Marco Aurélio Benini Paschoal

Genes expressed in dental enamel development are associated with molar-incisor hypomineralization

Genetic disturbances during dental development influence variation of number and shape of the dentition. In this study, we tested if genetic variation in enamel formation genes is associated with molar-incisor hypomineralization (MIH), also taking into consideration caries experience. DNA samples from 163 cases with MIH and 82 unaffected controls from Turkey, and 71 cases with MIH and 89 unaffected controls from Brazil were studied. Eleven markers in five genes [ameloblastin (AMBN), amelogenin (AMELX), enamelin (ENAM), tuftelin (TUFT1), and tuftelin-interacting protein 11 (TFIP11)] were genotyped by the TaqMan method. Chi-square was used to compare allele and genotype frequencies between cases with MIH and controls. In the Brazilian data, distinct caries experience within the MIH group was also tested for association with genetic variation in enamel formation genes. The ENAM rs3796704 marker was associated with MIH in both populations (Brazil: p=0.03; OR=0.28; 95% C.I.=0.06-1.0; Turkey: p=1.22e-012; OR=17.36; 95% C.I.=5.98-56.78). Associations between TFIP11 (p=0.02), ENAM (p=0.00001), and AMELX (p=0.01) could be seen with caries independent of having MIH or genomic DNA copies of Streptococcus mutans detected by real time PCR in the Brazilian sample. Several genes involved in enamel formation appear to contribute to MIH.

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 Tukeys 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.

Streptococcus mutans photoinactivation by combination of short exposure of a broad-spectrum visible light and low concentrations of photosensitizers

OBJECTIVE Investigate the photodynamic antimicrobial effect by the combination of a novel noncoherent broad spectrum visible light and low concentrations of curcumin and toluidine blue over suspensions of Streptococcus mutans. BACKGROUND DATA Long illumination times to activate photosensitizers (PS) and the use of high concentrations of these drugs in photodynamic antimicrobial chemotherapy (PACT) are limitations of its application as an antimicrobial technology in dental practice. MATERIALS AND METHODS Planktonic suspensions of S. mutans were standardized and submitted to PACT treatment at low concentrations of curcumin (C) (0.075; 0.75 and 7.5 μM) and toluidine blue (T) (0.25; 2.5 and 25 μM) exposed to 42 J/cm2 (12.2 sec; set power: 3.930 mW) of a white light (WL) (output wavelength range: 400-700 nm; beam diameter: 12 mm) (C+WL+ and T+WL+, PACT groups; incubation time, C: 60 sec; T: 5 min); isolated effect of both C (C+WL-) and T concentrations (T+WL-); effect of light source (C-WL+ and T-WL+) and suspensions neither submitted to PS nor to light-emitting diode (LED) illumination (control groups, C-WL- and T-WL-). Aliquots of each group were diluted and cultured on blood agar plates and the number of colony-forming units (CFU)/mL was recorded, transformed into log10 and analyzed by ANOVA and Tukeys test at a cutoff value at 0.05. RESULTS The groups submitted to PACT presented a bacterial reduction value of>5-log10 to both tested PS in comparison with control groups (p<0.05). PS or light source used alone demonstrated no antimicrobial effect on the number of viable bacterial counts. CONCLUSIONS The combination of a novel noncoherent light at short illumination exposure time with low concentrations of studied PS achieved a lethal photoinactivation of S. mutans, and can be considered an effective antimicrobial in vitro approach for reducing the number of micro-organisms involved with the dental caries process.

Therapeutic Effects of Low-Level Laser Therapy After Premolar Extraction in Adolescents: A Randomized Double-Blind Clinical Trial

OBJECTIVE The purpose of this study was to evaluate the effect of low-level laser therapy (LLLT) on wound healing process and pain levels after premolar extraction in adolescents. BACKGROUND DATA The advantage of using LLLT in oral surgeries is the reduction of inflammation and postoperative discomfort; however, the optimal dosing parameters and treatment effects in surgical procedures are inconclusive. METHODS A double-blind, randomized, controlled clinical trial was conducted with 14 patients who were to undergo surgical removal of premolars. Patients were randomly allocated to the LLLT (test) group and placebo (control) group. Patients in the test group received 5.1 J (60 J/cm(2)) of energy density of a gallium-aluminum-arsenide (GaAlAs) diode laser (wavelength, 830 nm; output power, 0.1 W) at three different points intraorally, 1 cm from the target tissue immediately and at 48 and 72 h after the surgical procedure. For patients in the placebo group, the laser device was applied to the same points without activating the hand piece. The wound healing process was evaluated by an independent examiner by visual inspection with the support of digital photographs at baseline and 2, 7, and 15 days postoperatively. Patients recorded the degree of pain using the visual analogue scale (VAS). RESULTS Compared with the placebo group, the test group showed a lower intensity of pain, but this difference was not statistically significant at any time point. The wound healing process was similar in both groups. CONCLUSIONS Within the limitations of this study, the LLLT parameters used neither increased the wound healing process nor significantly decreased pain intensity after premolar extraction in adolescents.

Noninvasive and Multidisciplinary Approach to the Functional and Esthetic Rehabilitation of Amelogenesis Imperfecta: A Pediatric Case Report

Case Report. An 8-year-old girl with amelogenesis imperfecta (AI) reported unsatisfactory aesthetics, difficulty in mastication, and dental hypersensitivity. The intraoral examination observed mixed dentition, malocclusion in anteroposterior relationships, anterior open bite, and dental asymmetry. A hypoplastic form of AI was diagnosed in the permanent dentition. A multidisciplinary planning was performed and divided into preventive, orthopedic, and rehabilitation stages. Initially, preventive treatment was implemented, with fluoride varnish applications, in order to protect the fragile enamel and reduce the dental sensitivity. In the second stage, the patient received an interceptive orthopedic treatment to improve cross-relationship of the arches during six months. Finally, the rehabilitation treatment was executed to establish the vertical dimension. In the posterior teeth, indirect composite resin crowns were performed with minimally invasive dental preparation. Direct composite resin restorations were used to improve the appearance of anterior teeth. Follow-Up. The follow-up was carried out after 3, 6, 12, and 18 months. After 18 months of follow-up, The restoration of integrity, oral hygiene, and patient satisfaction were observed . Conclusion. Successful reduction of the dental hypersensitivity and improvement of the aesthetic and functional aspects as well as quality of life were observed.

Longitudinal Evaluation of the Structural Integrity of Teeth Affected by Molar Incisor Hypomineralisation

The aim of this prospective cohort study was to evaluate the risk of posteruptive breakdown and the development of caries lesions in teeth with molar incisor hypomineralisation (MIH). A total of 367 permanent incisors and first molars, affected and not affected by MIH lesions, of 45 children with MIH from Araraquara, São Paulo, Brazil, were evaluated at intervals from 6 to 12 months by assessing the severity of MIH, the presence of tooth caries lesions and the treatment needed. During the study period, all patients received preventive care. The data were analysed using Fishers exact test and actuarial method survival analysis. Significant associations were also found in teeth between the presence of MIH and a DMFT index >0 in all periods and also between the need for treatment and the presence of MIH. The teeth affected by MIH opacities were healthy in 99% of incisors and 93% of molars at the end of the 12-month period. Due to the high likelihood of maintaining the tooth structure in opacities, the complete or premature removal of the affected area is not justified.

Streptococcus mutans photoinactivation using a combination of a high potency photopolymerizer and rose bengal

BACKGROUND Long treatment periods to reach a substantial inactivation of microorganisms are one of the critical challenges in the photodynamic therapy field. METHODS Planktonic suspensions of Streptococcus mutans were treated in different groups: presence of rose bengal (RB at 2μM) and light exposure by a new high potency photopolymerizer (L at wavelength=440-480nm; dosimetry=96J/cm(2) - 40s of irradiation; potency density=1200mW/cm(2); dosage=48J) - RB+L+ (PDT), just with dye - RB+L-, just with light - RB-L+ and absence of light and dye RB-L- (control group). Aliquots of each group were transferred to Petri dishes to colony counting (CFU/mL) with the data transformed in log10. The results were submitted to ANOVA and Tukey test at 5%. RESULTS PDT group presented total eradication of microorganisms showing statistical difference with all the other groups (5.82log reduction≅99%). CONCLUSION The high potency photopolymerizer in pulsed emission at an extra short irradiation and low concentration of rose bengal could be considered as a progressive alternative to the control of S. mutans suspensions.

Photodynamic therapy with high-power LED mediated by erythrosine eliminates Enterococcus faecalis in planktonic forms

BACKGROUND The failure of endodontic treatment is linked to the presence of microorganisms, particularly Enterococcus faecalis, in the root canals. This study evaluated the effectiveness of photodynamic therapy (PDT) using erythrosine irradiated by a high-power curing light on a planktonic suspension culture of E. faecalis. METHODS Bacterial suspensions of E. faecalis were adjusted and then mixed in a 1:1 proportion, in triplicate, in treatment groups by varying the length of irradiation time (120 and 240s) and the molarity of the erythrosine (5 and 10μM). In order to verify the post-treatment bactericidal effect, a count of the viable bacteria was performed (CFUmL-1) and transformed into Log10 CFU. The one-way ANOVA with Tukey post-hoc test was applied to check for differences between the groups. RESULTS The bacteria were completely eradicated in the groups that used PDT with 5μM 240s, 10μM 120s and 10μM 240s (p≪0.001). The effect of the PDT 5μM 120s group was significant (p≪0.05) in comparison with the groups using only light or only erythrosine. Positive control (exposure to 2.5% NaClO for 120 and 240s) completely eradicated E. faecalis. The negative control (PBS) did not alter the quantities of E. faecalis CFU with 9.605 Log10 CFU at 120s and 9.621 Log10 CFU at 240s. CONCLUSION PDT with erythrosine in a concentration of 10μM and high-power LED is capable of totally eliminating E. faecalis in planktonic suspension.

In vitro Effects of Lemongrass Extract on Candida albicans Biofilms, Human Cells Viability, and Denture Surface

The purpose of this study was to investigate whether immersion of a denture surface in lemongrass extract (LGE) has effects on C. albicans biofilms, human cell viability and denture surface. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were performed for LGE against C. albicans. For biofilm analysis, discs were fabricated using a denture acrylic resin with surface roughness standardization. C. albicans biofilms were developed on saliva-coated discs, and the effects of LGE at MIC, 5XMIC, and 10XMIC were investigated during biofilm formation and after biofilm maturation. Biofilms were investigated for cell counting, metabolic activity, and microscopic analysis. The cytotoxicity of different concentrations of LGE to peripheral blood mononuclear cells (PBMC) was analyzed using MTT. The effects of LGE on acrylic resin were verified by measuring changes in roughness, color and flexural strength after 28 days of immersion. Data were analyzed by ANOVA, followed by a Tukey test at a 5% significance level. The minimal concentration of LGE required to inhibit C. albicans growth was 0.625 mg/mL, while MFC was 2.5 mg/mL. The presence of LGE during biofilm development resulted in a reduction of cell counting (p < 0.05), which made the MIC sufficient to reduce approximately 90% of cells (p < 0.0001). The exposure of LGE after biofilm maturation also had a significant antifungal effect at all concentrations (p < 0.05). When compared to the control group, the exposure of PBMC to LGE at MIC resulted in similar viability (p > 0.05). There were no verified differences in color perception, roughness, or flexural strength after immersion in LGE at MIC compared to the control (p > 0.05). It could be concluded that immersion of the denture surface in LGE was effective in reducing C. albicans biofilms with no deleterious effects on acrylic properties at MIC. MIC was also an effective and safe concentration for use.

Photodynamic antimicrobial chemotherapy for prevention and treatment of dental caries: a critical review

Introduction Photodynamic antimicrobial chemotherapy studies regarding dental caries have been present more frequently in the literature. However, photodynamic antimicrobial chemotherapy depends on the adjustment of variables such as the type of light source and, photosensitisers target microorganism; this makes it difficult to draw meaningful comparisons. The purpose of this paper was to provide a critical review related to this coadjuvant approach in the prevention and treatment of dental caries. Materials and Methods A database search was made via Medline/PubMed (keywords: photodynamic therapy and dental caries) and 33 articles were found. Results Twelve articles were included after using the filter tool, being excluded reviews and manuscripts reporting works not related to the studied area. Conclusion The manuscripts showed that photodynamic therapy presents optimal results against dental caries, even though better understanding of photodynamic antimicrobial chemotherapy and its components are necessary before the clinical application of this alternative modality in the dental practice. Photodynamic antimicrobial chemotherapy for prevention and treatment of dental caries: a critical review MA Paschoal1, S Duarte2*, L Santos-Pinto1* Introduction The human oral cavity is heavily colonised by a complex, relatively specific and highly interrelated range of microorganisms (as many as 1000 different species have been detected) collectively known as normal oral microflora1. A peculiarity of this environment is that most of the bacteria found here are present in complex aggregates (known as biofilms)2. Microbial biofilms are composed of microorganisms adhered both to each other and to dental surfaces (or interfaces) and embedded in an extracellular polymeric matrix, which includes water and nutrient channels3. A change in a key environmental factor will trigger a shift in the balance of the resident microflora, which will promote the emergence of acidogenic/aciduric bacteria. The constant accumulation of these kind of bacteria change the equilibrium towards dental demineralisation (dental caries lesions)4. The constituents of diet present an important role in the development of dental caries. Sucrose is considered the most cariogenic dietary carbohydrate, because it is fermentable and serves as a substrate for the synthesis of extracellular polysaccharides (EPS) and intracellular polysaccharides in cariogenic dental plaque4. In addition, the presence of EPS (mainly insoluble glucan) promote bacterial adherence to the tooth surface and contribute to the structural integrity of dental biofilms. Yet, there is a clear evidence showing that sucrose exposure and insoluble EPS lead to a more cariogenic biofilm5. Dental caries is among the most significant human chronicle infectious diseases and results in the progressive dissolution of enamel. With the disease progression, it can lead the underlying dentine compromising the vitality of the element and its fixation in the maxillomandibular complex6. Prevention of dental caries can be achieved by controlling the accumulation of dental plaque by mechanical removal7. In cases of insufficient biofilm disorganisation, the association with antimicrobial chemical agents, such as chlorhexidine may help in the decreasing of pathogenic bacteria levels7. Unfortunately, this preventive approach does not reach the population as a whole, allowing dental cavity formation. Treatment of the carious lesion involves the removal of infected dentine with posterior restoration of the affected tooth with any of the variety of materials, for example mercury amalgam, resin composite and glass ionomer cements8. Due to emergence of antibiotic resistant strains, alteration in taste, burning sensation, increase of calculus formation and staining of the teeth and restorative materials stimulated a search for alternative treatments9. Recently, approaches that might offer the possibility of efficient intraoral bacterial count reduction with minimum damage to systemic health (preventive approach) and avoid secondary caries development reducing the chance of material substitution and pulp inflammation as well (curative approach) are necessary. For these circumstances, photodynamic antimicrobial chemotherapy (PACT) offers the possibility of a novel modality to reduce pathogenic bacteria, and consequently, prevent against (new) dental caries lesions10. * Corresponding authors Emails: marcobpaschoal@hotmail.com; lspinto@foar.unesp.br; sd84@nyu.edu 1 Department of Pediatric Dentistry, Araraquara School of Dentistry, Univ Estadual Paulista (UNESP), Araraquara, SP, Brazil 2 Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University (NYU), New York, NY, USA