The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

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.

Blood cells attachment after root conditioning and PRP application: An in vitro study

Aim: Root conditioning is aimed at smear layer removal and at dental matrix collagen exposure, which may promote periodontal regeneration. This in vitro study assessed smear layer removal, collagen fiber exposure and the influence of PRP (platelet-rich plasma) application on adhesion of blood cells to the root surface using scanning electron microscopy (SEM). Materials and methods: Scaled root samples (n = 160) were set in five groups and conditioned with: group I - control group (saline solution); group II (EDTA 24%); group III (citric acid 25%); group IV (tetracycline hydrochloride 50 mg/ml); group V (sodium citrate 30%). Eighty samples were assessed using the root surface modification index (RSMI). The other eighty samples were set in two groups. The first group (n = 40) received PRP gel application with a soft brush and the second group (n = 40) received PRP application and then a blood drop. The fibrin clot formation was assessed in the first group and the blood cells adhesion was assessed in the second group using the BEAI (blood elements adhesion index). A previously trained, calibrated, and blind examiner evaluated photomicrographs. Statistical analysis was performed using the Kruskal-Wallis's and Dunn's tests. Results: Group III attained the best results for RSMI and BEAI. Moreover, it was the only group showing fibrin clot formation. Conclusion: Citric acid was the most efficient conditioner for smear layer removal, collagen fiber exposure and blood cell adhesion. Moreover, it was the only group showing fibrin clot formation after PRP application. Clinical significance: This study demonstrated that root conditioning followed by PRP application may favor blood cell adhesion on root surface which may optimize periodontal healing.

Cytotoxic action of Brazilian propolis in vitro on canine osteosarcoma cells

Osteosarcoma (OSA) is a primary bone neoplasm frequently diagnosed in dogs. The biology of OSA in pet dogs is identical to that of pediatric patients, and it has been considered an excellent model in vivo to study human OSA. Since the individual response to chemotherapy is unpredictable and considering that propolis is a natural product with several biological properties, this work evaluated the cytotoxic action of propolis on canine OSA cells. The primary cell culture of canine OSA was obtained from the tumor of a dog with OSA. Cell viability was assessed after incubation with propolis, 70% ethanol (propolis solvent), and carboplatin after 6, 24, 48, and 72 h. Cell viability was analyzed by the crystal violet method. Data showed that canine OSA cells were sensitive to propolis in a dose- and time-dependent manner and had a distinct morphology compared to control. Its solvent (70% ethanol) had no effect on cell viability, suggesting that the cytotoxic action was exclusively due to propolis. Our propolis sample exerted a cytotoxic effect on canine OSA cells, and its introduction as a possible therapeutic agent in vivo could be investigated, providing a new contribution to OSA treatment. Copyright © 2012 John Wiley & Sons, Ltd.

AKT and AMPK Activation after High-Fat and High-Glucose In Vitro Treatment of Prostate Epithelial Cells

Considering the increasing consumption of saturated fat and glucose in diets worldwide and its possible association to carcinogenesis, this investigation analysed the proliferation profile of nonmalignant human prostate epithelial cells after exposure to elevated levels of fat and glucose. PNT1A cells were cultured with palmitate (100 or 200 mu M) and/or glucose (450mg/dl) for 24 or 48 h. Treated cells were evaluated for viability test and cell proliferation (MTS assay). AKT and AMPK phosphorylation status were analysed by Western blotting. After 24 h of high-fat alone or associated with high-glucose treatment, there was an increase in AMPK and AKT activation associated to unchanged MTS-cell proliferation. Following 48 h of high-fat but not high-glucose alone, cells decreased AMPK activation and maintained elevated AKT levels. These data were associated to increased cell proliferation after further high-fat treatment. After longer high-fat exposure, MTS revealed that cells remained proliferating. High-glucose alone or associated to high-fat treatment was not able to increase cell proliferation and AKT activation. A high-fat medium containing 100 mu M of palmitate stimulates proliferation in PNT1A cells by decreasing the activation of AMPK and increasing activation of AKT after longer exposure time. These findings improve the knowledge about the negative effect of high levels of this saturated fatty acid on proliferative disorders of prostate.

Mesenchymal stem cells modulate release of matrix proteins from tendon surfaces in vitro: a potential beneficial therapeutic effect

Aim: Injury of tendons contained within a synovial environment, such as joint, bursa or tendon sheath, frequently fails to heal and releases matrix proteins into the synovial fluid, driving inflammation. This study investigated the effectiveness of cells to seal tendon surfaces and provoke matrix synthesis as a possible effective injectable therapy. Materials & methods: Equine flexor tendon explants were cultured overnight in suspensions of bone marrow and synovium-derived mesenchymal stems cells and, as controls, two sources of fibroblasts, derived from tendon and skin, which adhered to the explants. Release of the most abundant tendon extracellular matrix proteins into the media was assayed, along with specific matrix proteins synthesis by real-time PCR. Results: Release of extracellular matrix proteins was influenced by the coating cell type. Fibroblasts from skin and tendon appeared less capable of preventing the release of matrix proteins than mesenchymal stems cells. Conclusion: The source of cell is an important consideration for cell therapy.

Characterisation of the methylation pattern in the intragenic CpG island of the IGF2 gene in Bos taurus indicus cumulus cells during in vitro maturation

The aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1-3 mm and a parts per thousand yenaEuro parts per thousand 8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern.Genomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised.There were no differences in the methylation pattern among groups (P > 0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17 +/- 14.11 % and 82.93 +/- 5.86 %, respectively, and those from large follicles showed methylation levels of 81.81 +/- 10.40 % and 79.64 +/- 13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17 +/- 12.01 % and 81.19 +/- 10.15 %.In conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis, and to be a non-invasive method for determining oocyte quality, the identification of new epigenetic markers in cumulus cells has great potential to be used to support reproductive biotechniques in humans and other mammals.

Bone marrow mesenchymal stem cells stimulated by bFGF up-regulated protein expression in comparison with periodontal fibroblasts in vitro

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

Toxicogenetic effects of low concentrations of the pesticides imidacloprid and sulfentrazone individually and in combination in in vitro tests with HepG2 cells and Salmonella typhimurium

The insecticide imidacloprid and the herbicide sulfentrazone are two different classes of pesticides that are used for pest control in sugarcane agriculture. To evaluate the genotoxic potential of low concentrations of these two pesticides alone and in mixture, the comet assay and the micronucleus (MN) test employing fluorescence in situ hybridization (FISH) with a centromeric probe were applied in human hepatoma cell lines (HepG2), in a 24-h assay. Mutagenicity was assessed by Salmonella/microsome assay with TA98 and TA100 strains in the absence and presence of an exogenous metabolizing system (S9). The results showed significant inductions of MN in HepG2 cells by both pesticides, for all the tested concentrations. As evidenced in the comet assay, only the imidacloprid presented significant responses. When the two pesticides were associated, a significant induction of damage was observed in the HepG2 cells by the comet assay, but not by the MN test. Moreover, the MN induced by the mixtures of the pesticides appeared at lower levels than those induced by sulfentrazone and imidacloprid when tested alone. According to the FISH results, the damage induced by imidacloprid in the HepG2 cells resulted from a clastogenic action of this insecticide (76.6% of the MN did not present a centromeric signal). For the herbicide sulfentrazone and for the mixture of the pesticides, a similar frequency of MN with and without the presence of the centromeric signal (herbicide: 52.45% of the MN without centromeric signal and 47.54% of the MN with centromeric signal; mixture: 48.71% of the MN without centromeric signal and 51.42% of the MN with centromeric signal) was verified. Based on these results, it was concluded that each one of the pesticides evaluated interacts with the DNA of HepG2 cells and causes irreparable alterations in the cells. However, the combination of the pesticides showed an antagonistic effect on the cells and the damage induced was milder and not persistent in HepG2 cells. The results obtained by the Ames test did not point out significant results.

Ribavirin has an in vitro antiviral effect in rabies virus infected neuronal cells but fails to provide benefit in experimental rabies in mice

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

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

In Vitro Photodynamic Inactivation of Cryptococcus neoformans Melanized Cells with Chloroaluminum Phthalocyanine Nanoemulsion

The selection of fungi resistant to currently used fungicides and the emergence of new pathogenic species make the development of alternative fungus-control techniques highly desirable. Photodynamic antimicrobial chemotherapy (PACT) is a promising method which combines a nontoxic photosensitizer (PS) with visible light to cause selective killing of microbial cells. The development of PACT to treat mycoses or kill fungi in the environment depends on identifying effective PS for the different pathogenic species and delivery systems able to expand and optimize their use. In the present study, the in vitro susceptibility of Cryptococcus neoformans melanized cells to the photodynamic effects of the PS agent ClAlPc in nanoemulsion (ClAlPc/NE) was examined. Cells were killed in a PS concentration- and light dose-dependent manner. Treatment with ClAlPc/NE, using PS concentrations (e.g. 4.5 mu m) and light doses (e.g. 10 J cm-2) compatible with PACT, resulted in a reduction of up to 6 logs in survival. Washing the cells to remove unbound PS before light exposure did not inhibit fungal photodynamic inactivation. Internalization of ClAlPc by C.neoformans was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on PS concentration.