A influência da criopreservação nas células mesenquimais indiferenciadas do ligamento periodontal de humanos análise comparativa in vitro

Cryopreservation is a process where cells or biological tissues are preserved by freezing at very low temperatures and aims to cease reversibly, in a controlled manner, all the biological functions of living tissues, i.e., maintain cell preservation so that it can recover with high degree of viability and functional integrity. This study aimed to evaluate the influence of cryopreservation on the mesenchymal stem cells originating from the periodontal ligament of human third molars by in vitro experiments. Six healthy teeth were removed and the periodontal cells grown in culture medium containing α-MEM supplemented with antibiotics and 15% FBS in a humidified atmosphere with 5% CO2 at 37° C. Cells isolated from each sample were divided into two groups: Group I - immediate cell culture (not fresh cryopreserved cells) and Group II - cell cryopreservation, during a period of 30 days. Analyses of rates of cell adhesion and proliferation in different groups were performed by counting the cells adhered to the wells, in intervals of 24, 48 and 72 hours after the start of cultivation. The number of cells in each well was obtained by counting viable cells with the use of hemocytometer and the method of exclusion of cells stained by trypan blue. The difference between groups for each of the times was analyzed by Wilcoxon test. Regarding the temporal evolution for each group, analysis was done by Friedman's test to verify the existence of differences between times and, when it existed, the Wilcoxon penalty was applied. The results showed no statistically significant difference between the two groups analyzed in this study. Therefore, we conclude that the cryopreservation process, after a period of 30 days, did not influence the cell type studied, and there was no difference in growth capacity in vitro between the groups

Avaliação preliminar da viabilidade de produção in vitro de um isolado brasileiro de baculovírus Spodoptera frugiperda MNPV

In vivo production of viral biopesticides is the major source of viral insecticides currently in the marketplace. However, this system presents limitations during production scale-up. For the Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV), the insect used for replication has cannibalistic characteristics, thus production is even more difficult. Insect cells are commonly used for in vitro baculovirus production. Most of these cell lines are derived from Lepidoptera species. The Sf21 cell line is derived from Spodoptera frugiperda caterpillar ovarian tissue, and its clonal isolate Sf9 has been used for biopesticide production due to its ease of growth in suspension cultures. In this work, the in vitro production capabilities of a Brazilian SfMNPV isolate obtained from cornfields was evaluated. Comparison of polyhedra production was carried out using both Sf21 and Sf9 cells, based on volumetric and specific yields. Both cell lines were cultivated in Hyclone medium supplemented with different fetal bovine serum concentrations (2,5 and 5%). The best results were obtained using Sf9 cells supplemented with 5% serum. These results were further confirmed quantitively through kinetic parameter estimation for both cells lines and different serum concentrations. After seven successive passages, this system still presented high specific polyhedra production

Efeito da adição de colesterol e ecdisona na produção in vitro do baculovírus spodoptera frugiperda MNPV

Among the pests that attack corn crop in Brazil, there is Spodoptera frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae), known as fall armyworm, which is the major corn pest. Due to genetic instability during serial passage of baculoviruses in insect cell culture, the viral bioinseticides in vitro production development is the greatest challenge for mass production of this bioproduct. Successive passages of virus using extracellular viruses (BVs), necessary during viral bioinseticides production scaling up, leads to the appearance of aberrant forms of virus, a process so called as "passage effect ". The main consequence of passage effect is the production of occlusion bodies (OB) decrease, preventing its production using in vitro process. In this study, it was carried out a serial passage of baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus, isolate 18, using Sf21 cells. A decrease in the production of occlusion bodies from 170 to 92 in the third to fourth passage was observed. A factorial experimental design (22) was employed to verify the influence of two input variables, concentration of the hormone 20 - hydroxyecdysone (CH) and cholesterol (CC) on the values of response variables (volumetric and the specific OB production) of the process, seeking to define the optimum operating ranges trying to reverse or minimize the passage effect. The result indicated a negative influence of the cholesterol addition and positive effect in the hormone supplementation which the optimum range found for the concentrations studied were 8 to 10μg/mL and 5 to 6.5 mg / mL, for cholesterol and hormone concentrations respectively. New experiments were performed with addition of hormone and cholesterol in order to check the influence of these additives on the OB production independently. While the best result obtained from the factorial experiment was 9.4 x 107 OB/mL and 128.4 specific OB/cell, with the addition of only 6μg/mL 20-hydroxyecdysone these concentrations increased to 1.9 x 108 OB/mL and 182.9 OB/cell for volumetric and specific OB production, respectively. This result confirms that the addition of the hormone 20-hydroxyecdysone enhances the SfMNPV in vitro production process performance using Sf21 cells

Avaliação in vitro da adesão e proliferação de células mesenquimais do ligamento periodontal humano em diferentes superfícies de titânio

In the last years, many scientific researches in implantology have been focused on alternatives that would provide higher speed and quality in the process of osseointegration. Different treatment methods can be used to modify the topographic and chemical properties of titanium surface in order to optimize the tissue-implant reactions by a positive tissue response. This study aimed to evaluate the adhesion and proliferation of mesenchymal cells from human periodontal ligament on two different titanium surfaces, using cell culture techniques. Grade II titanium discs received different surface treatments, forming two distinct groups: polished and cathodic cage plasma nitriding. Human periodontal ligament mesenchymal cells were cultured on titanium discs in 24-well cell culture plates, at a density of 2 x 104 cells per well, including wells with no discs as positive control. Data obtained by counting the cells that adhered to the titanium surfaces (polished group and cathodic cage group) and to the plastic surface (control group), in the 24, 48 and 72-hour periods after plating, were used to analyze cell adhesion and proliferation and to obtain the cell growing curve in the different groups. The data were submitted to nonparametric analysis and the differences between groups were compared by Kruskal-Wallis and Friedman statistical tests. No statistically significant differences were found in the cells counts between the groups (p>0.05). It was concluded that both treatments produced surfaces compatible with the adhesion and proliferation of human periodontal ligament mesenchymal cells

Estudo in vitro dos efeitos da BMP-2 e do seu antagonista Noggin sobre a proliferação e migração celulares em carcinoma epidermóide de língua

Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy in the oral cavity and reach a large number of individuals, has become an important public health problem. Studies have demonstrated changes in pathway components BMP in various types of cancers as prostate, colon, breast, gastric and OSCCs. Is the current knowledge that these proteins may exert pro-tumor effect in more advanced stages of neoplastic development coming to favor progression and invasion tumor. The inhibition of the signaling pathway BMP-2 through its antagonists, have shown positive results of antitumor activity and use of Noggin may be a novel therapeutic target for cancer. Given this evidence and the few studies with BMP-2, Noggin and OSCC, the objective of this research was to evaluate the effect of BMP-2 and its antagonist Noggin on proliferation and migration cell in line of cell cultures of human tongue squamous cell carcinoma (SCC25). The study was divided in three groups, a control group, where SCC25 cells suffered no treatment, a BMP-2 group, in which cells were treated with 100ng/ml of BMP-2 and a group of cells that were treated with 100ng/ml of Noggin. For the proliferation assay and cell cycle were established three time intervals (24, 48 and 72 hours). Proliferative activity was investigated by trypan blue and cell cycle analysis by staining with propidium iodide flow cytometry. The potential for migration / invasion of SCC25 cells was performing by a cell invasion assay using Matrigel in a 48-hour interval. The proliferation curve showed a higher proliferation in cells treated with BMP-2 in 72 hours (p < 0.05), and lower overgrowth and cell viability in Noggin group. Recombinant proteins favored a greater percentage of cells in cell cycle phase Go/G1 with a statistically significant difference in the interval of 24 hours (p < 0.05). BMP- 2 produced a greater invasion of cells studied as well as its antagonist Noggin inhibits invasion of cells (p < 0.05). Thus, these results indicate that BMP-2 promotes malignant phenotype, dues stimulates proliferation and invasion of SCC25 cells and, its antagonist Noggin may be an alternative treatment, due to inhibit the tumor progression

Avaliação da atividade anti-Helicobacter pylori e citotóxica in vitro de extratos orgânicos obtidos das folhas de Encholirium spectabile e Syzygium cumini

Helicobacter pylori is a spiral, Gram negative, mobile, and microaerophilic bacteria recognized as a major cause of gastritis, ulcer, gastric cancer, and gastric low grade, B cell, mucosa – associated lymphoid tissue (MALT) lymphoma, constituting an important microorganism in medical microbiology. Its importance comes from the difficulty of treatment because the requirement of multiple drugs use, besides the increasing emergence of resistant and multiresistant strains to antibiotics used in th e clinic. In order to expand safe and effective therapeutic options , chemical studies on medicinal plants by obtaining extracts, fractions, isolated compounds or essential oils with some biological activity has been intensified . Given the above, the objective was to evaluate the inhi bitory activity of organic extracts derived from Syzygium cumini and Encholirium spectabile, with antiulcer history, and the essential oil, obtained from S. cumini, against H. pylori (ATCC 43504) by the disk diffusion method, for qualitative evaluation, an d determination of minimum inhibitory concentration (MIC) using the broth microdilution method, for quantitative analysis. Also was evaluated the extracts in vitro toxicity by a hemolytic assay using sheep red blood cells, and VERO and HeLa cells using the MTT assay to analyze cell viability. The extracts of both plant used in antimicrobial assays did not inhibit bacterial growth, however the essential oil of S. cumini (SCFO) proved effective, showing MIC value of 205 μg/mL (0.024 % dilution of the original oil). In the hemolytic assay, the same oil shows moderate toxicity, by promote 25% hemolysis at 1000 μg/mL. Regarding the cytotoxicity in cell culture, the SCFO, at 260 μg/mL, affected the cell viability around 80% of HeLa and 50% of VERO cells. So the oi l obtained from S. cumini leaves has antimicrobial activity against H. pylori and cytotoxicity potential, suggesting a source of new molecule drug candidates, since new stages of toxicity in vitro and in vivo, as well, chemical characterization be evaluate d. Moreover, the development of a prospective drug delivery system can result in a prototype to be used in preclinical tests.