Antibiofilm effect of ozonized physiological saline solution on peri-implant-related biofilm.

BACKGROUND: Removal of dental plaque and local application of local chemical adjuncts, such as chlorhexidine (CHX), have been used to control and treat peri-implant disease. However, these methods can damage the surface properties of the implants or promote bacterial resistance. The application of ozone as an adjunctive treatment represents a new approach in the management of peri-implantitis. Thus, the purpose of this study was to evaluate the antimicrobial effect of ozonized physiological saline solution in different concentrations against oral biofilms developed on titanium surface. METHODS: Single and multi-species biofilms of Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus oralis were formed on titanium specimens for 5 days in anaerobic conditions. Biofilms were treated with ozonized saline solution at different concentrations (25, 50, and 80 µg/NmL), for 30 seconds and 1 minute. CHX (0.12%) and saline solution (0.89% NaCl) were used as positive and negative controls, respectively. Bacterial viability was quantified by colony forming units (CFU mL-1 ), and biofilm images were acquired by confocal laser scanning microscopy (CLSM). Data were analyzed by parametric test (ANOVA) with Tukey post-hoc test (P < 0.05). RESULTS: Ozonized saline solution showed antibiofilm activity at a concentration of 80 µg/NmL for 30 seconds and 1 minute, reducing, mainly, Porphyromonas gingivalis viability, with 2.78 and 1.7 log10 CFU mL-1 of reduction in both single and multi-species biofilms, respectively, when compared to the control (saline), whereas CHX reduced 1.4 and 1.2 log10 CFU mL-1 . CONCLUSION: Ozonized saline solution has antibiofilm activity, with better effect when applied for 1 minute at 80 µg/NmL, being a promising candidate therapy for the treatment of peri-implant diseases.

Synthesis of multifunctional chlorhexidine-doped thin films for titanium-based implant materials.

Our goal was to create bio-functional chlorhexidine (CHX)-doped thin films on commercially pure titanium (cpTi) discs using the glow discharge plasma approach. Different plasma deposition times (50, 35 and 20 min) were used to create bio-functional surfaces based on silicon films with CHX that were compared to the control groups [no CHX and bulk cpTi surface (machined)]. Physico-chemical and biological characterizations included: 1. Morphology, roughness, elemental chemical composition, film thickness, contact angle and surface free energy; 2. CHX-release rate; 3. Antibacterial effect on Streptococcus sanguinis biofilms at 24, 48 and 72 h; 4. Cytotoxicity and metabolic activity using fibroblasts cell culture (NIH-F3T3 cells) at 1, 2, 3 and 4 days; 5. Protein expression by NIH-F3T3 cells at 1, 2, 3 and 4 days; and 6. Co-culture assay of fibroblasts cells and S. sanguinis to assess live and dead cells on the confocal laser scanning microscopy, mitochondrial activity (XTT), membrane leakage (LDH release), and metabolic activity (WST-1 assay) at 1, 2 and 3 days of co-incubation. Data analysis showed that silicon films, with or without CHX coated cpTi discs, increased surface wettability and free energy (p < 0.05) without affecting surface roughness. CHX release was maintained over a 22-day period and resulted in a significant inhibition of biofilm growth (p < 0.05) at 48 and 72 h of biofilm formation for 50 min and 20 min of plasma deposition time groups, respectively. In general, CHX treatment did not significantly affect NIH-F3T3 cell viability (p > 0.05), whereas cell metabolism (MTT assay) was affected by CHX, with the 35 min of plasma deposition time group displaying the lowest values as compared to bulk cpTi (p < 0.05). Moreover, data analysis showed that films, with or without CHX, significantly affected the expression profile of inflammatory cytokines, including IL-4, IL-6, IL-17, IFN-y and TNF-α by NIH-F3T3 cells (p < 0.05). Co-culture demonstrated that CHX-doped film did not affect the metabolic activity, cytotoxicity and viability of fibroblasts cells (p > 0.05). Altogether, the findings of the current study support the conclusion that silicon films added with CHX can be successfully created on titanium discs and have the potential to affect bacterial growth and inflammatory markers without affecting cell viability/proliferation rates.

Comparative effects of photodynamic therapy mediated by curcumin on standard and clinical isolate of Streptococcus mutans.

AIM: The aim of this study was investigate the effect of photodynamic therapy (PDT) using curcumin (C) as a photosensitizing agent irradiated with an LED (L) in the blue wavelength as a light source on a standard and clinical isolate of Streptococcus mutans (S. mutans) in a planktonic suspension model. MATERIALS AND METHODS: Suspensions of both strains were divided into 4 groups as follows: absence of C and L (control group: C-L-), with C and without L (C group: C+L-), absence of C with L (L group: C-L+) and presence of C and L (PDT group: C+L+). Three different concentrations of curcumin (0.75 mg/ml, 1.5 mg/ml and 3 mg/ml) and three light fluences of studied light source (24, 48 and 72 J cm(-2)) were tested. Aliquots of each studied group was plated in BHI agar and submitted to colony forming units counting (CFU/ml) and the data transformed into logarithmical scale. RESULTS: A high photoinactivation rate of more than 70% was verified to standard S. mutans strain submitted to PDT whereas the clinical isolate showed a lower sensitivity to all the associations of curcumin and LED. A slight bacterial reduction was verified to C+L- and C-L+, demonstrating no toxic effects to the isolated application of light and photosensitizer to both S. mutans strains tested. CONCLUSION: Photodynamic therapy using a combination of curcumin and blue LED presented a substantial antimicrobial effect on S. mutans standard strain in a planktonic suspension model with a less pronounced effect on its clinical isolate counterparts due to resistance to this alternative approach. CLINICAL SIGNIFICANCE: Alternative antimicrobial approaches, as photodynamic therapy, should be encouraged due to optimal results against cariogenic bacteria aiming to prevent or treat dental caries.

Photodynamic potential of curcumin and blue LED against Streptococcus mutans in a planktonic culture.

BACKGROUND: The photodynamic therapy (PDT) involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent or dye in the presence of oxygen for eradication of target cells. In dentistry, this therapy is used to suppress the growth of microorganisms involved directly with dental decay and periodontitis process. There are evidences that curcumin dye is able to control microbial activity when illuminated with specific wavelength. The purpose of this study was to evaluate the in vitro efficacy of PDT using curcumin dye (Cur-C) in combination with a blue LED (L) device on a planktonic model of Streptococcus mutans (S. mutans). METHODS: Suspensions (0.5 mL) containing S. mutans at 1×10(7)CFU mL(-1) were prepared and divided into 4 groups: Group C-L- (control: no treatment and 1 experimental condition), Group C+L- (curcumin at 3 different concentrations: 2000; 4000 and 8000 µM and 3 experimental conditions), Group C-L+ (LED at 3 different dosages: 24, 48 and 72 Jcm(-2) and 3 experimental conditions), and Group C+L+ (PDT group: curcumin at respective concentrations combined to LED dosages and 9 experimental conditions). Samples of each experimental condition were cultured in Petri dishes of BHI agar. Incubation in micro-aerophilia at 37°C for 48 h was performed for subsequent visual counting of CFU/mL. Data were transformed into log10 and analyzed by two-way ANOVA and Tukey's test at p<0.05. RESULTS: Group C+L+, in specific experimental conditions, demonstrated a log bacterial reduction 70% higher than Group C-L-. Both groups C-L+ and C+L- presented a slight decrease in log bacterial counting. CONCLUSION: This in vitro method was able to reduce the number of S. mutans in a planktonic suspension.