Viability and cell cycle analysis of equine fibroblasts cultured in vitro

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD(A (R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

The Potential of p38 MAPK Inhibitors to Modulate Periodontal Infections

Periodontal disease initiation and progression occurs as a consequence of the host immune inflammatory response to oral pathogens. The innate and acquired immune systems are critical for the proper immune response. LPS, an outer membrane constituent of periodontal pathogenic bacteria, stimulates the production of inflammatory cytokines IL-1 beta TNF alpha IL-6 and RANKL either directly or indirectly. In LPS-stimulated cells, the induction of cytokine expression requires activation of several signaling pathways including the p38 MAPK pathway. This review will discuss the significance of the p38 MAPK pathway in periodontal disease progression and the potential therapeutic consequences of pharmacological antagonism of this pathway in the treatment of periodontal diseases.

Processo de reparo em feridas de extração dentária em camundongos tratados com o complexo Symphytum officinale e Calendula officinallis

Medicamentos homeopáticos como o Symphytum officinalle e a Calendula officinallis são dotados de propriedades anti-sépticas, antiinflamatória, cicatrizantes e também agem como promotores da consolidação de fraturas ósseas. Neste trabalho, uniram-se esses dois medicamentos similares em um complexo para verificar o seu efeito no reparo em feridas de extração dentária em camundongos. O complexo Symphytum officinalle e Calendula officinallis nas potências de 6CH e 3CH, respectivamente, foi ministrado por via oral ao grupo tratado durante 5 dias antes e após a extração do incisivo superior direito. No grupo controle, administraram-se 5ml de álcool etílico a 70% diluídos em 30 ml de soro fisiológico. Após a proservação, os animais foram sacrificados, a maxila direita separada da esquerda, fixada e processada para inclusão em parafina. Após a microtomia, os cortes obtidos foram corados pela H/E. A análise histológica mostrou que, tanto no grupo controle como no tratado, o alvéolo dentário estava preenchido por tecido de granulação e tecido ósseo neoformado, com graus variáveis de maturação, rico em osteócitos. No entanto, nos animais tratados, o processo de reparo em feridas após extração dentária do incisivo superior direito mostrou um avanço progressivo de neoformação óssea mais acentuado quando comparado ao grupo controle, em tempos equivalentes. Estes resultados enfatizam as propriedades biológicas do complexo Symphytum officinalle e Calendula officinallis e sua possível utilização como recurso terapêutico na Odontologia.

Determination of anaerobic threshold in rats using the lactate minimum test

The break point of the curve of blood lactate vs exercise load has been called anaerobic threshold (AT) and is considered to be an important indicator of endurance exercise capacity in human subjects. There are few studies of AT determination in animals. We describe a protocol for AT determination by the lactate minimum test in rats during swimming exercise. The test is based on the premise that during an incremental exercise test, and after a bout of maximal exercise, blood lactate decreases to a minimum and then increases again. This minimum value indicates the intensity of the AT. Adult male (90 days) Wistar rats adapted to swimming for 2 weeks were used. The initial state of lactic acidosis was obtained by making the animals jump into the water and swim while carrying a load equivalent to 50% of body weight for 6 min (30-s exercise interrupted by a 30-s rest). After a 9-min rest, blood was collected and the incremental swimming test was started. The test consisted of swimming while supporting loads of 4.5, 5.0, 5.5, 6.0 and 7.0% of body weight. Each exercise load lasted 5 min and was followed by a 30-s rest during which blood samples were taken. The blood lactate minimum was determined from a zero-gradient tangent to a spline function fitting the blood lactate vs workload curve. AT was estimated to be 4.95 ± 0.10% of body weight while interpolated blood lactate was 7.17 ± 0.16 mmol/l. These results suggest the application of AT determination in animal studies concerning metabolism during exercise.

Effectiveness of 2% peracetic acid for the disinfection of gutta-percha cones

The aim of this study was to evaluate the effectiveness of 2% peracetic acid for the disinfection of gutta-percha cones contaminated in vitro with Escherichia coli, Staphylococcus aureus, Streptococcus mutans, Candida albicans and Bacillus subtilus (in spore form). Two hundred and twenty-five gutta-percha cones were contaminated with standardized suspensions of each microorganism and incubated at 37 degrees C for 24 h. The cones were divided into 10 experimental groups (n = 15), according to the microorganism tested and disinfection testing times. The disinfection procedure consisted of immersing each cone in a plastic tube containing the substance. The specimens remained in contact with the substance for 1 or 2.5 minutes. Afterwards, each cone was transferred to a 10% sodium thiosulphate solution (Na2S2O3) to neutralize the disinfectant. Microbial biofilms adhering to the cones were dispersed by agitation. Aliquots of 0.1 ml of the suspensions obtained were plated on Sabouraud dextrose agar, or brain and heart infusion agar, and incubated at 37 degrees C for 24 h. The results were expressed in colony forming units (CFU/ml) and the data were submitted to the Wilcoxon Signed Rank Test (level of significance at 0.05). A significant reduction was observed, after 1 minute of exposure, in the test solution for C. albicans (p = 0.0190), S. aureus (p = 0.0001), S. mutans (p = 0.0001), B. subtilis (p = 0.0001), and E. coli (p = 0.0001). After 2.5 minutes of exposure, 100% of the microbial inocula were eliminated. It was concluded that the 2% peracetic acid solution was effective against the biofilms of the tested microorganisms on gutta-percha cones at 1 minute of exposure.

Effects of eating disorders on oral fungal diversity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of Airborne-Particle Abrasion and Mechanico-Thermal Cycling on the Flexural Strength of Glass Ceramic Fused to Gold or Cobalt-Chromium Alloy

Purpose: To evaluate the effect of airborne-particle abrasion and mechanico-thermal cycling on the flexural strength of a ceramic fused to cobalt-chromium alloy or gold alloy.Materials and Methods: Metallic bars (n = 120) were made (25 mm x 3 mm x 0.5 mm): 60 with gold alloy and 60 with Co-Cr. At the central area of the bars (8 mm x 3 mm), a layer of opaque ceramic and then two layers of glass ceramic (Vita VM13, Vita Zahnfabrick) were fired onto it (thickness: 1 mm). Ten specimens from each alloy group were randomly allocated to a surface treatment [(tungsten bur or air-particle abrasion (APA) with Al(2)O(3) at 10 mm or 20 mm away)] and mechanico-thermal cycling (no cycling or mechanically loaded 20,000 cycles; 10 N distilled water at 37 degrees C and then thermocycled 3000 cycles; 5 degrees C to 55 degrees C, dwell time 30 seconds) combination. Those specimens that did not undergo mechanico-thermal cyclingwere stored inwater (37 degrees C) for 24 hours. Bond strength was measured using a three-point bend test, according to ISO 9693. After the flexural strength test, failure types were noted. The data were analyzed using three factor-ANOVA and Tukey's test (alpha = 0.05).Results: There were no significant differences between the flexural bond strength of gold and Co-Cr groups (42.64 +/- 8.25 and 43.39 +/- 10.89 MPa, respectively). APA 10 and 20 mm away surface treatment (45.86 +/- 9.31 and 46.38 +/- 8.89 MPa, respectively) had similar mean flexural strength values, and both had significantly higher bond strength than tungsten bur treatment (36.81 +/- 7.60 MPa). Mechanico-thermal cycling decreased the mean flexural strength values significantly for all six alloy-surface treatment combinations tested when compared to the control groups. The failure type was adhesive in the metal/ceramic interface for specimens surface treated only with the tungsten bur, and mixed for specimens surface treated with APA 10 and 20 mm.Conclusions: Considering the levels adopted in this study, the alloy did not affect the bond strength; APA with Al(2)O(3) at 10 and 20 mm improved the flexural bond strength between ceramics and alloys used, and the mechanico-thermal cycling of metal-ceramic specimens resulted in a decrease of bond strength.

Polyurethane and PTFE membranes for guided bone regeneration: Histopathological and ultrastructural evaluation

Objective: The purpose of this study was to research a membrane material for use in guided bone regeneration. Study design: In this study, 25 male Wistar rats were used to analyze the biocompatibility and degradation process of biomembranes. The morphological changes in subcutaneous implantations were assessed after 7, 14, 21, 28 and 70 days. The materials were made of polyurethane polymer (AUG) obtained from vegetal oil (Ricinus communis) and polytetrafluoroethylene membrane (PTFE). The surface characteristics of the physical barriers in scanning electronic microscopic (SEM) were also evaluated. Results: In both groups, the initial histological analysis showed moderate inflammatory infiltrate, which was predominantly polymorphonuclear. There was also a presence of edema, which was gradually replaced by granulation tissue, culminating in a fibrous capsule. In the AUG group, some multinucleated giant cells were present in the contact interface, with the space previously occupied by the material. However, membrane degradation was not observed during the period studied. According to the present SEM findings, porosity was not detected in the AUG or PTFE membranes. Conclusion: The researched material is biocompatible and the degradation process is extremely slow or may not even occur at all.

Oral manifestations of diabetes mellitus in complete denture wearers

Statement of problem. The oral mucosa has been reported to show a variety of changes in subjects with diabetes mellitus.Purpose. The purpose of this study was to compare diabetic and nondiabetic subjects wearing complete dentures with regard to salivary flow, salivary buffering capacity, denture retention, and oral mucosal lesions.Material and methods. Sixty subjects, 30 with and 30 without a diagnosis of diabetes, were matched for gender, race, and age. Salivary flow, salivary buffering capacity, glycemia, blood pressure, presence of mucosal lesions, denture retention, use of medications, and behavioral factors (controlled or uncontrolled diet, alcohol consumption, and smoking) reported by the subjects, were evaluated. For the salivary buffering capacity test, 1 mL of saliva was pipetted into a test tube containing 3 mL 0.005 N of hydrochloric acid, and the pH was measured with indicator strips. Group differences were statistically analyzed using the Student t test and the Mann-Whitney test for quantitative variables and the chi-square test for qualitative variables (alpha = .05).Results. Mean (SD) salivary flow was 1.14 (0.87) mL/min in the nondiabetic subjects and 0.95 (0.61) mL/min in the diabetic subjects. Evaluation of self-reported denture retention revealed no significant difference between groups. Denture retention was observed in 66.7% (20/30) of the control group and in 50% (15/30) of the diabetic group. The prevalence of mucosal lesions was 90% (27/30) in the control group and 83.3% (25/30) in the diabetic group. Salivary buffering capacity was 5.80 (0.85) in the control group and 5.26 (0.83) in the diabetic group (P = .017).Conclusions. Within the limitations of this study, no significant differences were observed in salivary flow, denture retention, or oral lesions in diabetic and nondiabetic subjects.

Cytokeratin Profile in Exfoliative Cytology of Smokers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Experimental systemic virulence of oral Candida dubliniensis isolates in comparison with Candida albicans, Candida tropicalis and Candida krusei

There are no previous studies on the comparative virulence of Candida dubliniensis with other non-albicans species. The aim of this study was to compare the virulence and infection kinetics of C. dubliniensis and other species. Candida albicans, C. dubliniensis, Candida tropicalis and Candida krusei (reference strains) were inoculated intravenously in mice. For infection kinetics evaluation, a group of five animals were sacrificed after 6 h, 3, 7, 14 and 21 days. Microbiological evaluations (liver, spleen, kidneys, lungs and brain) and histopathological examination of the kidney were performed. The results of virulence evaluation were analysed using Kaplan-Meier survival analysis (5%). Candida dubliniensis-inoculated mice survived for longer periods compared with those with C. albicans (P = 0.005). No differences were detected in relation to C. tropicalis (P = 0.326) and C. krusei (P = 0.317). Most of the organs were persistently colonised by C. albicans and C. dubliniensis even by day 21. Tendency of C. krusei clearance was observed in all organs. Fungal masses and renal lesions were observed after inoculation of C. albicans, C. dubliniensis and C. tropicalis. Within the limits of the study, data on survival rate and dissemination capacity suggest that C. dubliniensis is less virulent than C. albicans.

Ex vivo evaluation of the effectiveness of bleaching agents on the shade alteration of blood-stained teeth

Aim To evaluate ex vivo effectiveness of the three formulations of bleaching materials for intracoronal bleaching of root filled teeth using the walking bleach technique.Methodology Extracted premolar teeth were stained artificially with human blood. After biomechanical preparation, the root canals were filled and a 3-mm thick intermediate base of zinc phosphate cement was placed at the level of the cementoenamel junction. The teeth were divided into four groups (n = 12): C (control, without bleaching material), A1 (sodium perborate + distilled water), A2 (sodium perborate + 10% carbamide peroxide) and A3 (sodium perborate + 35% carbamide peroxide). The bleaching materials were changed at 7 and 14 days. Evaluation of shade was undertaken with aid of the VITA Easyshade (TM) (Delta E*ab) and was performed after tooth staining and at 7, 14 and 21 days after bleaching, based on the CIELAB system. Data were analysed by ANOVA for repeated measurements, Tukey and Dunnett tests (alpha = 0.05).Results The Tukey test revealed that group A1 (10.58 +/- 4.83 Delta E*ab) was statistically different from the others (A2, 19.57 +/- 4.72 Delta E*ab and A3, 17.58 +/- 3.33 Delta E*ab), which were not different from each other. At 7 days: A1 was significantly different from A2; at 14 and 21 days: A2 and A3 were significantly better than A1; the Dunnett test revealed that the control group was different from A1, A2 and A3 at all periods (P < 0.05).Conclusion Sodium perborate associated with both 10% and 35% carbamide peroxide was more effective than when associated with distilled water.

Comparative Study of Oral Mucosa Micronuclei in Smokers and Alcoholic Smokers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)