with periodontopathogens or ligature induction of experimental periodontal disease in mice.

OBJECTIVE: To evaluate in long-term periods the destruction of periodontal tissues and bacterial colonization induced by oral gavage with periodontopathogens or ligature experimental periodontal disease models. MATERIAL AND METHODS: Forty-eight C57BL/6 J mice were divided into four groups: group C: negative control; group L: ligature; group G-Pg: oral gavage with Porphyromonas gingivalis; and group G-PgFn: oral gavage with Porphyromonas gingivalis associated with Fusobacterium nucleatum. Mice were infected by oral gavage five times in 2-day intervals. After 45 and 60 days, animals were sacrificed and the immune-inflammatory response in the periodontal tissue was assessed by stereometric analysis. The alveolar bone loss was evaluated by live microcomputed tomography and histometric analysis. qPCR was used to confirm the bacterial colonization in all the groups. Data were analyzed using the Kruskal-Wallis, Wilcoxon, and ANOVA tests, at 5 % of significance level. RESULTS: Ligature model induced inflammation and bone resorption characterized by increased number of inflammatory cells and decreased number of fibroblasts, followed by advanced alveolar bone loss at 45 and 60 days (p < 0.05). Bacterial colonization in groups G-Pg and G-PgFn was confirmed by qPCR but inflammation and bone resorption were not observed (p < 0.05). CONCLUSIONS: The ligature model but not the oral gavage models were effective to induce inflammation and bone loss in long-term periods. Pg colonization was observed in all models of experimental periodontal disease induction, independent of tissue alterations. These mice models of periodontitis validates, compliments, and enhances published PD models that utilize ligature or oral gavage and supports the importance of a successful colonization of a susceptible host, a bacterial invasion into vulnerable tissue, and host-bacterial interactions that lead to tissue destruction. CLINICAL RELEVANCE: The ligature model was an effective approach to induce inflammation and bone loss similar to human periodontitis, but the oral gavage models were not efficient in inducing periodontal inflammation and tissue destruction in the conditions studied. Ligature models can provide a basis for future interventional studies that contribute to the understanding of the disease pathogenesis and the complex host response to microbial challenge.

Structural and quantitative analysis of a mature anaerobic biofilm on different implant abutment surfaces.

STATEMENT OF PROBLEM: The longevity of dental implants depends on the absence of inflammation in the periimplant tissue. Similar to teeth, pathogenic bacteria can adhere on implant abutment surfaces and cause periimplant disease and consequently implant loss. PURPOSE: The purpose of this in vitro study was to evaluate the influence of physical and chemical properties of 2 common materials used as implant abutments, titanium (Ti) and zirconia (ZrO2), and the use of bovine enamel (BE) as a positive control on biofilm formation. MATERIAL AND METHODS: Biofilm formation was analyzed by growing Porphyromonas gingivalis and Fusobacterium nucleatum as monospecies and mixed species biofilms on the surfaces. The mean roughness (Ra) and surface free energy were evaluated for each material. Mature biofilm, formed after 7 days of incubation, was analyzed quantitatively and qualitatively by colony-forming unit and confocal laser scanning microscopy. RESULTS: The mean roughness in all disks was ≤0.21 µm and did not affect the bacterial adhesion. Titanium showed a greater degree of hydrophilicity compared with BE after 90 minutes of immersion in saliva. The surface free energy did not show differences, with the highest values for BE. Monospecies biofilms formed by P. gingivalis on Ti, and mixed species biofilm on ZrO2 exhibited small numbers of cells on disk surfaces. By confocal imaging, the mixed species biofilm appeared as a thin layer on ZrO2 surfaces. CONCLUSIONS: Material surfaces could have a significant impact on biofilm formation. ZrO2 implant abutment surfaces showed a decrease in anaerobic biofilm compared with Ti and BE.

Incorporation of chlorhexidine gluconate or diacetate into a glass-ionomer cement: porosity, surface roughness, and anti-biofilm activity.

PURPOSE: To evaluate the porosity, surface roughness and anti-biofilm activity of a glass-ionomer cement (GIC) after incorporation of different concentrations of chlorhexidine (CHX) gluconate or diacetate. METHODS: For the porosity and surface roughness tests, 10 test specimens were fabricated of the GIC Ketac Molar EasyMix (KM) and divided into the following groups: Control, GIC and 0.5% CHX diacetate; GIC and 1.0% CHX diacetate; GIC and 2.0% CHX diacetate; GIC and 0.5% CHX gluconate; GIC and 1.0% CHX gluconate; GIC and 2.0% CHX gluconate. To evaluate porosity, the test specimens were fractured. The fragments were photographed by scanning electron microscopy (SEM), and the images analyzed with the aid of the software program Image J. The surface roughness (Ra) was obtained by the mean value of three readouts performed on the surface of each specimen, always through the center. To analyze the anti-biofilm activity, strains of S. mutans ATCC 35688 were used, and the groups control and GIC +CHX diacetate 1% were divided as follows: GIC (1 day); GIC (7 days), GIC (14 days), GIC (21 days); GIC+CHX (1 day), GIC+CHX (7 days), GIC+CHX (14 days), GIC+CHX (21 days); GIC+ CHX (1 day), GIC+ CHX (7 days), GIC+ CHX (14 days) and GIC+ CHX (21 days) using 10 test specimens per group. For biofilm growth, the specimens were placed in a vertical position in 24-well plates and incubated overnight 10 times. The culture medium was renewed every 24 hours. The suspension was diluted and seeded on BHI agar for quantification of the bacteria present. For evaluation of all the tests the two-way ANOVA was used, and if necessary, the Tukey test was applied, with a level of significance of 5%. RESULTS: Regarding GIC porosity, the ANOVA showed that the presence of CHX increased the porosity (P < 0.001) proportionally to the increase in concentrations (P = 0.001), without however, presenting interaction between material and concentration (P = 0.705). Regarding the number of pores, a significant increase in pores was observed with the increase in CHX concentration (P = 0.003). The surface roughness test demonstrated no statistically significant effect as to increase or reduction in roughness at any of the CHX concentrations used (P > 0.05). Anti-biofilm activity analysis pointed out a significant effect of the factors material (P = 0.006) and time (P < 0.001), with CHX diacetate CHX presenting greater effectiveness in reducing microorganisms.

Testosterone replacement relieves ligature-induced periodontitis by mitigating inflammation, increasing pro-resolving markers and promoting angiogenesis in rats: A preclinical study.

OBJECTIVES: This study aimed to evaluate the inflammatory profile as well as the resolution of inflammation in a ligature-induced periodontal inflammation in rats with depletion and/or supraphysiological testosterone replacement. DESIGN: Sixty male rats (Holtzman) were used in the present study. Study groups were created as following: (1) Sham (no testicle removal); (2) Orchiectomy (OCX), 3) OCX + Testosterone (OCX + T); (4) Sham + Ligature (SH + L); (5) OCX+L; and 6) OCX + T + L. The surgeries were performed on day 1, and testosterone was administered weekly since day 1. On day 15, a cotton ligature was placed around the lower first molars and maintained for 15 days. Morphological changes in periodontal tissues were determined by histopathological analysis. Immunohistochemistry (factor VIII) and immunoenzymatic assay were performed to evaluate angiogenesis process and (pro- and anti-) inflammatory markers, respectively. RESULTS: Ligature promoted a marked inflammatory gingival infiltrate and bone loss (P < 0.05). Supraphysiological testosterone treatment increased the percentage of blood vessels, extracellular matrix and fibroblasts in the presence and absence of periodontal inflammation (P < 0.05). A high dose of testosterone increased factor VIII+ blood vessels and IL-10 expression in inflamed gingival tissue, while PGE2, LXA4 and MPO were reduced as a result of supraphysiological testosterone administration (P < 0.05). CONCLUSIONS: These results, in our experimental model, suggest that supraphysiological testosterone treatment stimulated gingival tissue repair during ligature-induced periodontitis, and it seems to be related to an anti-inflammatory and pro-resolutive mechanism resulting by the modulatory effect on PGE2 and IL-10 related to an enhanced angiogenesis.

Physical-chemical influences and cell behavior of natural compounds on titanium dental surfaces.

The present study evaluated the influence of carvacrol, terpinene-4-ol, and chlorhexidine on the physical-chemical properties of titanium surfaces, cell viability, proliferation, adhesion, and spreading of fibroblasts and osteoblasts in vitro. Titanium surfaces (Ti) were treated with Carvacrol (Cvc), Terpinen-4-ol (T4ol), Chlorhexidine (CHX), DMSO, and ultrapure water (Control group). Physical-chemical modifications were evaluated by surface wettability, the surface free energy (SFE) calculated from the contact angle values using the Owens-Wendt-Rabel-Kaeble (OWRK) equation, scanning electron microscopy (SEM) and energy dispersive spectrometry probe (EDS) system. Cells were seeded onto Ti-treated surfaces and incubated for 24 h and 72 h, then evaluated by Alamar blue assay and fluorescence microscopy. Surfaces treated with Cvc and T4ol showed the presence of Na, O, and Cl. All surfaces showed hydrophilic characteristics and SFE values between 5.5 mN/m and 3.4 mN/m. On the other hand, EDS peaks demonstrated the presence of O and Cl after CHX treatment. A reduction of cell viability and adhesion was noted on titanium surfaces treated with CHX after 24 and 72h. In conclusion, the results indicate that the decontamination with Cvc and T4ol on Ti surfaces does not alter the surface proprieties and allows an adequate interaction with cells involved in the re-osseointegration process such as fibroblasts and osteoblasts.

Improving antimicrobial activity against endodontic biofilm after exposure to blue light-activated novel curcumin nanoparticle.

New therapies involving natural products and nanobiotechnology open additional perspectives to reduce endodontic infections. Curcumin is a natural polyphenol extracted from the dry rhizome of curcuma long Linn with therapeutic properties for application in nanobiotechnology and as a photosensitizer for photodynamic therapy. This study aimed to synthesize a novel polymeric nanoparticle of poly (lactic-co-glycolic acid) (PLGA) loaded with curcumin (NP+Cur), and evaluate its antimicrobial activity against endodontic biofilms. Additionally, its biocompatibility using oral keratinocytes was assessed. The polymeric NP+Cur was prepared by the nanoprecipitation method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were calculated for the three endodontic bacteria (Enterococcus faecalis, Streptococcus oralis and Actinomyces viscosus). Antibacterial activity of NP+Cur against single- and multispecies biofilm pre-formed on the botton 24-well plate and into dentin tubules of bovine teeth were evaluated by colony forming units and confocal laser scanning microscopy. The pre-irradiation time was 5 min followed by exposure to blue light-emitting diode at 450 nm for the photodynamic treatment. Cell viability using oral keratinocytes was assessed by Alamar Blue assay. MIC and MBC showed antibacterial activity of NP+Cur against endodontic bacteria. A treatment of pre-formed biofilms of endodontic bacteria with NP+Cur also significantly decreased bacterial viability. The concentration of 325 µg/mL of photoactivated NP+Cur was the one that most reduced the viability of the endodontic bacteria evaluated. Regarding biocompatibility, NP+Cur 325 µg/mL and pure nanoparticles showed a cell viability greater than 80%. The novel polymeric nanoparticles loaded with curcumin may be a promising adjunct use to treatment of endodontic infections.

Antimicrobial activity of disinfectant agents incorporated into type IV dental stone.

PURPOSE: This study evaluated the antimicrobial activity of two disinfectant agents, 2% chlorhexidine digluconate solution (CHX) and 98% chlorhexidine hydrochloride powder (HYD), incorporated into type IV dental stone at the time of mixing. MATERIAL AND METHODS: Agar diffusion test was used for the following microorganisms: Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans. The specimens were grouped in: (1) dental stone mixed with sterile distilled water; (2) paper disc soaked with CHX; (3) dental stone mixed with CHX; and (4) dental stone with incorporation of HYD, in 1% proportion of the dental stone mass and mixed with sterile distilled water. The culture medium was inoculated with microbial suspensions 1 and 24 h after pouring of the dental stone. The antimicrobial activity was evaluated by the average diameter of microbial growth inhibition zones. The data were analysed with a nested anova (p < 0.05) and Tukey test for specific comparisons. RESULTS: The disinfectant agents demonstrated antimicrobial activity against all microorganisms, with the exception of C. albicans, against which the CHX was ineffective in two periods of analysis. Significant differences between disinfectants were found with all microorganisms. CONCLUSION: The disinfectant agents analysed were effective against most of the microorganisms tested, except C. albicans.

Effect of curcumin-loaded photoactivatable polymeric nanoparticle on peri-implantitis-related biofilm.

Curcumin has been used as a photosensitizer (PS) for antimicrobial photodynamic chemotherapy (PACT). However, its low solubility, instability, and poor bioavailability challenge its in vivo application. This study aimed to synthesize curcumin-loaded polymeric nanoparticles (curcumin-NP) and determine their antimicrobial and cytotoxic effects. Nanoparticles (NP) were synthesized using polycaprolactone (PCL) as a polymer by the nanoprecipitation method. Curcumin-NP was characterized by particle size, polydispersity index and zeta potential, scanning electron microscopy, and curcumin encapsulation efficiency (EE). Curcumin-NP was compared to free curcumin solubilized in 10% DMSO as photosensitizers for PACT in single and multispecies Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus oralis biofilms. Chlorhexidine 0.12% (CHX) and ultrapure water were used as positive and negative controls. The cytotoxic effect of curcumin-NP was evaluated on human periodontal ligament fibroblast cells (HPLF). Data were analyzed by ANOVA (α=0.05). Curcumin-NP exhibited homogeneity and stability in solution, small particle size, and 67.5% EE of curcumin. Curcumin-NP presented reduced antibiofilm activity at 500 µg/ml, although in planktonic cultures it showed inhibitory and bactericidal effect. Curcumin-NP and curcumin with and without photoactivation were not cytotoxic to HPLF cells. Curcumin-NP has antimicrobial and antibiofilm properties, with better effects when associated with blue light, being a promising therapy for preventing and treating peri-implant diseases.

Biosynthesis of silver nanoparticles from Syzygium cumini leaves and their potential effects on odontogenic pathogens and biofilms.

This study analyzed the antimicrobial and antibiofilm action and cytotoxicity of extract (HEScL) and silver nanoparticles (AgNPs-HEScL) from Syzygium cumini leaves. GC-MS, UV-Vis, EDX, FEG/SEM, DLS and zeta potential assays were used to characterize the extract or nanoparticles. Antimicrobial, antibiofilm and cytotoxicity analyses were carried out by in vitro methods: agar diffusion, microdilution and normal oral keratinocytes spontaneously immortalized (NOK-SI) cell culture. MICs of planktonic cells ranged from 31.2-250 (AgNPs-HEScL) to 1,296.8-10,375 µg/ml (HEScL) for Actinomyces naeslundii, Fusobacterium nucleatum, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Streptococcus oralis, Veillonella dispar, and Candida albicans. AgNPs-HEScL showed antibiofilm effects (125-8,000 µg/ml) toward Candida albicans, Streptococcus mutans and Streptococcus oralis, and Staphylococcus aureus and Staphylococcus epidermidis. The NOK-SI exhibited no cytotoxicity when treated with 32.8 and 680.3 µg/ml of AgNPs-HEScL and HEScL, respectively, for 5 min. The data suggest potential antimicrobial and antibiofilm action of HEScL, and more specifically, AgNPs-HEScL, involving pathogens of medical and dental interest (dose-, time- and species-dependent). The cytotoxicity of HEScL and AgNPs-HEScL detected in NOK-SI was dose- and time-dependent. This study presents toxicological information about the lyophilized ethanolic extract of S. cumini leaves, including their metallic nanoparticles, and adds scientific values to incipient studies found in the literature.

Typing Candida albicans oral isolates from healthy brazilian schoolchildren using multilocus enzyme electrophoresis reveals two highly polymorphic taxa.

The genetic diversity of C. albicans oral isolates from 75 healthy schoolchildren from eight schools located in different geographic areas of Piracicaba city, São Paulo state, Brazil, was established using isoenzymes marker (Multilocus Enzyme Electrophoresis - MLEE) and cluster analysis. Patterns of monoclonal and polyclonal oral colonization by C. albicans within and between groups of schoolchildren were identified. However, significant divergence between the observed and the expected genotypic frequencies (Hardy-Weinberg equilibrium test) was not detected in the geographically adjacent groups, suggesting the hypothesis that populations of healthy schoolchildren do not correspond to the selection factor (differential survival) of strains. Two highly polymorphic and distantly genetically related taxa (A and B) were identified within the total population of yeasts, each contained subgroups (A1, A2, A3, A4, B1 and B2) and clusters of moderately related strains (from I to X), suggesting the existence of strains restricted or not to certain groups of geographically limited, healthy students. However, the coexistence of identical strains in healthy schoolchildren from the same school (geographically related) reinforces the hypothesis of oral transmission, where the sources of propagation could be explored. Furthermore, this could also be used in current and retrospective analyses of C. albicans isolated from immunocompetent and immunocompromised people, in order to detect commensal or potentially pathogenic yeast groups, predominantly in candidiasis, and in the development of strategies to prevent transmission or human propagation.

Antimicrobial effects of silver nanoparticles and extracts of Syzygium cumini flowers and seeds: Periodontal, cariogenic and opportunistic pathogens.

OBJECTIVE: This study aimed to analyze the antimicrobial effects of lyophilized hydroalcoholic extract (HEScSeed and HEScFlower) and silver nanoparticles (AgNPs-HEScSeed and AgNPs-HEScFlower) of S. cumini seed and flower, and to characterize some compounds of these extracts and their NPs. DESIGN: Phytochemical screening was performed by GC-MS. Nanoparticles were characterized by UV-vis spectroscopy, energy-dispersive X-ray (EDX) spectrophotometry, scanning electron microscopy (SEM) and field emission gun (FEG), dynamic light scattering (DLS) and zeta potential (ZP). Antimicrobial susceptibility tests were analyzed by broth microdilution and agar diffusion methods. RESULTS: HEScSeed and HEScFlower showed 7 and 17 phytochemical compounds, respectively. AgNPs-plant extracts were reported as stable and with variable shapes and sizes. All studied species (A. naeslundii, C. albicans, F. nucleatum, S. aureus, S. epidermidis, S. mutans, S. oralis and V. dispar) were susceptible to extracts and AgNPs-plant extracts, with varying degrees of antimicrobial activities (extract: 648.4-5,187.5 µg/mL; AgNPs-plant: 31.2-2,000 µg/mL). CONCLUSION: The extracts of S. cumini seed and flower have antimicrobial action against pathogens of medical and dental interest, whose MIC and MMC are species-dependent. The AgNPs-HEScSeed and AgNPs-HEScFlower have different shapes, sizes, organic compounds, stability and electronegativity (capping), characteristics that contribute to their bacteriostatic and fungistatic effects, but at significantly lower concentrations than plant extracts.

Impact of citrus flavonoid supplementation on inflammation in lipopolysaccharide-induced periodontal disease in mice.

In general, the consumption of flavonoid-rich foods may influence the control/dysregulation of the magnitude and duration of inflammation and oxidative stress, which are known to contribute to multiple pathologies. Information regarding the impact of citrus flavonoid dietary supplementation on periodontal disease is still scarce. Herein, we investigated whether a diet supplemented with eriocitrin and eriodictyol could alter the course of the inflammatory response associated with LPS-induced periodontal disease in mice. Sixty BALB/c mice received a standard diet or a diet supplemented with different concentrations of eriocitrin or eriodictyol. After 30 days of food supplementation, a solution containing LPS from Escherichia coli was injected into the gingival tissues three times per week for four weeks. Neutrophils, mononuclear cells and eosinophils were assessed using a severity analysis system in H&E-stained sections and modified picrosirius red. The activities of myeloperoxidase (MPO), a marker of granulocyte infiltration, and eosinophil peroxidase (EPO) were determined spectrophotometrically. The oxidative damage was determined by measuring the malondialdehyde (MDA) content and anti-oxidative activity through the assessment of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Interleukin (IL)-1ß, TNF-α, and IL-10 were quantified by multiplex immunoassay. Periodontal inflammation was significantly inhibited by citrus flavonoid supplementation, including reduced flatness of the gingival epithelium and chronic and acute inflammatory cell infiltration, as well as loss of connective tissue in the gingival papillae. Both eriocitrin and eriodictyol inhibited gingival IL-1ß and TNF-α and increased IL-10 secondary to periodontitis. Significant protection and decreased MPO and EPO activity were detected in the periodontal tissue of citrus flavonoid-treated animals. In comparison with the LPS group, SOD, CAT and GPx activities were increased, while the MDA content was reduced, indicating decreased oxidative damage. These results suggest that a diet supplemented with the citrus flavonoids eriocitrin or eriodictyol may aid in the prevention of periodontitis, representing a potential method to enhance local immunity and host defense.

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans.

Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Terpinen-4-ol and nystatin co-loaded precursor of liquid crystalline system for topical treatment of oral candidiasis.

This study was performed to develop a liquid crystalline system (LCS) incorporated with terpinen-4-ol and nystatin to evaluate its antifungal, antibiofilm, and synergistic/modulatory activity against Candida albicans. The LCS was composed of a dispersion containing 40% propoxylated and ethoxylated cetyl alcohol, 40% oleic acid, and 0.5% chitosan dispersion. According to analysis by polarized light microscopy, rheology, and mucoadhesion studies, the incorporation of 100% artificial saliva increased the pseudoplasticity, consistency index, viscosity, and mucoadhesion of the formulation. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity; the LCS containing terpinen-4-ol and nystatin effectively inhibited C. albicans growth at a lower concentration, displaying a synergistic action. Therefore, LCS incorporated with terpinen-4-ol and nystatin is a promising alternative for preventing and treating infections and shows potential for the development of therapeutic strategies against candidiasis.

Streptococcus mutans and Actinomyces naeslundii Interaction in Dual-Species Biofilm.

The study of bacterial interaction between Streptococcus mutans and Actinomyces naeslundii may disclose important features of biofilm interspecies relationships. The aim of this study was to characterize-with an emphasis on biofilm formation and composition and metabolic activity-single- and dual-species biofilms of S. mutans or A. naeslundii, and to use a drip flow reactor (DFR) to evaluate biofilm stress responses to 0.2% chlorhexidine diacetate (CHX). Single- and dual-species biofilms were grown for 24 h. The following factors were evaluated: cell viability, biomass and total proteins in the extracellular matrix, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide-"XTT"-reduction and lactic acid production. To evaluate stress response, biofilms were grown in DFR. Biofilms were treated with CHX or 0.9% sodium chloride (NaCl; control). Biofilms were plated for viability assessment. Confocal laser-scanning microscopy (CLSM) was also performed. Data analysis was carried out at 5% significance level. S. mutans viability and lactic acid production in dual-species biofilms were significantly reduced. S. mutans showed a higher resistance to CHX in dual-species biofilms. Total protein content, biomass and XTT reduction showed no significant differences between single- and dual-species biofilms. CLSM images showed the formation of large clusters in dual-species biofilms. In conclusion, dual-species biofilms reduced S. mutans viability and lactic acid production and increased S. mutans' resistance to chlorhexidine.