Antimicrobial endodontic compounds do not modulate alkylation-induced genotoxicity and oxidative stress in vitro

Objective: To investigate if formocresol, paramonochlorophenol, or calcium hydroxide modulate the genotoxic effects induced by the oxidatively damaging agent hydrogen peroxide (H 2O 2) or the alkylating agent methyl methanesulfonate (MMS) in vitro by using single cell gel (comet) assay. Study design: Chinese hamster ovary (CHO) cells in culture were exposed directly to formocresol, paramonochlorophenol, or calcium hydroxide (adjusted to 100 μg/mL) for 1 hour at 37°C. Subsequently the cultures were incubated with increasing concentrations (0-10 μmol/L) of MMS in phosphate-buffered solution (PBS) for 15 minutes at 37°C or of H 2O 2 at increasing concentrations (0-100 μmol/L) in distilled water for 5 minutes on ice. The negative control cells were treated with PBS for 1 hour at 37°C. The parameter from the comet assay (tail moment) was assessed by the Kruskal-Wallis nonparametric test followed by a post hoc analysis (Dunn test). Results: Clear concentration-related effects were observed for the genotoxin-exposed CHO cells. Increase of MMS-induced DNA damage was not significantly altered by the presence of the compounds tested. Similarly, no significant changes were observed when hydrogen peroxide was used with the endodontic compounds evaluated. Conclusion: Formocresol, paramonochlorophenol, and calcium hydroxide are not able to modulate alkylation-induced genotoxicity or oxidative DNA damage as depicted by the single cell gel (comet) assay. © 2006 Mosby, Inc. All rights reserved.

Efeito da sacarose e sorbitol na conservação in vitro de Passiflora giberti N. E. Brown

This work objectified the study of sucrose and sorbitol effect in the in vitro conservation for Passiflora giberti N. E. Brown, access. Therefore, an experiment was conducted in a completely randomized design to compare control treatment (standard MS) to MS medium supplemented with three sucrose concentrations (0, 15 and 30 g L -1) combined with three sorbitol concentrations (10, 20 and 40 g L -1), in a total of 10 treatments with 20 replicas. The experiment evaluation was carried out at 30, 60, 90 and 120 days of incubation, whereas the height of shoots (cm), number of roots, number and color of leaves were observed. The results showed the possibility to maintain passion-fruit microplants for a four months period under slow growth in MS medium supplemented with 10 or 20 g L -1 of sorbitol, without sucrose, and kept under 16 hours photoperiod (22 μ E m -2 s -1) and temperature of 27 ± 1°C. Sucrose sustained the longest development of the microplants. Root formation was affected by the sorbitol in the concentration of 40 g L -1 and by the absence of sucrose in the culture medium.

Enraizamento in vitro e aclimatização de mudas micropropagadas de Aloe vera L.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Efeito de poliaminas exógenas no crescimento inical in vitro e nos teores de fenóis, poliaminas e atividade da peroxidase em Aloe vera (L.) Burm.

Lateral shoots of the Aloe vera (L.) Burm. cultivated in vitro, without addition vegetal regulators, for 90 days, were inoculated in MS culture-medium, containing or not spermine and/or spermidine. After 30 days of cultivation, the plants were submitted to biochemical analysis together with micropropagated plants - that were under in vitro cultivation for 90 days - (denominated as characterization), and matrix plants (in vivo). The levels of free polyamines, total phenols, total flavonoids, and the activity of peroxidase were evaluated in the biochemical analyses. The exogenous application of spermidine have promoted large number of shoots. Spermidine and spermine have promoted, when associated, an increase in the number of shoots as well as an increase of the contents of putrescine and and flavonoids. The putrescine has presented the most significant alterations, enabling to be utilized as marker of morphogenesis in the micropropagated Aloe vera. Tissues under active growth have presented high activity of peroxidase as well as those with greater rate of oxidation. In these tissues, there were noticed also higher contents of total flavonoids, indicating the need of antioxidative compounds. The action of polyamines jointly tseemed to be benefic for the shooting of micropropagated Aloe vera.

Efeito do alumínio no conteúdo de poliaminas livres e atividade da fosfatase ácida durante o crescimento de brotações de Eucalyptus grandis × E. urophylla cultivadas in vitro

This paper is aiming to evaluate the effects of different levels of the aluminum on the growth of the Eucalyptus grandis x E. urophylla shoots cultivated in vitro. Evaluations were carried out on pH and chemicals modifications of the culture medium by Geochem program, polyamines contents (putrescine, espermidine e espermine) and acid phosphatase ativity on the shoots. The trial had a totally randomized design with four treatments and four replicates. The treatments were: 0.0, 6.75,13.50 and 27 mg.L -1 of AlCl 3.6H 2. Evaluations were carried out on the 4th, 8th, 12th, 16th, 20th, 24th and 28th day of culture. The addition of the aluminium, in all concentrations, affected the culture medium ionic equilibrium, the morphology of the shoots, reduced the pH on the medium, induced an increase in polyamines content and higher acid phosphatase activity.

Enraizamento de plantas cultivadas in vitro

The adventitious rooting process of in vitro cultured plantlets is a technique that has been employed for the vegetative propagation of a significant number of native and exotic species. Many factors are associated with the rooting stage influencing positive and/or negatively the establishment of micropropagation protocols. The objective of this work was a literature review of the main inherent factors concerning in vitro rooting process including the correlation among others the endogenous and exogenous auxins levels, juvenility, genotype, mineral nutrition, culture medium conditions, addition of growth regulators and other substances as phenolic compounds and active coal besides growth environmental conditions of in vitro cultures. Although the complete elucidation of all processes involved with rooting of in vitro cultured plants has not been achieved so far, a comprehensive study of the main factors that interfere on rooting is fundamental for the establishment of new researches that might contribute for the rooting of economically important plants.

Meio de cultura e tipo de explante no estabelecimento in vitro de espécies de maracujazeiro

Brazil is one of the main centers of genetic variability dispersion of the Passiflora genera. Its self incompatibility as well as disease incidence in its leaves and root system and, deforestation and monocultivation, promote loss of genetic material. Considering the risk of genetic erosion, the conservation of the variability in germplasm banks, which is of great interest in plant breeding, is necessary. Studies regarding the type of expiant and concentration of the culture media are necessary in order to determine protocols of establishment and in vitro conservation of passion-fruit germplasm. The objective of the present work was to evaluate the influence of the salt and nutrient concentration in the MS culture medium and types of expiants in the establishment and growth of the Passion fruit species: Passiflora giberti N. E.Brown, P. edulis Sims and P. laurifolia L. Each Passiflora species presented its own characteristics regarding in vitro development. The complete MS medium and nodal segments the second axilliary bud promoted better development of the genotypes studied.

Efeitos de diferentes sistemas de cultivo in vitro sobre o crescimento de folículos pré-antrais isolados de ovários de fetos bovinos

The objective of this study is to use different in vitro culture systems of preantral follicles from Nelore breed bovine fetuses in the last gestation quarter. The evaluation of treatments considered the time of growth of isolated follicles. Preantral follicles were mechanically isolated and submitted to the individual culture, for 9 days, in media no supplemented or supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or synthetic defined supplement substitute of serum KnockoutSR (KNO). We have also evaluated the effects of collagen gel or fetal calf fibroblast monolayer as substratum for in vitro cultures. The increase on the follicular diameter was followed in the first day (0 h), at the 72 h, 144 h and 216 h. Considering cultures of isolated follicles, the results have shown that the association between media supplemented with FCS and collagen gel was significantly more efficient on the increase of the follicular diameter than other treatments. It is not still established a system of appropriate cultivation that sustains the differentiation and multiplication of the granular cells and that maintains the contact of the same ones with the oocyte to provide molecules and factors that supply the metabolic demand. We also understand that our results also represent another promising step on the search for the ultimate system of in vitro culture of preantral follicles from bovines.

Carotenóides totais em Pothomorphe umbellata (L.) Miq. cultivadas in vitro

The aim of the present work was to compare the content of carotenoids between callus and regenerated plants of Pothomorphe umbellate. Germinated seeds (40 days old) were inoculated in different concentrations and combinations of BAP (benzylaminopurine) and NAA (naphthalene acetic acid) in order to stimulate the callus' production. After 60 days of culture, the callus containing some shoots were transferred to organogenesis medium (GA 3 0.1 mg L -1, BAP 0.5 mg L -1) for 40 days. Next, they were subcultivated in a medium for seedling growth (without regulators) for 40 days. Callus (collected after 60 days) and seedlings (collected after 140 days) were frozen in liquid nitrogen and kept under -80°C for future carotenoids' analysis. The highest concentration of carotenoids was found in plants cultivated in medium without regulators. The callus did not showed difference concerning the culture medium; however, they presented lower content of carotenoids in relation to plants.

Estado físico do meio de cultura na propagação in vitro de bromeliaceae

The objective of this study was to evaluate the effect of the physical state of the culture medium on the behavior in vitro of the bromeliaceas Neoregelia cruenta, Tillandsia stricta, Vriesea gigantea, V. guttata e V. incun/ata. Micropropagated shoots had been cultivated in culture medium MS, with 30 g sucrose, 2,5 mg dm-3 BAP, and 0,5 mg dm-3 ANA, establishing the treatments: T1- half-solid with 7 g agar; T2- static liquid; T3- liquid under-agitation of 90 rpm; and T4-static liquid with bridge of paper filter. After 30 days, the use of static way of liquid culture presented better results in relation of proliferative average rate in all the species (9.4 shoots in N. cruenta, 5.6 in T. stricta, 11.5 in V. gigantea, 9.2 in V. guttata and 3.9 in V. incurvata). In relation the average height of the shoots, was distinguished for N. cruenta the liquid under-agitation (2.83 cm), T. stricta and V. incurvata the semisolid (1.76 cm and 2.02 cm, respectively) and V. gigantea and V. guttata the static liquid (0.61 cm and 1.48 cm, respectively). The use of medium MS static liquid was more suitable for in vitro culture of bromeliads species studied.

Characterization and in vitro cytocompatibility of an acid-etched titanium surface

The aims of this study were to characterize the microstructure of a commercially pure titanium (cpTi) surface etched with HCl/H 2SO 4 (AE-cpTi) and to investigate its in vitro cytocompatibility compared to turned cpTi (T-cpTi). T-cpTi showed a grooved surface and AE-cpTi revealed a surface characterized by the presence of micropits. Surface parameters indicated that the AE-cpTi surface is more isotropic and present a greater area compared to T-cpTi. The oxide film thickness was similar between both surfaces; however, AE-cpTi presented more Ti and O and less C. Osteoblastic cell proliferation, alkaline phosphatase activity, and bone-like nodule formation were greater on T-cpTi than on AE-cpTi. These results show that acid etching treatment produced a surface with different topographical and chemical features compared to the turned one, and such surface modification affected negatively the in vitro cytocompatibility of cpTi as demonstrated by decreasing culture growth and expression of osteoblastic phenotype.

In vitro analysis with human bone marrow stem cells on Ti-15Mo alloy for dental and orthopedic implants application

Aim: Nowadays, research on orthopedic and dental implants is focused on titanium alloys for their mechanical properties and corrosion resistance in the human body environment. Another important aspect to be investigated is their surface topography, which is very important to osseointegration. With laser beam irradiation for roughening the implants surface an easier control of the microtopography is achieved, and surface contamination is avoided. The aim of this study was to assess human bone marrow stem cells response to a newly developed titanium alloy, Ti-15Mo, with surface topography modified by laser beam irradiation. Materials and methods: A total of 10 Ti machined disks (control), 10 Ti-15Mo machined disks and 10 Ti-15Mo disks treated by laser beam-irradiation were prepared. To study how Ti-15Mo surface topografy can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed, using real time Reverse Transcription-Polymerase Chain Reaction. Results: In Test 1 (comparison between Ti-15Mo machined disks and Ti-machined disks) quantitative real-time RT-PCR showed a significant induction of ALPL, FOSL1 and SPP1, which increase 20% or more. In Test 2 (comparison between Ti-15Mo laser treated disks and Ti-machined disks) all investigated genes were up-regulated. By comparing Test 1 and Test 2 it was detected that COL1A1, COL3A1, FOSL1 and ENG sensibly increased their expression whereas RUNX2, ALPL and SPP1 expression remained substantially unchanged. Conclusion: The present study demonstrated that laser treated Ti-15Mo alloys are promising materials for implants application.

In vitro susceptibility of oral Candida albicans strains to different pH levels and calcium hydroxide saturated aqueous solution

Candida albicans is present in the oral cavity and in the whole digestive tract of humans and other animals, being frequently related to endodontic treatment failure. The present study determined the incidence of C. albicans in the oral cavity and the susceptibility of isolates to different pH values and saturated calcium hydroxide aqueous solution at pH 12.5. Sixty-five patients attending the Endodontic Clinic at the Sagrado Coração University participated in the study. The collected samples were cultivated in selective media for C. albicans and the isolates were tested in terms of resistance to both alkaline pH and saturated aqueous solution of calcium hydroxide. In relation to time variables, yeast viability was assessed by the Sabouraud's agar culture and fluorescein diacetate and ethidium bromide fluorescent staining method. Results from the different pHs and experimental times, including those from different techniques measuring fungal viability, were compared using the chi-square and Fisher's exact tests (α=0.05). The yeasts became completely inviable after 48 h of contact with the calcium hydroxide solution. On the other hand, when exposed to the alkaline culture broth, the yeasts were found to be viable at pHs 9.5 and 10.5 for up to 7 days. In conclusion, C. albicans can only be completely inhibited by direct contact with saturated calcium hydroxide aqueous solution after 48 h of exposure.

In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts

The aim of this study was to determine adequate energy doses using specific parameters of LLLT to produce biostimulatory effects on human gingival fibroblast culture. Cells (3 10 4 cells/cm 2) were seeded on 24-well acrylic plates using plain DMEM supplemented with 10 fetal bovine serum. After 48-hour incubation with 5 CO2 at 37C, cells were irradiated with a InGaAsP diode laser prototype (LASERTable; 780 3 nm; 40mW) with energy doses of 0.5, 1.5, 3, 5, and 7J/cm 2. Cells were irradiated every 24h totalizing 3 applications. Twenty-four hours after the last irradiation, cell metabolism was evaluated by the MTT assay and the two most effective doses (0.5 and 3J/cm 2) were selected to evaluate the cell number (trypan blue assay) and the cell migration capacity (wound healing assay; transwell migration assay). Data were analyzed by the Kruskal-Wallis and Mann-Whitney nonparametric tests with statistical significance of 5. Irradiation of the fibroblasts with 0.5 and 3J/cm 2 resulted in significant increase in cell metabolism compared with the nonrradiated group (P 0.05). Both energy doses promoted significant increase in the cell number as well as in cell migration (P 0.05). These results demonstrate that, under the tested conditions, LLLT promoted biostimulation of fibroblasts in vitro. Copyright © 2012 Fernanda G. Basso et al.