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.

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of São Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical sips of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Aracatuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick (R)-stained for optical microscopy. Direct immumofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick (R)-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific. (C) 2006 Elsevier B.V. All rights reserved.

Tooth replantation after use of euro-coffins solution or bovine milk as storage medium: a histomorphometric analysis in dogs

Purpose: Euro-Collins solution was developed for the preservation of organs for transplantation, whose characteristics have raised interest for its use as a storage medium for avulsed teeth before replantation. This study evaluated histologically and morphometrically the healing process of dog teeth replanted after storage in Euro-Collins solution or bovine milk.Materials and Methods: Eighty roots of 4 young adult mongrel clogs were randomly assigned to 4 groups (n = 20) and the root canals were instrumented and obturated with gutta-percha and a calcium hydroxide-based sealer. After 2 weeks, the teeth were extracted and subjected to the following protocols: GI (negative control), replantation immediately after extraction; GII (positive control), bench-drying for 2 hours before replantation; GIII and GIV, immersion in 10 mL of whole bovine milk and Euro-Collins solution at 4 C, respectively, for 8 hours before replantation. The animals were sacrificed 90 days postoperatively. The pieces containing the replanted teeth were subjected to routine processing for histologic and histometric analyses under light microscopy and polarized light microscopy.Results: Root resorption was observed in all groups. GII exhibited the greatest loss of dental structure (P < .01), and inflammatory resorption was predominant in this group. Storage in milk showed poorer results than immediate replantation and storage in Euro-Collins solution (P < .01). The teeth stored in Euro-Collins solution presented similar extension of root resorption and periodontal ligament reorganization to those of immediately replanted teeth.Conclusions: The findings of this study suggest that the Euro-Collins solution is an adequate storage medium for keeping avulsed teeth for up to 8 hours before replantation.

Effect of low-level laser therapy on the healing process after tooth replantation: a histomorphometrical and immunohistochemical analysis

Success of tooth replantation is limited because part of the replanted tooth is lost because of progressive root resorption. This study used histomorphometry and immunohistochemistry to evaluate the effect of low-level laser therapy (LLLT) on the healing process of rat teeth replanted after different extra-oral periods, simulating immediate and delayed replantation. Sixty Wistar rats (Rattus norvegicus albinus) had their maxillary right incisors extracted and randomly assigned to six groups (n = 10): C4, C30 and C45, in which the teeth were replanted 4 min (immediate), 30 min (delayed) and 45 min (delayed) after extraction, respectively, and L4, L30 and L45, in which the teeth were replanted after the same extra-alveolar times, but the root surfaces and the alveolar wounds were irradiated with a gallium-aluminum-arsenate (GaAlAs) diode laser before replantation. The animals were sacrificed after 60 days. The anatomic pieces containing the replanted teeth were obtained and processed for either histomorphometrical analysis under optical microscopy or immunohistochemical expression of receptor activator of nuclear factor Kappa-B (RANK), and its ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP) proteins. Areas of external replacement and inflammatory root resorption were observed in all groups, without statistically significant differences (P > 0.05). Ankylosis was more frequent in L30 than in C30 (P < 0.05). RANKL immunostaining predominated over RANK and OPG immunostaining in both groups with immediate tooth replantation (P < 0.05). For the 45-min extra-alveolar time, however, there was greater evidence of RANK immunostaining compared to RANKL for both control and laser-treated groups (P < 0.05). Positive TRAP immunostaining predominated in L4 and L30 (P < 0.05). In conclusion, under the tested conditions, the treatment of the root surface and the alveolar wound with LLLT did not improve the healing process after immediate and delayed tooth replantation in rats.

Hybrid layer thickness and resin tag length of a self-etching adhesive bonded to sound dentin

Objectives: the purpose of this study is to employ optical microscopy to measure the thickness of the hybrid layer and the penetration (tags) of an aggressive self-etching adhesive system into sound dentin.Methods: occtusat cavities were prepared in 40 extracted human posterior teeth. The prepared teeth were randomly assigned to four experimental groups with 10 specimens each. The self-etching adhesive system Adper Prompt L-Pop was applied to the dentin surface as follows: Group 1: cavosurface enamel was etched for 60 s and dentin for 20 s with 35% phosphoric acid get, immediately followed by application of the self -etching adhesive with a brush to the entire cavity for 15 s; Groups 2, 3, and 4: no pre-etching was performed, and the self -etching adhesive was applied to both enamel and dentin for 15, 30 and 45 s, respectively. After curing, the cavities were fitted with composite resin Fittek Z250. Afterwards, the teeth were decalcified and the restorations were carefully removed for later embedding in paraffin. The specimens were serially sectioned at 6 mu m of thickness and sequentially mounted in glass slides. These sections were stained with Brown and Brenn staining for posterior analysis and measurement of the hybrid layer and resin tags on a tight microscope with a micrometric ocular 40/075. The results were submitted to analysis of variance at the 5% level.Results: whenever there was significance, the Tukey test was applied at the 5% level. The specimens receiving application of acid etching before the selfetching. adhesive displayed a larger thickness of the hybrid layer; on the other hand, specimens receiving only application of the self -etching adhesive on dentin for 15, 30 and 45 s exhibited similar thickness of the hybrid layer. As regards the resin tags, no statistically significant differences could be found between the study groups.Conclusions: it could be concluded that the increase in the time of application of the self-etching adhesive Adper Prompt L-Pop did not significantly influence the formation and thickness of hybrid layer, as well as its penetration into the sound dentin surface. (c) 2005 Elsevier Ltd. All rights reserved.

Exposed collagen in aged resin-dentin bonds produced on sound and caries-affected dentin in the presence of chlorhexidine

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

Histomorphometric analysis of tissue responses to bioactive glass implants in critical defects in rat calvaria

The aim of this study was to evaluate the osteogenic behavior of two chemically similar bioactive glass products (Biogran (R) and Perioglas (R)) implanted in critical bone defects in rat calvaria. Thirty-six transfixed bone defects of 8 mm diameter were made surgically in adult male Wistar rats. The animals were distributed equally into three groups: Biogran (GI), Perioglas (GII) and without implant material (control; GIII). The morphology and composition of both bioactive glasses were analyzed by scanning electron microscopy and energy-dispersive spectrometry. Tissue specimens were analyzed at the biological time points of 15, 30 and 60 days by optical microscopy and morphometry, demonstrating biocompatibility for the tested materials with moderate chronic inflammation involving their particles. Bone neoformation resulted only as a reparative reaction to an intentionally produced defect and was limited to the defect's edges. No statistically significant differences among the groups were observed. At the scar interstice, abundant deposits of collagenous fibers enveloping the particles were noted. The present results indicated that the bioactive glasses, under the experimental conditions analyzed, did not show osteogenic behavior. Copyright (c) 2007 S. Karger AG, Basel.

Morphological characterization of rat incisor fluorotic lesions

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

Comparative Histopathological Analysis of Human Pulps after Class I Cavity Preparation with a High-Speed Air-Turbine Handpiece or Er:YAG Laser

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

THE INFLUENCE of SHORT-HEATING-CYCLE INVESTMENTS on THE QUALITY of COMMERCIALLY PURE TITANIUM CASTINGS

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

Response of mice connective tissue to intracanal dressings containing chlorhexidine

Substances containing chlorhexidine (CHX) have been studied as intracanal medicaments. The aim of the present study was to characterize the response of mouse subcutaneous connective tissue to CHX-containing medications by conventional optical microscopy. The tissue response was evaluated by implanting polyethylene tubes containing one of the substances evaluated: Calen paste + 0.5% CHX, Calen + 2% CHX, 2% CHX gel, and Calen paste (control). After experimental periods of 7, 21, and 63 days, the implants (n = 10) were removed along with the subcutaneous connective tissue. Tissue samples were subjected to histological processing, and sections were stained with hematoxylin and eosin. Qualitative and quantitative analyses of the number of inflammatory cells, blood vessels, and vascularized areas were performed. Results were analyzed by ANOVA and Tukey tests with the significance level set at 5%. We concluded that Calen + 0.5% CHX led to reparative tissue response in contrast with Calen + 2% CHX and 2% CHX gel, which induced persistent inflammatory response, pointing to the aggressive nature of this mixture. When Calen + 2% CHX and 2% CHX gel were compared, the latter induced more intense inflammatory response. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Evaluation of different protocols for anatomical studies in Parmeliaceae (lichenized Ascomycota)

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

Silk formation mechanisms in the larval salivary glands of Apis mellifera (Hymenoptera : Apidae)

The mechanism of silk formation in Apis mellifera salivary glands, during the 5th instar, was studied. Larval salivary glands were dissected and prepared for light and polarized light microscopy, as well as for scanning and transmission electron microscopy. The results showed that silk formation starts at the middle of the 5th instar and finishes at the end of the same instar. This process begins in the distal secretory portion of the gland, going towards the proximal secretory portion; and from the periphery to the center of the gland lumen. The silk proteins are released from the secretory cells as a homogeneous substance that polymerizes in the lumen to form compact birefringent tactoids. Secondly, the water absorption from the lumen secretion, carried out by secretory and duct cells, promotes aggregation of the tactoids that form a spiral-shape filament with a zigzag pattern. This pattern is also the results of the silk compression in the gland lumen and represents a high concentration of macromolecularly well-oriented silk proteins.

Morphological and Structural Characteristics of Zein Biofilms with Added Xanthan Gum

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

Effect of modified clays on the structure and functional properties of biofilms produced with zein

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