Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste

Aim. To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. Design. Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. Results. Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P < 0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). Conclusion. Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

Effects of the Association between a Calcium Hydroxide Paste and 0.4% Chlorhexidine on the Development of the Osteogenic Phenotype In Vitro

The present study aimed to evaluate whether the association between a calcium hydroxide paste (Calen paste) and 0.4% chlorhexidine (CHX) affects the development of the osteogenic phenotype in vitro. With rat calvarial osteogenic cell cultures, the following parameters were assayed: cell morphology and viability, alkaline phosphatase activity, total protein content, bone sialoprotein immunolocalization, and mineralized nodule formation. Comparisons were carried out by using the nonparametric Kruskal-Wallis test (level of significance, 5%). The results showed that the association between Calen paste and 0.4% CHX did not affect the development of the osteogenic phenotype. No significant changes were observed in terms of cell shape, cell viability, alkaline phosphatase activity, and the total amount of bone-like nodule formation among control, Calen, or Calen + CHX groups. The strategy to combine Ca(OH)(2) and CHX to promote a desirable synergistic antibacterial effect during endodontic treatment in vivo might not significantly affect osteoblastic cell biology. (J Endod 2008;34:1485-1489)

Effect of a calcium hydroxide-based paste associated to chlorhexidine on RAW 264.7 macrophage cell line culture

Objective. The objective of this study was to evaluate the effect of a calcium hydroxide Ca(OH)(2)-based paste (Calen) associated or not to 0.4% chlorhexidine digluconate (CHX) on RAW 264.7 macrophage cell line culture. Study design. The cell viability (MTT assay), immunostimulating properties (NO dosage), and anti-inflammatory properties (NO, TNF-alpha, and IL-1 alpha dosage) were evaluated after cell exposure to the materials. Data were analyzed statistically by Kruskal-Wallis test at 5% significance level. Results. There was low immunostimulating activity of the Calen paste associated or not to 0.4% CHX in the different materials` concentrations evaluated (P > .05). Anti-inflammatory activity with inhibition of NO and cytokine (TNF-alpha and IL1-alpha) release was observed only with Ca(OH)(2) + CHX at the highest concentration (25 mu g/mL). Conclusion. As the Calen paste associated to 0.4% CHX did not alter cell viability or the immunostimulating and anti-inflammatory properties, the addition of CHX brought no benefits to the Ca(OH)(2)-based paste with regard to the tested parameters. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:e44-e51)

Study of the pozzolanic reaction kinetics in sugar cane bagasse-clay ash/calcium hydroxide system: kinetic parameters and pozzolanic activity

This paper presents a study of the pozzolanic reaction kinetics between calcium hydroxide and a mixture of sugar cane bagasse with 20 and 30% of clay, burned at 800 and 1000 degrees C (SCBCA) by electrical conductivity measurements. A kinetic-diffusive model produced in previous studies by some of the authors was used. The model was fitted to the experimental data, which allowed the computation of the kinetic parameters of the pozzolanic reaction (reaction rate constant and free energy of activation) that rigorously characterised the pozzolanic activity of the materials. The results show that SCBCA demonstrated reactivity and good pozzolanic qualities in the range 800-1000 degrees C.

Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

Introduction: The objective of this study was to investigate the expression of matrix metalloproteinases (MM Ps) in apical periodontitis and during the periapical healing phase after root canal treatment. Methods: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. Results: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusions: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes. (J Endod 2010;36:231-237)

Nitric oxide detection in cell culture exposed to LPS after Er:YAG laser irradiation

P>Aim To evaluate in vitro the effect of calcium hydroxide [Ca(OH)(2)] and Er:YAG laser on bacterial endotoxin [also known as lipopolysaccharide (LPS)] as determined by nitric oxide (NO) detection in J774 murine macrophage cell line culture. Methodology Samples of LPS solution (50 mu gmL-1), Ca(OH)(2) suspension (25 mg mL-1) and LPS suspension with Ca(OH)(2) were prepared. The studied groups were: I - LPS (control); II - LPS + Ca(OH)(2); III - LPS + Er:YAG laser (15 Hz 140 mJ); IV - LPS + Er:YAG laser (15 Hz 200 mJ); V - LPS + Er:YAG laser (15 Hz 250 mJ), VI - Pyrogen-free water; VII - Ca(OH)(2). Murine macrophage J774 cells were plated and 10 mu L of the samples were added to each well. The supernatants were collected for NO detection by the Griess reaction. Data were analysed statistically by one-way anova and Tukey`s test at 5% significance level. Results The mean and SE (in mu mol L-1) values of NO release were: I - 10.48 +/- 0.58, II - 6.41 +/- 0.90, III - 10.2 +/- 0.60, IV - 8.35 +/- 0.40, V - 10.40 +/- 0.53, VI - 3.75 +/- 0.70, VII - 6.44 +/- 0.60; and the values for the same experiment repeated after 1 week were: I - 21.20 +/- 1.50, II - 9.10 +/- 0.60, III - 19.50 +/- 1.00, IV - 18.50 +/- 0.60, V - 21.30 +/- 0.90, VI - 2.00 +/- 0.20, VII - 6.80 +/- 1.70. There was no significant difference (P > 0.05) between the control and the laser-treated groups (III, IV and V), or comparing groups II, VI and VII to each other (P > 0.05). Group I had significantly higher NO release than group II (P < 0.05). Groups II and VI had similar NO release (P > 0.05). Conclusions Calcium hydroxide inactivated the bacterial endotoxin (LPS) whereas none of the Er:YAG laser parameter settings had the same effectiveness.

Cone-beam computerized tomographic, radiographic, and histologic evaluation of periapical repair in dogs` post-endodontic treatment

Objective. To evaluate the periapical repair after root canal treatment in the teeth of dogs using CT and conventional radiography and to compare these findings with the gold standard microscopic evaluation. Study design. The animals were divided into three groups according to endodontic treatment performed: Group 1, single-visit endodontic treatment in teeth without apical periodontitis; Group 2, single-visit endodontic treatment in teeth with apical periodontitis; and Group 3, endodontic treatment in teeth with apical periodontitis using calcium hydroxide as a root canal dressing. Group 4 consisted of teeth with apical periodontitis not submitted to root canal treatment and Group 5 consisted of healthy teeth without periapical disease. Radiographic, tomographic, and microscopic evaluations were performed by blind examiners. At 180 days experimental time, CT and radiographic measurements of periapical disease were compared with the gold standard microscopic measurement using intraclass correlation coefficient. Intergroup comparisons considering different methods of periapical lesions measurement or different clinical protocols of root canal treatment were performed by Kruskal Wallis test followed by Dunn. Integrity of lamina dura, presence of radiolucent areas, and presence of root resorption were analyzed by Fisher`s exact test. Results. There was discontinuity of the lamina dura and CPD in all teeth from Groups 2, 3, and 4 evaluated by tomography and radiography 45 days after CPD induction. Radiographically, 180 days after root canal treatment, there was no periapical lesion in teeth from Groups 1 and 3, different from groups 2 and 4 (p < .05). The highest reduction in the CPD size was observed on Group 3 (p < .05). According to the tomographic results, there was decrease of the size of the CPD on Group 3 but not on Groups 2 or 4. However, in all groups the periapical lesions presented larger mesio-distal extension if compared with radiography, both 45 days after CPD induction and 180 days after root canal treatment. At 180 days, CT measurements were closely related to microscopic results (ICC = 0.95) differently from radiographic evaluation (ICC = 0.86). Conclusion. CT Scan evaluation of periapical repair following root canal treatment provided similar information than that obtained by microscopic analysis, whereas radiographic evaluation underestimated the size do periapical lesion. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108:796-805)

Scanning Electron Microscopic Preliminary Study of the Efficacy of SmearClear and EDTA for Smear Layer Removal after Root Canal Instrumentation in Permanent Teeth

This study aimed to evaluate the efficacy of SmearClear (SybronEndo, Orange, CA) and EDTA for smear layer removal from root canals of permanent teeth after instrumentation. Thirty extracted human permanent teeth (n = 10) were randomly assigned to the following groups: group 1 = 14.3% EDTA, group 2 = SmearClear, and group 3 = no smear layer removal procedure was undertaken (control). The specimens were submitted to scanning electron microscopy analysis. Magnifications of 200x and 750x were used to evaluate cleaning at the apical, middle, and cervical thirds according to a three-point scoring system. Data were analyzed statistically by the Mann-Whitney U test (5% significance level). Groups 1 and 2 differed significantly from group 3 (p < 0.01). However, there was no statistically significant difference (p > 0.05) between groups 1 and 2. In conclusion, SmearClear was able to remove the smear layer from the root canals of permanent teeth similarly as 14.3% EDTA, suggesting that both solutions may be indicated for such purpose. (J Endod 2008,34:1541-1544)

Preliminary study of the inflammatory response to subcutaneous implantation of three root canal sealers

P>Aim To evaluate the kinetics of the inflammatory tissue response to three root canal sealers using a physicochemical method for quantification of the enhanced vascular permeability and histopathological analysis. Methodology Twenty-eight male Wistar rats randomly assigned to four groups according to the evaluation periods (1, 3, 7 and 14 days) were used to assess the vascular permeability and histopathological reaction to RoekoSeal, AH Plus and Sealapex (new formulation) sealers, using saline and Chloropercha as negative and positive controls, respectively. Seven rats were sacrificed per period. The biocompatibility of the sealers was evaluated spectrophotometrically and histopathologically. Results At day 14, Sealapex produced significantly more inflammatory exudate than AH Plus and RoekoSeal (P < 0.05); however, there was no significant difference between AH Plus and RoekoSeal (P > 0.05). Sealapex (new formulation) was the most irritating sealer, producing severe inflammation with the presence of multinucleated giant cells. RoekoSeal was the most biocompatible sealer, producing the least amount of inflammatory exudate. Conclusions RoekoSeal root canal sealer was biocompatible when implanted in connective tissue.

Direct pulp capping with a self-etching adhesive system: Histopathologic evaluation in dogs` teeth

Objective. This study evaluated histopathologically the response of pulp and periradicular tissues after pulp capping with an all-in-one self-etching adhesive system in dogs` teeth. Study design. Forty teeth of 4 dogs were assigned to 3 groups according to the pulp capping material: G1 (n = 20): self-etching adhesive system; G2 (n = 10): Ca(OH)(2); G3 (n = 10): zinc oxide-eugenol. The animals were killed 7 and 70 days after pulp capping. The pieces containing the pulp-capped teeth were removed and processed for histologic analysis. Results. At 7 days, no dentin bridge formation was observed; G1 and G3 exhibited inflammatory pulpal alterations, whereas G2 presented only mild inflammatory infiltrate in the pulp tissue adjacent to the capping material, the remainder being intact. At 70 days, no specimen in G1 or G3 presented dentin bridge formation. The remaining pulp tissue exhibited severe inflammatory alterations and areas of necrosis. In G2, all specimens showed dentin bridge formation and absence of inflammation and mineralized tissue resorption. No bacteria were identified using Brown and Brenn staining techniques in all 3 groups at any observation period. Conclusion. According to the conditions of this study, direct pulp capping with the self-etching adhesive system did not allow pulp tissue repair and failed histopathologically in 100% of the cases. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e34-e40)

Pozzolanic behavior of bamboo leaf ash: Characterization and determination of the kinetic parameters

The paper presents a characterization and study of the pozzolanic behavior between calcium hydroxide (CH) and bamboo leaf ash (BLAsh), which was obtained by calcining bamboo leaves at 600 degrees C for 2 h in a laboratory electric furnace. To evaluate the pozzolanic behavior the conductometric method was used, which is based on the measurement of the electrical conductivity in a BLAsh/CH solution with the reaction time. Later, the kinetic parameters are quantified by applying a kinetic-diffusive model. The kinetic parameters that characterize the process (in particular, the reaction rate constant and free energy of activation) were determined with relative accuracy in the fitting process of the model. The pozzolanic activity is quantitatively evaluated according to the values obtained of the kinetic parameters. Other experimental techniques, such as X-ray diffraction (XRD) and scanning electron microscopy (SEM), were also employed. The results show that this kind of ash is formed by silica with a completely amorphous nature and a high pozzolanic activity. The correlation between the values of free energy of activation (Delta G(#)) and the reaction rate constants (K) are in correspondence with the theoretical studies about the rate processes reported in the literature. (C) 2010 Elsevier Ltd. All rights reserved.

Evaluation of dental pulp repair using low level laser therapy (688 nm and 785 nm) morphologic study in capuchin monkeys

The aim of this study was to evaluate the hypothesis that low-level laser therapy (LLLT) 688 nm and 785 nm accelerate dentin barrier formation and repair process after traumatic pulp exposure. The sample consisted of 45 premolars of capuchin monkeys (Cebus apella) with pulp exposure Class V cavities. All premolars were treated with calcium hydroxide (Ca(OH)(2)), divided in groups of 15 teeth each, and analyzed on 7(th), 25(th), and 60(th) day. Group GI - only Ca(OH)(2), GIF- laser 688 nm, and GIII - laser 785 nm. Laser beam was used in single and punctual dose with the parameters: continuous, 688 nm and 785 nm wavelength, tip`s area of 0.00785 cm(2), power 50 mW, application time 20 s, dose 255 J/cm(2), energy 2 J. Teeth were capped with Ca(OH)(2), Ca(OH)(2) cement and restored with amalgam. All groups presented pulp repair. On 25(th) day the thickness of the formed dentin barrier was different between the groups GI and GII (p < 0.05) and between groups GI and GIII (p < 0.01). On 60(th) day there was difference between GI and GIII (p < 0.01). It may be concluded that, LLLT 688 nm and 785 nm accelerated dentin barrier formation and consequently pulp repair process, with best results using infrared laser 785 nm. (c) 2009 by Astro Ltd. Published exclusively by WLLEY-VCH Verlag GmbH & Co. KGaA

Effects of calcium supplementation on body weight reduction in overweight calcium stone formers

A randomized, placebo-controlled trial was conducted in overweight calcium stone-forming (CSF) patients, to evaluate the effect of calcium supplementation associated with a calorie-restricted diet on body weight (BW) and fat reduction and its potential changes upon serum and urinary parameters. Fifteen patients were placed on a hypocaloric diet for 3 months, supplemented with either calcium carbonate (CaCO(3), n = 8) or placebo (n = 7), 500 mg bid. Blood and 24-h urine samples were collected and body composition was assessed at baseline and after the intervention. At the end of the study, final BW was significantly lower vs baseline in both CaCO(3) (74 +/- A 14 vs. 80 +/- A 14 kg, P = 0.01) and placebo groups (80 +/- A 10 vs. 87 +/- A 9 kg, P = 0.02) but the mean percentage of loss of body weight and body fat did not differ between CaCO(3) and placebo (7.0 +/- A 2.0 vs. 8.0 +/- A 3.0%, P = 0.40 and 13.0 +/- A 7.0 vs. 13.0 +/- A 10.0%; P = 0.81, respectively). After CaCO(3) or placebo, no significant differences versus baseline were observed for urinary parameters in both CaCO(3) and placebo, except for a higher mean urinary citrate in placebo group. These data suggest that increasing calcium intake by calcium carbonate supplementation did not contribute to a further reduction of BW and fat in overweight CSF patients submitted to a hypocaloric diet nor altered urinary lithogenic parameters.

Dietary calcium intake and overweight: An epidemiologic view

Objective: We evaluated the relation between overweight and calcium intake in adults living in the municipality of Sao Paulo, Brazil. Methods: This was a cross-sectional population-based Study on a sample of 1459 adults that was obtained by multistage cluster sampling. Dietary intake was measured by the 24-h recall method. Poisson`s and linear regression analyses were performed to evaluate the relation between overweight and quartiles of calcium intake adjusted for energy. Results: The prevalence of overweight was 43.1% and the average adjusted calcium intake was 448.6 mg. In the linear regression. analyses, the regression coefficient for adjusted calcium was significant and negative (P = 0.019, beta(1) = -0.0001). Although evaluated by quartiles, the prevalence ratio for overweight in the first quartile of calcium intake was 1.24 (95% confidence interval 1.00-1.54) and that in the second quartile was 1.24 (95% confidence interval 1.03-1.49). Conclusion: In the present study, calcium intake showed a significant negative association with body mass index. (C) 2008 Elsevier Inc. All rights reserved.