Consequences of tooth luxation associated with complicated crown fracture: a case report.

The objective of this clinical case report is to describe the late treatment performed in a 10-year-old patient who suffered a complicated crown fracture associated with luxation of the maxillary left central incisor. The patient-s parents reported that the patient fell and was immediately taken to a hospital, where radiographs, splinting, and suturing were performed and an anti-inflammatory prescription was provided, but no treatment was given for the exposed pulp. The parents and patient sought treatment at a pediatric dentistry clinic 16 days post-trauma. The clinical examination revealed the presence of a left central incisor with a fracture of enamel and dentin involving the pulp, which was necrotic, and a splint extending from the right lateral incisor to the left lateral incisor. However, it was not possible to diagnose the type of luxation that had occurred. The fractured incisor was treated with periodic changes of calcium hydroxide dressing for apexification for 10 months, and the root was subsequently obturated with gutta percha and root canal sealer. The tooth fragment was stored in physiologic solution during this time. After obturation, tooth darkening was observed, and LED-assisted tooth bleaching was accomplished prior to reattachment of the fractured fragment. The tooth was reexamined every 6 months. Two years after the traumatic event, the left central incisor was infraoccluded in relation to the right central incisor. A radiograph confirmed ankylosis of the traumatized tooth. When trauma affects the enamel, dentin, pulp, and supporting tissues, the prognosis can be unfavorable even when late treatment is adequate, especially when a tooth in a growing patient has points of ankylosis.

In vitro treatment of Enterococcus faecalis with calcium hydroxide impairs phagocytosis by human macrophages.

OBJECTIVE: Monocyte-derived macrophages (MDMs) ability to phagocytize and produce nitric oxide (NO) was tested against root-canal strains of Enterococcus faecalis submitted to alkaline stress. Root-canal strains were also compared with urine Enterococci. MATERIALS AND METHODS: Enterococcus faecalis were stressed with alkaline-BHI broth and incubated in vitro at a cell/bacteria ratio of 1:5. Phagocytosis was analyzed by fluorescence microscopy using acridine orange stain, and NO concentration was measured in supernatants. RESULTS AND CONCLUSIONS: Alkaline-stress significantly impaired MDMs phagocytosis of E. faecalis strains analyzed, except in ATCC4083 isolated from a pulpless tooth, but NO production was unchanged. Comparison of different strains showed the urine isolate had higher NO levels than root canal strains. Alterations in the bacterial cell wall structures after alkaline-stress possibly made bacteria less recognizable and phagocytized by MDMs but did not affect their ability to activate NO production. Furthermore, root canal strains elicited different responses by immune cells compared with strains from urine. Clinically, impaired phagocytosis of E. faecalis could contribute to their persistence in root canal systems previously treated with calcium hydroxide.

Effect of ultrasound streaming on the disinfection of flattened root canals prepared by rotary and reciprocating systems.

New technical and scientific developments have been advocated to promote the success of the endodontic treatment. In addition to rotary and reciprocating systems, irrigating solution agitation has been suggested and passive ultrasonic irrigation (PUI) is the most used. OBJECTIVE: To evaluate, in vitro, the effect of ultrasound streaming (US) in the disinfection of flattened root canal systems prepared by the ProTaper, BioRaCe and Reciproc systems, utilizing the microbiological culture. METHODOLOGY: Extracted human mandibular incisors (n=84) were used. Suspensions of Enterococcus faecalis (ATCC 29212) were standardized and inserted along with the teeth immersed in brain-heart infusion (BHI) broth. The contamination was made following a protocol during 5 days. The teeth were randomly divided into six groups: G1, ProTaper Universal; G2, ProTaper Universal with US; G3, BioRaCe; G4, BioRaCe with US; G5, Reciproc; and G6, Reciproc with US. Irrigation was performed with saline solution. After biomechanical preparation, microbiological samples were performed with sterilized paper points, which were diluted and spread on BHI agar; after 48 h, the colony forming units (CFU/mL) were counted for each sample. RESULTS: Groups using ultrasonic agitation presented a greater antibacterial effect than the other ones, even using saline solution as irrigant. The ProTaper Universal system showed the best antibacterial activity of the tested systems (median of 0 CFU/mL with and without surfactant or ultrasonic activation [PUI]). Even with PUI, Reciproc (median of 2.5 CFU/mL with PUI and 5 without it) could not reduce as many colonies as ProTaper Universal without US. The BioRaCe system had greater bacterial reduction when using US (median of 0 CFU/mL with PUI and 30 without it). CONCLUSIONS: US promoted greater reduction in the number of bacteria in the flattened root canals prepared with nickel-titanium mechanized systems. Regarding the instruments used, the ProTaper Universal system was the most effective in reducing the bacterial number.

Effect of ultrasonic streaming on intra-dentinal disinfection and penetration of calcium hydroxide paste in endodontic treatment.

OBJECTIVE: The antimicrobial effect of ultrasonic agitation of calcium hydroxide (CH) pastes in infected bovine dentin and their penetrability were evaluated using confocal laser scanning microscopy (CLSM) and microbiological culture. MATERIAL AND METHODS: Fifty-two bovine teeth were infected with Enterococcus faecalis using a new contamination protocol; then they received CH paste and were divided into groups with or without ultrasound. Ultrasonic agitation was conducted for 1 min with a plain point insert. After 15 d, the CLSM analyzed the viable and dead bacteria with Live and Dead assay. The dentinal wall debris was collected by burs, and the colony forming units (CFU/mL) were counted. The penetrability of the paste inside dentinal tubules was tested using the B-rodamine dye. RESULTS: The calcium hydroxide paste showed better results with the use of ultrasonic agitation (p<0.05). CONCLUSION: The ultrasonic agitation of CH paste increased its antimicrobial action and was responsible for intradentinal penetration with the fulfilment of the tubules.

Rat subcutaneous tissue response to calcium silicate containing different arsenic concentrations.

OBJECTIVE: To evaluate the response of rat subcutaneous tissue in implanted polyethylene tubes that were filled with GMTA Angelus and Portland cements containing different arsenic concentrations. MATERIAL AND METHODS: Atomic absorption spectrophotometry was utilized to obtain the values of the arsenic concentration in the materials. Thirty-six rats were divided into 3 groups of 12 animals for each experimental period. Each animal received two implants of polyethylene tubes filled with different test cements and the lateral of the tubes was used as a control group. After 15, 30 and 60 days of implantation, the animals were killed and the specimens were prepared for descriptive and morphometric analysis considering: inflammatory cells, collagen fibers, fibroblasts, blood vessels and other components. The results were analyzed utilizing the Kuskal-Wallis test and the Dunn's Multiple test for comparison (p<0.05). RESULTS: The materials showed, according to atomic absorption spectrophotometry, the following doses of arsenic: GMTA Angelus: 5.01 mg/kg, WPC Irajazinho: 0.69 mg/kg, GPC Minetti: 18.46 mg/kg and GPC Votoran: 10.76 mg/kg. In a 60-day periods, all specimens displayed a neoformation of connective tissue with a structure of fibrocellular aspect (capsule). Control groups and MTA Angelus produced the lower amount of inflammatory reaction and GPC Minetti, the highest reaction. CONCLUSIONS: There was no direct relationship between the concentration of arsenic present in the composition of the materials and the intensity of the inflammatory reactions. Higher values, as 18.46 mg/kg of arsenic in the cement, produce characteristics of severe inflammation reaction at the 60-day period. The best results were found in MTA angelus.

Analysis of the reaction of subcutaneous tissues in rats and the antimicrobial activity of calcium hydroxide paste used in association with different substances.

The aim of this study was to evaluate the subcutaneous tissue response in rats and the antimicrobial activity of intracanal calcium hydroxide dressings mixed with different substances against E. faecalis. Fifty four rats were divided into three experimental groups according to the vehicle in the calcium hydroxide treatment: 0.4% chlorohexidine in propylene glycol (PG),Casearia sylvestris Sw in PG and calcium hydroxide+PG (control group). The pastes were placed into polyethylene tubes and implanted into the subcutaneous tissue. After 7, 14 and 30 days, the samples were processed and histologically evaluated (hematoxylin and eosin). The tissue surface in contact with the material was analyzed, and the quantitative analysis determined the volume density occupied by the inflammatory infiltrate (giant cells, polymorphonuclear cells and mononuclear cells), fibroblasts, collagen fibers and blood vessels. For the antimicrobial analysis, 20 dentin blocks infected with E. faecalis were treated with calcium hydroxide pastes in different vehicles; 0.4% chlorhexidine in PG, PG, extract from Casearia sylvestris Sw in PG and a positive control (infection and without medication) for 7 days. The efficiency of the pastes was evaluated by the live/dead technique and confocal microscopy. The results showed that 0.4% chlorhexidine induced a higher inflammatory response than the other groups. The Casearia sylvestris Sw extract showed satisfactory results in relation to the intensity of the inflammatory response. In the microbiological test, there were no statistical differences between the evaluated intracanal dressings and the percentage of bacterial viability was between 33 and 42%. The control group showed an 86% viability. Antimicrobial components such as chlorhexidine or Casearia sylvestris Sw did not improve the antimicrobial activity against E. faecalis in comparison to the calcium hydroxide+PG treatment. In addition, the incorporation of chlorhexidine in the calcium hydroxide paste promoted the highest inflammatory response.

A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy.

OBJECTIVES: To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM). MATERIAL AND METHODS: Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalisduring the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn's tests were used to evaluate the live and dead cells rates, and the scores obtained. RESULTS: The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05). CONCLUSION: The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.

Influence of bismuth oxide concentration on the pH level and biocompatibility of white Portland cement.

OBJECTIVES: To investigate if there is a relation between the increase of bismuth oxide and the decrease of pH levels and an intensification of toxicity in the Portland cement. MATERIAL AND METHODS: White Portland cement (WPC) was mixed with 0, 15, 20, 30 and 50% bismuth oxide, in weight. For the pH level test, polyethylene tubes were filled with the cements and immersed in Milli-Q water for 15, 30 and 60 days. After each period, the increase of the pH level was assessed. For the biocompatibility, two polyethylene tubes filled with the cements were implanted in ninety albino rats (n=6). The analysis of the intensity of the inflammatory infiltrate was performed after 15, 30 and 60 days. The statistical analysis was performed using the Kruskal-Wallis, Dunn and Friedman tests for the pH level and the Kruskal-Wallis and Dunn tests for the biological analysis (p<0.05). RESULTS: The results showed an increase of the pH level after 15 days, followed by a slight increase after 30 days and a decrease after 60 days. There were no significant statistical differences among the groups (p>0.05). For the inflammatory infiltrates, no significant statistical differences were found among the groups in each period (p>0.05). The 15% WPC showed a significant decrease of the inflammatory infiltrate from 15 to 30 and 60 days (p<0.05). CONCLUSIONS: The addition of bismuth oxide into Portland cement did not affect the pH level and the biological response. The concentration of 15% of bismuth oxide resulted in significant reduction in inflammatory response in comparison with the other concentrations evaluated.

Avaliação in vitro da atividade antifúngica da pasta de hidróxido de cálcio associada a diferentes fármacos, frente à Candida albicans / In vitro evaluation of antifungal activity of calcium hydroxide paste mixed to different drugs, against Candida albicans

Introdução: a frequência de micoses invasivas causadas por patógenos fúngicos oportunistas tem aumentado significativamente nas últimas décadas. Entre os principais agentes etiológicos de micoses oportunistas está a Candida albicans. Essa levedura apresenta vários fatores de virulência importantes para produção de doença. Alguns estudos têm demonstrado que C. albicans coloniza canais radiculares e túbulos dentinários. O espectro de atividade antimicrobiana do curativo de hidróxido de cálcio deve incluir essa levedura. Objetivos: o objetivo da presente pesquisa foi determinar a suscetibilidade in vitro de quatro linhagens de C. albicans coletadas da cavidade oral e uma estirpe padrão ATCC 10231 a pastas de hidróxido de cálcio associadas a drogas antifúngicas, antibióticas e anti-inflamatórias. A eficiência das pastas de Ca(OH)2 associadas às drogas sobre as leveduras foram analisadas pelo método da difusão radial e, também, pela técnica do contato direto com os medicamentos. Os dados foram analisados pelo teste de Kruskal-Wallis e o pós-teste de Dunn, para indicar as diferenças entres os grupos, com um nível de significância de 5%. Resultados: todas as medicações antifúngicas potencializaram a ação das pastas de hidróxido de cálcio contra a Candida albicans. Conclusões: a associação de medicações antifúngicas às pastas de Ca(OH)2 pode ser uma alternativa como medicação intracanal. (AU)

Introduction: the frequency of invasive mycoses caused by opportunistic fungal pathogens has increased significantly in the last decades. Among the main etiological agents of opportunistic mycoses is Candida albicans. This yeast has several important virulence factors for producing disease. Some studies have demonstrated that C. albicans colonized root canals and dentinal tubules. The spectrum of antimicrobial activity of calcium hydroxide dressing should include this yeast. Objectives: the aim of the present study was to determine the in vitro susceptibility of four C. albicans strains collected from the oral cavity and a standard strain ATCC 10231 to calcium hydroxide paste associated with antifungal, antibiotic and anti-inflammatory drugs. The efficiency of the Ca(OH)2 paste associated with the drugs on the yeasts was analyzed by the radial diffusion method and also by the direct contact method. Data were analyzed by Kruskal Wallis test and the Dunn post-test were used to indicate the differences between the groups with a significance level of 5%. Results: all antifungal drugs increased the action of the calcium hydroxide pastes against Candida albicans. Conclusions: the association of antifungals with Ca(OH)2 may be considered for use as intracanal medicaments (AU).

Effect of ultrasound streaming on the disinfection of flattened root canals prepared by rotary and reciprocating systems

Abstract New technical and scientific developments have been advocated to promote the success of the endodontic treatment. In addition to rotary and reciprocating systems, irrigating solution agitation has been suggested and passive ultrasonic irrigation (PUI) is the most used. Objective: To evaluate, in vitro, the effect of ultrasound streaming (US) in the disinfection of flattened root canal systems prepared by the ProTaper, BioRaCe and Reciproc systems, utilizing the microbiological culture. Methodology: Extracted human mandibular incisors (n=84) were used. Suspensions of Enterococcus faecalis (ATCC 29212) were standardized and inserted along with the teeth immersed in brain-heart infusion (BHI) broth. The contamination was made following a protocol during 5 days. The teeth were randomly divided into six groups: G1, ProTaper Universal; G2, ProTaper Universal with US; G3, BioRaCe; G4, BioRaCe with US; G5, Reciproc; and G6, Reciproc with US. Irrigation was performed with saline solution. After biomechanical preparation, microbiological samples were performed with sterilized paper points, which were diluted and spread on BHI agar; after 48 h, the colony forming units (CFU/mL) were counted for each sample. Results: Groups using ultrasonic agitation presented a greater antibacterial effect than the other ones, even using saline solution as irrigant. The ProTaper Universal system showed the best antibacterial activity of the tested systems (median of 0 CFU/mL with and without surfactant or ultrasonic activation [PUI]). Even with PUI, Reciproc (median of 2.5 CFU/mL with PUI and 5 without it) could not reduce as many colonies as ProTaper Universal without US. The BioRaCe system had greater bacterial reduction when using US (median of 0 CFU/mL with PUI and 30 without it). Conclusions: US promoted greater reduction in the number of bacteria in the flattened root canals prepared with nickel-titanium mechanized systems. Regarding the instruments used, the ProTaper Universal system was the most effective in reducing the bacterial number.

Effect of ultrasonic streaming on intra-dentinal disinfection and penetration of calcium hydroxide paste in endodontic treatment

ABSTRACT Objective The antimicrobial effect of ultrasonic agitation of calcium hydroxide (CH) pastes in infected bovine dentin and their penetrability were evaluated using confocal laser scanning microscopy (CLSM) and microbiological culture. Material and Methods Fifty-two bovine teeth were infected with Enterococcus faecalis using a new contamination protocol; then they received CH paste and were divided into groups with or without ultrasound. Ultrasonic agitation was conducted for 1 min with a plain point insert. After 15 d, the CLSM analyzed the viable and dead bacteria with Live and Dead assay. The dentinal wall debris was collected by burs, and the colony forming units (CFU/mL) were counted. The penetrability of the paste inside dentinal tubules was tested using the B-rodamine dye. Results The calcium hydroxide paste showed better results with the use of ultrasonic agitation (p<0.05). Conclusion The ultrasonic agitation of CH paste increased its antimicrobial action and was responsible for intradentinal penetration with the fulfilment of the tubules.

Rat subcutaneous tissue response to calcium silicate containing different arsenic concentrations

Objective: To evaluate the response of rat subcutaneous tissue in implanted polyethylene tubes that were filled with GMTA Angelus and Portland cements containing different arsenic concentrations. Material and Methods: Atomic absorption spectrophotometry was utilized to obtain the values of the arsenic concentration in the materials. Thirty-six rats were divided into 3 groups of 12 animals for each experimental period. Each animal received two implants of polyethylene tubes filled with different test cements and the lateral of the tubes was used as a control group. After 15, 30 and 60 days of implantation, the animals were killed and the specimens were prepared for descriptive and morphometric analysis considering: inflammatory cells, collagen fibers, fibroblasts, blood vessels and other components. The results were analyzed utilizing the Kuskal-Wallis test and the Dunn's Multiple test for comparison (p<0.05). Results: The materials showed, according to atomic absorption spectrophotometry, the following doses of arsenic: GMTA Angelus: 5.01 mg/kg, WPC Irajazinho: 0.69 mg/kg, GPC Minetti: 18.46 mg/kg and GPC Votoran: 10.76 mg/kg. In a 60-day periods, all specimens displayed a neoformation of connective tissue with a structure of fibrocellular aspect (capsule). Control groups and MTA Angelus produced the lower amount of inflammatory reaction and GPC Minetti, the highest reaction. Conclusions: There was no direct relationship between the concentration of arsenic present in the composition of the materials and the intensity of the inflammatory reactions. Higher values, as 18.46 mg/kg of arsenic in the cement, produce characteristics of severe inflammation reaction at the 60-day period. The best results were found in MTA angelus. .

Analysis of the reaction of subcutaneous tissues in rats and the antimicrobial activity of calcium hydroxide paste used in association with different substances

The aim of this study was to evaluate the subcutaneous tissue response in rats and the antimicrobial activity of intracanal calcium hydroxide dressings mixed with different substances against E. faecalis. Fifty four rats were divided into three experimental groups according to the vehicle in the calcium hydroxide treatment: 0.4% chlorohexidine in propylene glycol (PG),Casearia sylvestris Sw in PG and calcium hydroxide+PG (control group). The pastes were placed into polyethylene tubes and implanted into the subcutaneous tissue. After 7, 14 and 30 days, the samples were processed and histologically evaluated (hematoxylin and eosin). The tissue surface in contact with the material was analyzed, and the quantitative analysis determined the volume density occupied by the inflammatory infiltrate (giant cells, polymorphonuclear cells and mononuclear cells), fibroblasts, collagen fibers and blood vessels. For the antimicrobial analysis, 20 dentin blocks infected with E. faecalis were treated with calcium hydroxide pastes in different vehicles; 0.4% chlorhexidine in PG, PG, extract fromCasearia sylvestris Sw in PG and a positive control (infection and without medication) for 7 days. The efficiency of the pastes was evaluated by the live/dead technique and confocal microscopy. The results showed that 0.4% chlorhexidine induced a higher inflammatory response than the other groups. The Casearia sylvestris Sw extract showed satisfactory results in relation to the intensity of the inflammatory response. In the microbiological test, there were no statistical differences between the evaluated intracanal dressings and the percentage of bacterial viability was between 33 and 42%. The control group showed an 86% viability. Antimicrobial components such as chlorhexidine or Casearia sylvestris Sw did not improve the antimicrobial activity against E. faecalis in comparison to the calcium hydroxide+PG treatment. In addition, the incorporation of chlorhexidine in the calcium hydroxide paste promoted the highest inflammatory response.

A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy

Objectives To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM). Material and Methods Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalisduring the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD® for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn’s tests were used to evaluate the live and dead cells rates, and the scores obtained. Results The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05). Conclusion The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.

Influence of bismuth oxide concentration on the pH level and biocompatibility of white Portland cement

Objectives: To investigate if there is a relation between the increase of bismuth oxide and the decrease of pH levels and an intensification of toxicity in the Portland cement. Material and Methods: White Portland cement (WPC) was mixed with 0, 15, 20, 30 and 50% bismuth oxide, in weight. For the pH level test, polyethylene tubes were filled with the cements and immersed in Milli-Q water for 15, 30 and 60 days. After each period, the increase of the pH level was assessed. For the biocompatibility, two polyethylene tubes filled with the cements were implanted in ninety albino rats (n=6). The analysis of the intensity of the inflammatory infiltrate was performed after 15, 30 and 60 days. The statistical analysis was performed using the Kruskal-Wallis, Dunn and Friedman tests for the pH level and the Kruskal-Wallis and Dunn tests for the biological analysis (p<0.05). Results: The results showed an increase of the pH level after 15 days, followed by a slight increase after 30 days and a decrease after 60 days. There were no significant statistical differences among the groups (p>0.05). For the inflammatory infiltrates, no significant statistical differences were found among the groups in each period (p>0.05). The 15% WPC showed a significant decrease of the inflammatory infiltrate from 15 to 30 and 60 days (p<0.05). Conclusions: The addition of bismuth oxide into Portland cement did not affect the pH level and the biological response. The concentration of 15% of bismuth oxide resulted in significant reduction in inflammatory response in comparison with the other concentrations evaluated. .

Evaluation of pH and calcium ion release of calcium hydroxide pastes containing different substances.

INTRODUCTION: The objective of this study was to evaluate the pH and calcium ion release of calcium hydroxide pastes associated with different substances. METHODS: Forty acrylic teeth with simulated root canals were divided into 4 groups according to the substance associated to the calcium hydroxide paste: chlorhexidine (CHX) in 2 formulations (1% solution and 2% gel), Casearia sylvestris Sw extract, and propylene glycol (control). The teeth with pastes and sealed coronal accesses were immersed in 10 mL deionized water. After 10 minutes, 24 hours, 48 hours, and 7, 15, and 30 days, the teeth were removed to another container, and the liquid was analyzed. Calcium ion release was measured by atomic absorption spectrophotometry, and pH readings were made with a pH meter. Data were analyzed statistically by analysis of variance and Tukey test (alpha = 0.05). RESULTS: Calcium analysis revealed significant differences (P < .05) for 1% CHX solution and 2% CHX gel at 10 minutes. After 24 hours, 2% CHX gel x Control and 2% CHX gel x 1% CHX solution differed significantly (P < .05). After 48 hours, there were significant differences (P < .05) for 2% CHX gel x Control and Extract x Control. No differences (P > .05) were observed among groups in the other periods. Regarding the pH, there were significant differences (P < .05) for 2% CHX gel x Control and 2% CHX gel x 1% CHX solution after 48 hours and for 2% CHX gel x Control after 15 days. In the other periods, no differences (P > .05) were observed among groups. CONCLUSIONS: All pastes behaved similarly in terms of pH and calcium ion release in the studied periods.

Análise, com microscopia confocal de varredura a laser, da formação de biofilme de Bifidobacterium dentium e Fusobacterium nucleatum sobre diferentes substratos / Analysis of Bifidobacterium dentium and Fusobacterium nucleatum biofilm formation on different substrates by confocal laser scanning microscopy

Objetivo: o objetivo do presente estudo foi avaliar, por meio da microscopia confocal de varredura a laser (MCVL), a influência de diferentes substratos (dentina bovina em blocos, blocos de vidro) sobre o desenvolvimento de biofilmes de F. nucleatum e B. dentium. Métodos: placas para cultura de células com 24 poços foram usadas para induzir o biofilme sobre os substratos, com uma linhagem de F. nucleatum ATCC 25586 e uma de B. dentium ATCC 27534, durante 7 dias. Após o período de indução do crescimento, os espécimes foram corados com Live/Dead e analisados por MCVL. Os resultados obtidos pela MCVL foram analisados no software BioImage_L. Resultados: todos os resultados foram analisados pelo teste não paramétrico de Kruskal-Wallis, para comparação entre os grupos (p < 0,05). Houve formação de biofilme em todos os grupos experimentais. O biovolume total e a porcentagem de bactérias viáveis no biofilme de B. dentium nos blocos de dentina e nos blocos de vidro não mostraram diferenças estatísticas. O biovolume de bactérias viáveis não revelou diferença entre os substratos no biofilme de F. nucleatum nos blocos de dentina e nos blocos de vidro. Já o biovolume total mostrou-se maior no biofilme formado em blocos de dentina. B. dentium e F. nucleatum são capazes de formar biofilme em todos os substratos estudados. Conclusão: pela metodologia adotada, o tipo de substrato influencia as características do biofilme formado, sendo o bloco de dentina mais propício para a formação do biofilme dos microrganismos estudados.

Suscetibilidade antimicrobiana e protocolo de purificação de RNA para análise de expressão gênica de isolados clínicos de Fusobacterium nucleatum / Antimicrobial susceptibility and RNA purification protocol for gene expression analysis of clinical isolates of Fusobacterium nucleatum

Fusobacterium nucleatum é uma espécie bacteriana Gram-negativa, anaeróbia estrita e uma das espécies frequentemente encontradas nas infecções primárias do sistema de canais radiculares. Esta espécie tem grande importância na formação de biofilmes por ser uma ponte de união entre espécies que não são capazes de interagir. Os micro-organismos constituintes de biofilmes trocam material genético, aumentando a tolerancia dos mesmos e é quase impossível um isolado clínico ter os genes totalmente iguais à cepa padrão de coleções de cultura como da ATCC (American Type Culture Colection). O presente estudo investigou a espécie bacteriana anaeróbia Fusobacterium nucleatum isolada de canais radiculares, comparando-a com sua cepa de referência. Foi feito a comparação da suscetibilidade microbiana in vitro por meio de cultura microbiológica pelo método do E-test, com as cepas em crescimento planctônico e em biofilme. Também foi definido um protocolo de Purificação de RNA para esta espécie em ambas as condições de crescimento. As cepas clínicas de F. nucelatum foram isoladas por meio da cultura microbiológica de 23 pacientes que apresentavam dentes com infecção primária e periodontite apical visível em radiografia. Foi feito isolamento e identificação da espécie por série bioquímica com testes comerciais (Sistema Api, Bio-Meriéux, França) e PCR convencional, sendo no total 4 isolados clínicos investigados. Foi verificada a suscetibilidade antimicrobiana dos seguintes antibióticos: Amoxicilina, Amoxicilina + ácido clavulânico, Ampicilina, Azitromicina, Clindamicina, Eritromicina e Metronidazol. O protocolo para purificação de RNA foi feito com microesferas de zircônia, dispositivo bead beater, kit comercial RNeasy (Qiagen) e transcrição para DNA complementar (cDNA). A qualidade da purificação foi testada quanto a sua capacidade de amplificação pela reação em cadeia da polimerase em tempo real (qPCR) utilizando primer para o gene 16s RNA específico para F. nucelatum...

Fusobacterium nucleatum is a Gram-negative bacterial species, strict anaerobic and one of the species often found in primary infection of the root canal system. This species has great importance in biofilm formation to be a union bridge between species which are not able to act alone. The constituent microorganisms of the biofilm exchange each tother genetic material, increasing the strength of them. It is almost impossible for a clinical isolate have genes totally equal to a standard strain, such as strains of culture collections like ATCC (American Type Culture Collection). The present study investigated anaerobic bacterial species Fusobacterium nucleatum isolated from root canals, comparing them to the ATCC strain. The microbial in vitro susceptibility of biofilm and planktonic growth of the strains was compared by means of microbiological culture and the E-test method, with the antibiotics Amoxicillin, Amoxicillin + clavulanic acid, Ampicillin, Azithromycin, Clindamycin, Eritromycin and Metronidazole.. Also, a RNA Purification protocol for the strains under the same growth conditions was defined. Clinical isolates were obtained by microbiological samples of patients with teeth with pulp necrosis and apical periodontitis visible on radiographs. The species isolation and identification were performed using commercial biochemical tests (Sistema Api, Bio-Meriéux, France) and conventional PCR, obtaining four clinical isolates. The protocol for RNA purification was done with zirconia beads, bead beater device and commercial kit RNeasy (Qiagen) and transcribed into complementary DNA (cDNA). The quality of purification was tested for its ability of amplification by real time polymerase chain reaction (qPCR) using primer for the gene 16s RNA specific for F. nucleatum. All tested trains were 100% susceptible to amoxicillin + clavulanic acid, ampicillin, azithromycin, clindamycin and metronidazole. Both types of bacterial growth showed resistance to...

Análise da reação do tecido subcutâneo de ratos à implantação de pastas de hidróxido de cálcio associadas a diferentes substâncias / Analysis of the reaction of rat subcutaneous tissue to the implantation of calcium hydroxide pastes associated with different substances

O presente estudo teve como objetivo avaliar e comparar, morfologicamente, a resposta do tecido subcutâneo de ratos frente ao de hidróxido de cálcio associado a diferentes substâncias, sendo elas: clorexidina 0,2% em propilenoglicol, clorexidina 0,4% em propilenoglicol, Casearia sylvestris Sw (Guaçatonga) em propilenoglicol e propilenoglicol (que serviu como grupo controle). Para isso, foram utilizados 72 ratos Wistar. As pastas foram preparadas e colocadas em tubos de polietileno, que foram implantados no tecido subcutâneo do dorso de cada animal. Os períodos experimentais foram de 7, 14 e 30 dias após o procedimento cirúrgico, quando então, os animais foram mortos. As amostras coletadas passaram por processamento histotécnico e foram feitos cortes de 5µm de espessura e coloração com Hematoxilina e Eosina (H.E.). A avaliação das respostas do tecido conjuntivo em contato com as pastas testadas foirealizada de forma descritiva e quantitativa subjetiva.Os dados obtidos após análise quantitativa foram submetidos ao teste estatístico de Kruskal-Wallis e Dunn com nível de significância p<0,05. Os resultados mostraram que a clorexidina 0,4%, de modo geral, se mostrou mais irritante que os demais grupos. O extrato de Casearia sylvestris Sw apresentou comportamento satisfatório em relação à intensidade da reação inflamatória, igualando-se aos grupos 1 e 4. Com base na metodologia aplicada, foi possível concluir que aos 30 dias todos os grupos mostraram não interferir no processo de reparo e que todas as substâncias experimentais (clorexidina a 0,4%, a 0,2% e o extrato de Casearia sylvestris Sw) podem ser associadas ao hidróxido de cálcio.

This study aimed to evaluate the response of rat subcutaneous tissue against calcium hydroxide associated with different substances: chlorhexidine 0.2% in propylene glycol, 0,4% chlorhexidine in propylene glycol, Casearia sylvestris (guaçatonga) in propylene glycol and propylene glycol (control group). The pastes were prepared and placed in polyethylene tubes, which were implanted in the dorsum of each animal (N=18). The experimental periods were 7, 14 and 30 days after surgery, at which time the animals were killed. The samples were histologically processed and stained with hematoxylin and eosin. The evaluation of the responses of connective tissue in contact with the pastes was performed using descriptive and quantitative analyses. The data obtained after the quantitative analysis were subjected to statistical test of Kruskal-Wallis and Dunn with significance level p <0.05. In overall, chlorhexidine 0,4%/calcium hydroxide showed the more intense inflamatory reaction in comparison to the other groups. The extract of Casearia sylvestris showed satisfactory performance in relation to the intensity of the inflammatory reaction, similar to groups 1 and 4. Based on the methodology, it was concluded that at 30 days all the materials do not to interfere with the repair process and that all experimental substances (chlorhexidine 0.4%, 0.2% and the extract of Casearia sylvestris) can be associated with calcium hydroxide.