Dual nanocarrier of chlorhexidine and fluconazole: Physicochemical characterization and effects on microcosm biofilms and oral keratinocytes.

OBJECTIVES: This study assembled and characterized a dual nanocarrier of chlorhexidine (CHX) and fluconazole (FLZ), and evaluated its antibiofilm and cytotoxic effects. METHODS: CHX and FLZ were added to iron oxide nanoparticles (IONPs) previously coated by chitosan (CS) and characterized by physical-chemical analyses. Biofilms from human saliva supplemented with Candida species were grown (72 h) on glass discs and treated (24 h) with IONPs-CS carrying CHX (at 39, 78, or 156 µg/mL) and FLZ (at 156, 312, or 624 µg/mL) in three growing associations. IONPs and CS alone, and 156 µg/mL CHX + 624 µg/mL FLZ (CHX156-FLZ624) were tested as controls. Next, microbiological analyses were performed. The viability of human oral keratinocytes (NOKsi lineage) was also determined (MTT reduction assay). Data were submitted to ANOVA or Kruskal-Wallis, followed by Fisher's LSD or Tukey's tests (α=0.05). RESULTS: Nanocarriers with spherical-like shape and diameter around 6 nm were assembled, without compromising the crystalline property and stability of IONPs. Nanocarrier at the highest concentrations was the most effective in reducing colony-forming units of Streptococcus mutans, Lactobacillus spp., Candida albicans, and Candida glabrata. The other carriers and CHX156-FLZ624 showed similar antibiofilm effects, and significantly reduced lactic acid production (p<0.001). Also, a dose-dependent cytotoxic effect against oral keratinocytes was observed for the dual nanocarrier. IONPs-CS-CHX-FLZ and CHX-FLZ significantly reduced keratinocyte viability at CHX and FLZ concentrations ≥7.8 and 31.25 µg/mL, respectively (p<0.05). CONCLUSION: The nanotherapy developed outperformed the effect of the combination CHX-FLZ on microcosm biofilms, without increasing the cytotoxic effect of the antimicrobials administered. CLINICAL SIGNIFICANCE: The dual nanocarrier is a promising topically-applied therapy for the management of oral candidiasis considering that its higher antibiofilm effects allow the use of lower concentrations of antimicrobials than those found in commercial products.

In vitro antimicrobial effects of chitosan on microcosm biofilms of oral candidiasis.

OBJECTIVE: This study assessed the effects of chitosan (CS) on microcosm biofilms derived from saliva of patients with Candida-associated denture stomatitis. METHODS: Five removable denture wearers with denture stomatitis were included in the study. The minimum inhibitory concentration (MIC) of CS against clinical isolates of Candida albicans was determined according to the broth microdilution method. Pooled saliva from the donors was used as an inoculum for the formation of biofilms, which were developed during 72 h on acrylic surfaces in the Amsterdam Active Attachment model. The biofilms were then treated with different concentrations of CS, and the antibiofilm effects were evaluated through the quantification of colony-forming units (CFUs), total biomass (TB), metabolic activity (MA), lactic acid production (LAP), and cell viability (by confocal laser scanning microscopy). Chlorhexidine, miconazole, and nystatin were tested as positive controls, while the negative control (NC) was the untreated biofilm. Data were analyzed by 1-way ANOVA and Fischer LSD's post hoc test (α=0.05). RESULTS: MIC values of CS ranged from 500 to 800 µg/mL. For CFUs, 2500 µg/mL CS was the most effective treatment in reducing total anaerobes, mutans streptococci, and Lactobacillus spp., significantly differing from the controls. For C. albicans CFUs, CS and positive controls did not differ from each other but led to significant reductions compared to NC. Regarding TB, MA, LAP, and cell viability, 2500 µg/mL CS promoted the greatest reductions compared to NC. CONCLUSION: CS has similar or superior effects to conventional active principles on important parameters of oral candidiasis microcosm biofilms. CLINICAL RELEVANCE: The antibiofilm effects of CS show that this compound has great potential to improve the clinical condition of denture stomatitis patients, and formulations containing this natural polymer could be useful for controlling oral candidiasis.

Nanocarriers of Miconazole or Fluconazole: Effects on Three-Species Candida Biofilms and Cytotoxic Effects In Vitro.

The contribution of different Candida species in oral fungal infections has stimulated the search for more effective therapies. This study assessed the antibiofilm effects of nanocarriers of miconazole (MCZ) or fluconazole (FLZ) on Candida biofilms, and their cytotoxic effects on murine fibroblasts. Three-species biofilms (Candida albicans/Candida glabrata/Candida tropicalis) were formed on 96-well plates, and they were treated with nanocarriers (iron oxide nanoparticles coated with chitosan-"IONPs-CS") of MCZ or FLZ at 39/78/156 µg/mL; antifungals alone at 156 µg/mL and artificial saliva were tested as positive and negative controls, respectively. Biofilms were analyzed by colony forming units (CFU), biomass, metabolic activity, and structure/viability. The cytotoxicity (L929 cells) of all treatments was determined via 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Data were submitted to one- or two-way ANOVA, followed by Tukey's or Fisher LSD's tests (p < 0.05). IONPs-CS-MCZ at 78 µg/mL promoted similar antibiofilm and cytotoxic effects compared with MCZ at 156 µg/mL. In turn, IONPs-CS-FLZ at 156 µg/mL was overall the most effective FLZ antibiofilm treatment, surpassing the effects of FLZ alone; this nanocarrier was also less cytotoxic compared with FLZ alone. It can be concluded that both nanocarriers are more effective alternatives to fight Candida biofilms compared with their respective positive controls in vitro, being a promising alternative for the treatment of oral fungal infections.

Effects of Antifungal Carriers Based on Chitosan-Coated Iron Oxide Nanoparticles on Microcosm Biofilms.

Resistance of Candida species to conventional therapies has motivated the development of antifungal nanocarriers based on iron oxide nanoparticles (IONPs) coated with chitosan (CS). This study evaluates the effects of IONPs-CS as carriers of miconazole (MCZ) or fluconazole (FLZ) on microcosm biofilms. Pooled saliva from two healthy volunteers supplemented with C. albicans and C. glabrata was the inoculum for biofilm formation. Biofilms were formed for 96 h on coverslips using the Amsterdam Active Attachment model, followed by 24 h treatment with nanocarriers containing different concentrations of each antifungal (78 and 156 µg/mL). MCZ or FLZ (156 µg/mL), and untreated biofilms were considered as controls. Anti-biofilm effects were evaluated by enumeration of colony-forming units (CFUs), composition of the extracellular matrix, lactic acid production, and structure and live/dead biofilm cells (confocal laser scanning microscopy-CLSM). Data were analyzed by one-way ANOVA and Fisher LSD's test (α = 0.05). IONPs-CS carrying MCZ or FLZ were the most effective treatments in reducing CFUs compared to either an antifungal agent alone for C. albicans and MCZ for C. glabrata. Significant reductions in mutans streptococci and Lactobacillus spp. were shown, though mainly for the MCZ nanocarrier. Antifungals and their nanocarriers also showed significantly higher proportions of dead cells compared to untreated biofilm by CLSM (p < 0.001), and promoted significant reductions in lactic acid, while simultaneously showing increases in some components of the extracellular matrix. These findings reinforce the use of nanocarriers as effective alternatives to fight oral fungal infections.

Novel Colloidal Nanocarrier of Cetylpyridinium Chloride: Antifungal Activities on Candida Species and Cytotoxic Potential on Murine Fibroblasts.

Nanocarriers have been used as alternative tools to overcome the resistance of Candida species to conventional treatments. This study prepared a nanocarrier of cetylpyridinium chloride (CPC) using iron oxide nanoparticles (IONPs) conjugated with chitosan (CS), and assessed its antifungal and cytotoxic effects. CPC was immobilized on CS-coated IONPs, and the nanocarrier was physico-chemically characterized. Antifungal effects were determined on planktonic cells of Candida albicans and Candida glabrata (by minimum inhibitory concentration (MIC) assays) and on single- and dual-species biofilms of these strains (by quantification of cultivable cells, total biomass and metabolic activity). Murine fibroblasts were exposed to different concentrations of the nanocarrier, and the cytotoxic effect was evaluated by MTT reduction assay. Characterization methods confirmed the presence of a nanocarrier smaller than 313 nm. IONPs-CS-CPC and free CPC showed the same MIC values (0.78 µg mL-1). CPC-containing nanocarrier at 78 µg mL-1 significantly reduced the number of cultivable cells for all biofilms, surpassing the effect promoted by free CPC. For total biomass, metabolic activity, and cytotoxic effects, the nanocarrier and free CPC produced statistically similar outcomes. In conclusion, the IONPs-CS-CPC nanocarrier was more effective than CPC in reducing the cultivable cells of Candida biofilms without increasing the cytotoxic effects of CPC, and may be a useful tool for the treatment of oral fungal infections.

Antimicrobial, antibiofilm and cytotoxic effects of a colloidal nanocarrier composed by chitosan-coated iron oxide nanoparticles loaded with chlorhexidine.

OBJECTIVES: This study evaluated the antimicrobial and antibiofilm effects of a colloidal nanocarrier for chlorhexidine (CHX) on Candida glabrata and Enterococcus faecalis, as well as tested its cytotoxic effect on murine fibroblasts. METHODS: Iron oxide nanoparticles (IONPs) were coated with chitosan (CS) and loaded with CHX at 31.2, 78 and 156 µg/mL. Antimicrobial effects were assessed by determining the minimum inhibitory concentration (MIC), using the broth microdilution method, and fractional inhibitory concentration index (FICI). Preformed biofilms (48 h) were treated with different concentrations of the nanocarrier (24 h) and quantified by colony-forming units (CFUs), total biomass and metabolic activity. For cytotoxicity, the viability of L929 cells was evaluated by MTT assay after 24 and 48 h of exposure to the nanocarrier. Data were submitted to ANOVA and Fisher LSD or Tukey post-hoc tests (α = 0.05). RESULTS: MIC and FICI results showed an indifferent interaction among the components of the nanocarrier for all strains evaluated. CHX alone and nanocarrier containing 156 µg/mL CHX did not differ from each other in reducing the number of CFUs. However, the nanocarrier containing 156 µg/mL CHX promoted the highest reductions in total biofilm biomass and metabolism, surpassing the effect of CHX alone. After 24 and 48 h of exposure, the nanocarrier reduced CHX toxicity to the L929 cell at low concentrations. CONCLUSION: These findings suggest that the CHX nanocarrier has potential to be used in the control of oral diseases associated with C. glabrata and E. faecalis. CLINICAL RELEVANCE: CHX has improved the antibiofilm effect and reduced the cytotoxicity (at low concentrations) when conjugated to CS-coated IONPs. This new colloidal formulation has potential as an alternative antimicrobial agent to pure CHX for the control of biofilm-related oral diseases, such as oral candidiasis and endodontic infections.

Atenolol increases dental mineralization in male offspring of treated hypertensive rats and normotensive rats.

This study evaluates how atenolol affects dental mineralization in offspring of female spontaneously hypertensive rats (fSHR) and normotensive Wistar rats (fW). fSHR and fW were treated with atenolol (100 mg/Kg/day, orally) during pregnancy and lactation. Non-treated fSHR and fW were the control groups. Enamel and dentin hardness were analyzed (Knoop, 15 g load, 10s) in mandibular incisor teeth (IT) and molar teeth (MT) obtained from the male offspring of atenolol-treated and non-treated fWistar and fSHR. Data were analyzed by ANOVA, followed by Tukey post hoc test (p < 0.05). Atenolol reduced the arterial blood pressure (SBP) in fSHR, but it did not change the SBP in fW. The offspring of non-treated fSHR had lower enamel (IT and MT) and dentin (IT) hardness than the offspring of non-treated fW (p < 0.05). Atenolol increased enamel and dentin hardness in the IT obtained from the offspring of fSHR and fW (p<0.05), but the offspring of fSHR presented higher values (p < 0.05). Atenolol did not alter enamel width in the IT obtained from any of the groups, but it increased enamel and dentin hardness in the IT obtained from the offspring of fSHR and fW. Atenolol affected the IT obtained from the offspring of fSHR. Atenolol increased only enamel hardness in the MT obtained from the offspring of fW. In conclusion, maternal hypertension reduces tooth hard tissues, and treatment with atenolol increases tooth hardness in male offspring of hypertensive and normotensive female rats.

Carious Lesion Severity Induces Higher Antioxidant System Activity and Consequently Reduces Oxidative Damage in Children's Saliva.

Oxidative stress biomarkers can be found at detectable concentrations in saliva. These salivary biomarkers reflect specific oxidation pathways associated with caries and periodontitis. Our study evaluated the influence of dental caries severity (assessed using the ICCMS™ criteria) on the levels of oxidative stress biomarkers in saliva from children. Unstimulated saliva samples were collected from patients (from one to three years old) in a day care center in Birigui, SP, Brazil, two hours after fasting. Children were divided into four groups (n = 30/group), according to caries severity: caries free (group A), early carious lesions (group B), moderate carious lesions (group C), and advanced carious lesions (group D). The following salivary biomarkers were determined: total proteins (TP), measured by the Lowry method; oxidative damage, measured by the TBARS method; total antioxidant capacity (TAC); superoxide dismutase (SOD) enzymatic antioxidant activity; and uric acid (UA) non-enzymatic antioxidant activity. Data were analyzed by ANOVA, followed by the Student-Newman-Keuls test, Pearson and Spearman correlation coefficients, and multivariable linear regression (p < 0.05). TP, TAC, SOD enzymatic antioxidant activity, and UA non-enzymatic antioxidant activity increased with caries severity, consequently reducing salivary oxidative damage. It was concluded that higher caries severity increases salivary antioxidant system activity, with consequent reduction in salivary oxidative damage.

Atenolol increases dental mineralization in male offspring of treated hypertensive rats and normotensive rats

Abstract This study evaluates how atenolol affects dental mineralization in offspring of female spontaneously hypertensive rats (fSHR) and normotensive Wistar rats (fW). fSHR and fW were treated with atenolol (100 mg/Kg/day, orally) during pregnancy and lactation. Non-treated fSHR and fW were the control groups. Enamel and dentin hardness were analyzed (Knoop, 15 g load, 10s) in mandibular incisor teeth (IT) and molar teeth (MT) obtained from the male offspring of atenolol-treated and non-treated fWistar and fSHR. Data were analyzed by ANOVA, followed by Tukey post hoc test (p < 0.05). Atenolol reduced the arterial blood pressure (SBP) in fSHR, but it did not change the SBP in fW. The offspring of non-treated fSHR had lower enamel (IT and MT) and dentin (IT) hardness than the offspring of non-treated fW (p < 0.05). Atenolol increased enamel and dentin hardness in the IT obtained from the offspring of fSHR and fW (p<0.05), but the offspring of fSHR presented higher values (p < 0.05). Atenolol did not alter enamel width in the IT obtained from any of the groups, but it increased enamel and dentin hardness in the IT obtained from the offspring of fSHR and fW. Atenolol affected the IT obtained from the offspring of fSHR. Atenolol increased only enamel hardness in the MT obtained from the offspring of fW. In conclusion, maternal hypertension reduces tooth hard tissues, and treatment with atenolol increases tooth hardness in male offspring of hypertensive and normotensive female rats.

Traumatic dental injuries in young children / Traumatismos dentais em crianças pequenas / Traumatismos dentales en niños pequenos

Introduction: Dental trauma is a frequent occurance in Dentistry. They often occur in early childhood, between 0 to 3 years old, when children begin to learn their first steps, to play and run, because their habilities are not refined. Objective: The purpose of this article is to verify the occurance of traumatic dental injury in primary teeth in a children population. Material and Method: seventy-eight child from 0 to 6 years old male and female were evaluated. The study was conducted in Regional Hospital of Presidente Prudente. Children were evaluated by a questionary about occurance of traumatic injury, cause and search for attendance. The data were collected and submited to Spearman correlation test. Results: Traumatic dental injury occurs in deciduous teeth in (44,8%) of the population, more common in males (68,5%), at two years old (28,5%) and in front superior region (91,5%) in the upper central incisors (48,5%). Conclusion: By the use of correlation test of Spearman, it was possible to verify that there is a positive correlation between the factors: darkness and mobility, darkness and search for treatment, early loss and satisfactory treatment and search for treatment and satisfactory treatment(AU)

Introdução: O traumatismo dentário é uma ocorrência frequente em odontologia. Muitas vezes ocorrem na primeira infância, entre 0 a 3 anos, quando as crianças começam a aprender seus primeiros passos, a brincar e correr, porque suas habilidades não são refinadas. Objetivo: O objetivo deste artigo é verificar a ocorrência de lesões dentárias traumáticas em dentes primários em uma população infantil. Material e Método: Foram selecionadas 78 crianças de 0 a 6 anos do sexo masculino e feminino do Hospital Regional de Presidente Prudente, 35 crianças foram avaliadas, pois haviam sofrido trauma, e seus pais preencheram um questionário sobre ocorrência de lesão traumática, causas e atendimento. Os dados foram coletados por meio de questionário e submetidos ao teste de correlação de Spearman (P<0,05). Resultados: ocorreu lesão dentária traumática em dentes decíduos em (44,8%) da população, mais comum nos homens (68,5%), aos dois anos de idade (28,5%) e na região frontal superior (91,5%) nos incisivos centrais superiores (48,5%). Conclusão: Foi possível verificar que há uma correlação positiva entre os fatores: escurecimento e mobilidade, escurecimento e busca de tratamento, perda precoce e tratamento satisfatório e busca tratamento e tratamento satisfatório(AU)

Introducción: El traumatismo dental es una ocurrencia frecuente en odontología. Muchas veces ocurren en la primera infancia, entre 0 a 3 años, cuando los niños empiezan a aprender sus primeros pasos, a jugar y correr, porque sus habilidades no son refinadas. Objetivo: El objetivo de este artículo es verificar la ocurrencia de lesiones dentales traumáticas en dientes primarios en una población infantil. Material Y Métodos: Se seleccionaron 78 niños de 0 a 6 años del sexo masculino y femenino del Hospital Regional de Presidente Prudente, 35 niños fueron evaluados, pues habían sufrido trauma, y sus padres llenaron un cuestionario sobre ocurrencia de lesión traumática, causas y atención. Los datos fueron recolectados en un cuestionario y se sometió a la prueba de correlación de Spearman (P <0,05). En la mayoría de los casos, se observó un aumento en la incidencia de la enfermedad de Chagas, %) en los incisivos centrales superiores (48,5%). Conclusión: Fue posible verificar que hay una correlación positiva entre los factores: oscurecimiento y movilidad, oscurecimiento y búsqueda de tratamiento, pérdida precoz y tratamiento satisfactorio y busca tratamiento y tratamiento satisfactorio(AU)

Microorganisms related to early childhood caries in a sample of an oral preventive-educative program: a longitudinal study / Microorganismos relacionados a cárie precoce da infância em uma amostra de um programa preventivo de cárie dentária: um estudo longitudinal

Objetivo: Premature acquisition of cariogenic microorganisms seems to be related to higher prevalence and activity of caries lesions. The aim of the study was to evaluate the prevalence of Streptococcus mutans and Streptococcus sobrinus in infants enrolled in a dental preventive program and in their mothers, as well as to assess the influence of bacterial prevalence, diet and oral hygiene in dental caries prevalence. Material and methods: After clinical examinations (n = 50), saliva and oral biofilms were collected and stored prior to real-time PCR at 6, 12, 18 and 24 months of age. Results: No correlation was observed between the presence of cariogenic pathogens and diet or hygiene habits at all ages; however, association increased with number of erupted teeth. Salivary levels of bacteria were lower in children than in their mothers at all ages, and children with carious lesions had high ingestion of sugared food. Conclusion: As the levels of cariogenic pathogens were low in the patients that were enrolled in a preventive program, we can conclude that control of oral biofilm as eruption of infants' teeth occurs and sugar ingestion should be considered of great importance in preventive dentistry, because the association between them was highly positive. (AU)

Objetivo: A aquisição precoce de microorganismos cariogênicos está correlacionada a alta prevalência e atividade de lesões de cárie. O objetivo deste estudo foi avaliar a prevalência de Streptococcus mutans e Streptococcus sobrinus em bebês e pares de mães, matriculados no programa educativo e preventivo da Bebê Clínica da Faculdade de Odontologia de Araçatuba (UNESP), como também foi avaliada influência da prevalência bacteriana, dieta e higiene oral na prevalência de cárie dentária. Material e métodos: Após exame clínico (n=50), foram coletados saliva e biofilme e submetidos a real-time PCR aos 6, 12, 18 e 24 meses de idade. Resultados: Não foi observada correlação entre a presence de patógenos cariogênicos e hábitos de dieta e higiene em todas as idades avaliadas. Níveis salivares foram mais baixos nas crianças do que em suas mães em todas as idades, e as crianças portadoras de lesões cariosas tinham maior ingestão de alimentos açúcarados. Conclusão: Uma vez que os níveis de patógenos cariogênicos foram baixos nos pacientes que frequentam um programa preventivo, podemos concluir que o controle de biofilme durante a erupção dentária e da ingestão de açúcares pode ser considerada medida de grande relevância em prevenção odontológica, uma vez que a associação foi altamente positiva. (AU)

A severidade da lesão de cárie induz maior atividade de sistemas antioxidantes e consequente redução do estresse oxidativo na saliva de crianças / Carious lesion severity induces higher activity of antioxidant systems and consequent reduction of oxidative stress in saliva of children

O objetivo deste estudo foi avaliar a relação entre os níveis de biomarcadores de estresse/ dano oxidativo na saliva de crianças e a severidade de cárie dentária classificada pelo Sistema Internacional de Classificação e Gerenciamento de Cárie (ICCMS™, termo em inglês International Caries Classification and Management System). Amostras de saliva não estimuladas foram coletadas de pacientes de 1-3 anos de idade, após 2 horas de jejum, no período da manhã, em uma creche do município de Birigui, SP, Brasil. As crianças foram divididas em 4 grupos (n=30/grupo), de acordo com a severidade de cárie, sendo livre de cárie (grupo A), lesão de cárie em estágio inicial (grupo B), lesão de cárie intermediaria (grupo C) e lesão de cárie em estágio avançado (grupo D). Foram avaliados os seguintes biomarcadores salivares de estresse oxidativo: dano oxidativo ou malonaldeído (MDA), mensurado pelo método de TBARS; capacidade antioxidante total (TAC), medida pelo ensaio do poder antioxidante férrico redutor; atividade antioxidante enzimática da superóxido dismutase (SOD) e atividade antioxidante não enzimática do ácido úrico (UA). Os dados foram analisados por ANOVA, seguido do teste de Student-Newman-Keuls, coeficientes de correlação de Pearson e Spearman, e regressão linear multivariada (p <0,05). Os resultados demonstraram que a quantidade de proteína total, TAC, atividade antioxidante enzimática e não-enzimática salivar aumentaram de acordo com a severidade das lesões de cárie, levando a redução do dano oxidativo salivar. Podemos concluir que quanto maior a severidade das lesões de cárie, maior atividade dos sistemas antioxidantes salivares, havendo, consequentemente, diminuição gradual do dano oxidativo salivar(AU)

The objective of this study was to evaluate the relationship between the levels of biomarkers of oxidative stress / oxidative damage in children's saliva and the severity of dental caries classified by the International Caries Classification and Management System (ICCMSTM). Unstimulated saliva samples were collected from patients 0-3 years old after 2 hours of fasting in the morning in a day care center in Birigui municipality. The children were divided into 4 groups (n = 30 / group), according to caries severity, being caries free (group A), early caries lesion (group B), intermediate caries lesion (group C) and advanced carious lesion (group D). The following salivary biomarkers of oxidative stress were evaluated: oxidative or malonaldehyde damage (MDA), measured by the TBARS method; total antioxidant capacity (TAC), as measured by the ferric reducing antioxidant power test; enzymatic antioxidant activity of superoxide dismutase (SOD) and non-enzymatic antioxidant activity of uric acid (UA). Data were analyzed by ANOVA, followed by Student-Newman-Keuls test, Pearson and Spearman correlation coefficients, and multivariate linear regression (p <0.05). The results showed that the concentration of total protein, TAC, enzymatic antioxidant and non-enzymatic salivary activity increased according to the severity of caries lesions, leading to reduction of salivary oxidative damage. We can conclude that the greater the severity of caries lesions, the greater the activity of the salivary antioxidant systems, and consequently the gradual decrease of salivary oxidative damage(AU)