Biocompatibilidade e bioatividade de cimentos à base de silicato tricálcico: estudo in vitro e in vivo

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

Avaliação dos fatores de virulência, atividade antimicrobiana e viabilidade celular de bactéria cariogênica na presença do Terpinen-4-ol: estudo in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biocompatibilidade e bioatividade de cimentos à base de silicato tricálcico: estudo in vitro e in vivo

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

Prediction of apparent protein digestibility of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei

Aquafeed production faces global issues related to availability of feed ingredients. Feed manufacturers require greater flexibility in order to develop nutritional and cost-effective formulations that take into account nutrient content and availability of ingredients. The search for appropriate ingredients requires detailed screening of their potential nutritional value and variability at the industrial level. In vitro digestion of feedstuffs by enzymes extracted from the target species has been correlated with apparent protein digestibility (APD) in fish and shrimp species. The present study verified the relationship between APD and in vitro degree of protein hydrolysis (DH) with Litopenaeus vannamei hepatopancreas enzymes in several different ingredients (n = 26): blood meals, casein, corn gluten meal, crab meal, distiller`s dried grains with solubles, feather meal, fish meals, gelatin, krill meals, poultry by-product meal, soybean meals, squid meals and wheat gluten. The relationship between APD and DH was further verified in diets formulated with these ingredients at 30% inclusion into a reference diet. APD was determined in vivo (30.1 +/- 0.5 degrees C, 32.2 +/- 0.4%.) with juvenile L vannamei (9 to 12 g) after placement of test ingredients into a reference diet (35 g kg(-1) CP: 8.03 g kg(-1) lipid; 2.01 kcal g(-1)) with chromic oxide as the inert marker. In vitro DH was assessed in ingredients and diets with standardized hepatopancreas enzymes extracted from pond-reared shrimp. The DH of ingredients was determined under different assay conditions to check for the most suitable in vitro protocol for APD prediction: different batches of enzyme extracts (HPf5 or HPf6), temperatures (25 or 30 degrees C) and enzyme activity (azocasein): crude protein ratios (4 U: 80 mg CP or 4 U: 40 mg CP). DH was not affected by ingredient proximate composition. APD was significantly correlated to DH in regressions considering either ingredients or diets. The relationships between APD and DH of the ingredients could be suitably adjusted to a Rational Function (y = (a + bx)/(1 + cx + dx2), n = 26. Best in vitro APD predictions were obtained at 25 degrees C, 4 U: 80 mg CP both for ingredients (R(2) = 0.86: P = 0.001) and test diets (R(2) = 0.96; P = 0.007). The regression model including all 26 ingredients generated higher prediction residuals (i.e., predicted APD - determined APD) for corn gluten meal, feather meal. poultry by-product meal and krill flour. The remaining test ingredients presented mean prediction residuals of 3.5 points. A model including only ingredients with APD>80% showed higher prediction precision (R(2) = 0.98: P = 0.000004; n = 20) with average residual of 1.8 points. Predictive models including only ingredients from the same origin (e.g., marine-based, R(2) = 0.98; P = 0.033) also displayed low residuals. Since in vitro techniques have been usually validated through regressions against in vivo APD, the DH predictive capacity may depend on the consistency of the in vivo methodology. Regressions between APD and DH suggested a close relationship between peptide bond breakage by hepatopancreas digestive proteases and the apparent nitrogen assimilation in shrimp, and this may be a useful tool to provide rapid nutritional information. (C) 2009 Elsevier B.V. All rights reserved.

Evaluation of an Experimental Gel Containing Euclea natalensis: An In Vitro Study

Objective. To evaluate the effect of an experimental gel containing Euclea natalensis extract on dentin permeability. Methods. Thirty-six dentin discs, 1-mm-thick. The discs were prepared from the coronal dentin of extracted human third molars that were divided into 3 groups (n = 10). The dentin discs in each group were treated with the groups following experimental materials: (FG): 1.23% fluoride gel, pH 4.1; (EG): Euclea natalensis extract gel, pH 4.1; (CG): control gel, pH 4.1. The gels were applied to the occlusal slide of the dentin under the following conditions: after 37% phosphoric acid and before 6% citric acid. The hydraulic conductance (HC) of each condition was determined four times using a fluid flow apparatus (Flodec). The data were analyzed using Two-way ANOVA and Tukey's test (P < 0.05). Results. The greatest mean reduction in HC was produced in group EG dentin discs (61.2%; P < 0.05). Even after acid challenge with 6% citric acid the great reduction occurred in group EG (66.0%; P < 0.05) than other groups (CG-77.1%, FG-90.8%). Conclusion. E. natalensis gel not only reduced dentin permeability, but also resisted posttreatment citric acid challenge without changing its permeability. Further research has to confirm this promising result in the clinical situation.

Stability of Carotenoids, Total Phenolics and In Vitro Antioxidant Capacity in the Thermal Processing of Orange-Fleshed Sweet Potato (Ipomoea batatas Lam.) Cultivars Grown in Brazil

Intervention strategies regarding the biofortification of orange-fleshed sweet potato, which is a rich source of carotenoids for combating vitamin A deficiency, are being developed in Brazil. This study was conducted to evaluate the concentrations of individual carotenoids, total phenolic compounds and antioxidant capacity in the roots of four biofortified sweet potato cultivars that were raw or processed by four common heat treatments. HPLC, Folin-Ciocalteu, DPPH and ABTS assays were used. All cultivars showed high levels of carotenoids in raw roots, predominantly all-trans-beta-carotene (79.1-128.5 mg.100 g(-1) DW), suggesting a high estimated vitamin A activity. The CNPH 1194 cultivar reported carotenoids values highest than those of other cultivars (p < 0.05). The total phenolic compounds varied among cultivars and heat treatments (0.96-2.05 mg.g(-1) DW). In most cases, the heat treatments resulted in a significant decrease in the carotenoids and phenolic compounds contents as well as antioxidant capacity. Processing of flour presented the greatest losses of major carotenoids and phenolics. The phenolic compounds showed more stability than carotenoids after processing. There were significant correlations between the carotenoids and phenolic compounds and the antioxidant capacity.

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10-14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Preliminary in vitro evaluation of N '-(benzofuroxan-5-yl)methylene benzohydrazide derivatives as potential anti-Trypanosoma cruzi agents

A set of benzofuroxan derivatives was tested in vitro against Trypanosoma cruzi epimastigote forms. The influence of physicochemical properties on these benzofuroxan derivatives' activity was observed, and the presence of electron-withdrawing and hydrophobic groups attached to the benzene ring seems to make a favorable contribution at lower concentrations.

ANALYSIS OF IN VITRO AND IN VIVO ANTIOXIDANT PROPERTIES OF HYDROPHILIC FRACTIONS FROM THE SEAWEED HALIMEDA MONILE L.

In this work, in vitro and in vivo antioxidant properties of the marine algae Halimeda monile were assessed and the levels of some of its compounds likely to be responsible for such properties were determined. The estimated contents of total polyphenols, chlorophylls a and b and carotenoids were 179.5, 356.3, 452.8 and 42.2 mu g/g dry weight seaweed, respectively. The presence of terpenoids and flavonoids was also observed. The antioxidant activity of two polar fractions from H. monile (lyophilized aqueous extract and free phenolic acid fraction) was evaluated using three antioxidant assays: ferric reducing antioxidant power, 1,1-diphenyl-2-picrylhydrazyl and lipid peroxidation. Treatment of CCl4-induced liver damage in rats with extracts resulted in lower serum thiobarbituric acid-reactive substances levels and higher hepatic glutathione concentrations compared to those observed in the CCl4-treated group. Also, a significant increase in catalase activity was detected after treatment with the extracts. These results suggest that the seaweed H. monile could be a potential source for natural antioxidants.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

Introduction: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. Methods: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36 degrees C with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. Results: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075 mu M and an LLC MK2 toxicity of 2.003 mu M. Pyrimethamine was effective at an IC50 of 0.482 mu M and a toxicity of 11.178 mu M. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. Conclusions: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

E series prostaglandins alter the proliferative, apoptotic and migratory properties of T98G human glioma cells in vitro

Background: In many types of cancer, prostaglandin E-2 (PGE(2)) is associated with tumour related processes including proliferation, migration, angiogenesis and apoptosis. However in gliomas the role of this prostanoid is poorly understood. Here, we report on the proliferative, migratory, and apoptotic effects of PGE(1), PGE(2) and Ibuprofen (IBP) observed in the T98G human glioma cell line in vitro. Methods: T98G human glioma cells were treated with IBP, PGE(1) or PGE(2) at varying concentrations for 24-72 hours. Cell proliferation, mitotic index and apoptotic index were determined for each treatment. Caspase-9 and caspase-3 activity was measured using fluorescent probes in live cells (FITC-LEHD-FMK and FITC-DEVD-FMK respectively). The migratory capacity of the cells was quantified using a scratch migration assay and a transwell migration assay. Results: A significant decrease was seen in cell number (54%) in the presence of 50 mu M IBP. Mitotic index and bromodeoxyuridine (BrdU) incorporation were also decreased 57% and 65%, respectively, by IBP. The apoptotic index was increased (167%) and the in situ activity of caspase-9 and caspase-3 was evident in IBP treated cells. The inhibition of COX activity by IBP also caused a significant inhibition of cell migration in the monolayer scratch assay (74%) and the transwell migration assay (36%). In contrast, the presence of exogenous PGE(1) or PGE(2) caused significant increases in cell number (37% PGE(1) and 45% PGE(2)). When mitotic index was measured no change was found for either PG treatment. However, the BrdU incorporation rate was significantly increased by PGE(1) (62%) and to a greater extent by PGE(2) (100%). The apoptotic index was unchanged by exogenous PGs. The addition of exogenous PGs caused an increase in cell migration in the monolayer scratch assay (43% PGE(1) and 44% PGE(2)) and the transwell migration assay (28% PGE(1) and 68% PGE(2)). Conclusions: The present study demonstrated that treatments which alter PGE(1) and PGE(2) metabolism influence the proliferative and apoptotic indices of T98G glioma cells. The migratory capacity of the cells was also significantly affected by the change in prostaglandin metabolism. Modifying PG metabolism remains an interesting target for future studies in gliomas.

IN VITRO TRYPANOCIDAL EVALUATION OF PINANE DERIVATIVES FROM ESSENTIAL OILS OF RIPE FRUITS FROM Schinus terebinthifolius RADDI (ANACARDIACEAE)

Essential oils of ripe fruits from Schinus terebinthifolius (Anacardiaceae), obtained using a pilot extractor and a Clevenger apparatus were chemically characterized. Due the high amount of (-)-alpha-pinene in both oils, this monoterpene was tested against the protozoan parasite Trypanosoma cruzi, showing a moderate potential (IC50 63.56 mu g/mL) when compared to benznidazole (IC50 43.14 mu g/mL). Otherwise, (-)-alpha-pinene oxide did not showed anti-trypanosomal activity (IC50 > 400 mu g/mL) while (-)-pinane showed an IC50 of 56.50 mu g/mL. The obtained results indicated that the epoxydation of a-pinene results to the loss of the anti-parasitic activity while its hydrogenation product, contributed slightly to the increased activity.

Low-Level Laser Therapy Influences Mouse Odontoblast-Like Cell Response in Vitro

Objective: The purpose of this study was to analyze the influence of two different irradiation times with 85mW/cm(2) 830nm laser on the behavior of mouse odontoblast-like cells. Background data: The use of low-level laser therapy (LLLT) to stimulate pulp tissue is a reality, but few reports relate odontoblastic responses to irradiation in in vitro models. Methods: Odontoblast-like cells (MDPC-23) were cultivated and divided into three groups: control/nonirradiated (group 1); or irradiated with 85mW/cm(2), 830nm laser for 10 sec (0.8 J/cm(2)) (group 2); or for 50 sec (4.2 J/cm(2)) (group 3) with a wavelength of 830 nm. After 3, 7, and 10 days, it was analyzed: growth curve and cell viability, total protein content, alkaline phosphatase (ALP) activity, calcified nodules detection and quantification, collagen immunolocalization, vascular endothelial growth factor (VEGF) expression, and real-time polymerase chain reaction (PCR) for DMP1 gene. Data were analyzed by Kruskall-Wallis test (alpha = 0.05). Results: Cell growth was smaller in group 2 (p < 0.01), whereas viability was similar in all groups and at all periods. Total protein content and ALP activity increased on the 10th day with 0.8 J/cm(2) (p < 0.01), as well as the detection and quantification of mineralization nodules (p < 0.05), collagen, and VEGF expression (p < 0.01). The expression of DMP1 increased in all groups (p < 0.05) compared with control at 3 days, except for 0.8 J/cm(2) at 3 days and control at 10 days. Conclusions: LLLT influenced the behavior of odontoblast-like cells; the shorter time/smallest energy density promoted the expression of odontoblastic phenotype in a more significant way.

Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Phos has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma. Published by Elsevier Masson SAS.

In Vitro Evaluation of the Probiotic Potential of Bacteriocin Producer Lactobacillus sakei 1

Lactobacillus sakei 1 is a food isolate that produces a heat-stable antimicrobial peptide (sakacin 1, a class ha bacteriocin) inhibitory to the opportunistic pathogen Listeria monocytogenes. Bacterial isolates with antimicrobial activity may be useful for food biopreservation and also for developing probiotics. To evaluate the probiotic potential of L. sakei I, it was tested for (i) in vitro gastric resistance (with synthetic gastric juice adjusted to pH 2.0, 2.5, or 3.0); (ii) survival and bacteriocin production in the presence of bile salts and commercial prebiotics (inulin and oligofructose); (iii) adhesion to Caco-2 cells; and (iv) effect on the adhesion of L. monocytogenes to Caco-2 cells and invasion of these cells by the organism. The results showed that L. sakei I survival in gastric environment varied according to pH, with the maximum survival achieved at pH 3.0, despite a 4-log reduction of the population after 3 h. Regarding the bile salt tolerance and influence of prebiotics, it was observed that L. sakei 1 survival rates were similar (P > 0.05) for all de Man Rogosa Shame (MRS) broth formulations when tests were done after 4 h of incubation. However, after incubation for 24 h, the survival of L. sakei 1 in MRS broth was reduced by 1.8 log (P < 0.001), when glucose was replaced by either inulin or oligofructose (without Oxgall). L. sakei 1 was unable to deconjugate bile salts, and there was a significant decrease (1.4 log) of the L. sakei 1 population in regular MRS broth plus Oxgall (P < 0.05). In spite of this, tolerance levels of L. sakei 1 to bile salts were similar in regular MRS broth and in MRS broth with oligofructose. Lower bacteriocin production was observed in MRS broth when inulin (3,200 AU/ml) or oligofructose (2,400 AU/ml) was used instead of glucose (6,400 AU/ml). L. sakei I adhered to Caco-2 cells, and its cell-free pH-neutralized supernatant containing sakacin I led to a significant reduction of in vitro listerial invasion of human intestinal Caco-2 cells.