Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of similar to 120 kDa that could be solubilized by phospholipase C or Polidocanol. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10. 25, and 50 mu M, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage. (C) 2007 Elsevier Ltd. All rights reserved.

Human breast tumor slices as an alternative approach to cell lines to individualize research for each patient

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

Análise do cultivo de fibroblastos de pterígios primários e recidivados e da cápsula de Tenon normal

OBJETIVO: Avaliar a atividade proliferativa dos fibroblastos da cápsula de Tenon, provenientes de explantes de pterígios primários e recidivados e da conjuntiva normal. MÉTODOS: Foi realizado estudo prospectivo, randomizado, avaliando-se 43 peças cirúrgicas, produto da exérese de 30 pterígios primários e 13 recidivados, além de fragmentos da cápsula de Tenon normal, obtida dos próprios portadores de pterígio. Foram avaliadas a taxa de proliferação, migração e confluência, analisadas segundo dados dos portadores como: idade, localização da lesão, tipo de lesão (tamanho, involutivo ou carnoso), primário ou recidivado. Os dados obtidos foram submetidos à análise estatística. RESULTADOS: Dentre os 30 pterígios primários cultivados, 70% migraram e proliferaram e 60% chegaram à confluência. A migração, proliferação e confluência iniciaram-se mais precocemente nas culturas de fibroblastos provenientes de pterígios em comparação àquelas provenientes da Tenon normal. Os pterígios recidivados apresentaram migração mais precoce que os primários. Não houve diferença estatisticamente significativa quanto ao início da migração, proliferação e confluência entre os pterígios carnosos ou involutivos, assim como entre os pterígios de graus I-II e os de graus III-IV. CONCLUSÃO: O cultivo celular de fibroblastos de pterígios é mais viável que o de Tenon normal. A migração, proliferação e confluência diferem em pterígios primários e recidivados. Pterígios carnosos e involutivos são semelhantes em cultura, assim como não existe diferença entre os pterígios segundo o tamanho da lesão.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

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

Nutritional stress enhances cell viability of odontoblast-like cells subjected to low level laser irradiation

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

Expression of annexin A1 mRNA in peripheral blood from oral squamous cell carcinoma patients

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biocompatibility of denture base acrylic resins evaluated M culture of L929 cells. Effect of polymerisation cycle and postpoymerisation treatments

Objective: the purpose of this study was to evaluate the effect of two post-polymerisation treatments and different cycles of polymerisation on the cytotoxicity of two denture base resins.Materials and methods: the resins tested were Lucitone 550 and QC 20. Discs of resins were fabricated following the manufacturer's instructions. Lucitone 550 was processed by long cycle or short cycle. The resin QC 20 was processed by reverse cycle or normal cycle. The specimens were divided into groups: (i) post-polymerised in microwave for 3 min at 500 W; (ii) post-polymerised in water-bath at 55 degrees C for 60 min and (iii) without post-polymerisation. Eluates were prepared by placing three discs into a sterile glass vial with 9 ml of Eagle's medium and incubated at 37 degrees C for 24 hours. L929 cells were seeded into 96 3 well culture plates and DNA synthesis was assessed by H-thymidine incorporation assay.Results: the results were submitted to two-way ANOVA and Tukey HSD test. QC 20 specimens polymerised by the normal cycle and submitted to microwave post-polymerisation were graded as moderately cytotoxic. Similar results were observed for Lucitone 550 processed by long cycle without post-polymerisation. The other experimental groups were graded as not cytotoxic. After water-bath post-polymerisation, specimens of Lucitone 550 processed by long cycle produced significantly lower inhibition of DNA synthesis than the other groups.Conclusion: the long cycle increased the cytotoxicity of Lucitone 550 and water-bath post-polymerisation reduced the cytotoxicity of Lucitone 550 processed by long cycle.

Effect of curing regime on the cytotoxicity of resin-modified glass-ionomer lining cements applied to an odontoblast-cell line

Objective: The aim of this in vitro study was to evaluate the cytotoxicity of resin-modified glass-ionomer lining cements submitted to different curing regimes and applied to an immortalized odontoblast-cell line (MDPC-23). Methods: Forty round-shaped specimens of each experimental material (Fuji Lining LC and Vitrebond) were prepared. They were light-cured for the manufacturers' recommended time (MRT = 30 s), under-cured (0.5 MRT = 15 s), over-cured (1.5 MRT = 45 s) or allowed to dark cure (0 MRT). Sterilized filter papers soaked with either 5 μL of PBS or HEMA were used as negative and positive control, respectively. After placing the specimens individually in wells of 24-well dishes, odontoblast-like cells MDPC-23 (30,000 cells/cm2) were plated in each well and incubated for 72 h in a humidified incubator at 37 °C with 5% CO2 and 95% air. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). Results: Fuji Lining LC was less cytotoxic than Vitrebond (p < 0.05) in all the experimental conditions. However, the cytotoxicity of Fuji Lining LC was noticeably increased in the absence of light-curing while the same was not observed for Vitrebond. The length of light-curing (15, 30 or 45 s) did not influence the toxicity of both lining materials when they were applied on the odontoblast-cell line MDPC-23. Significance: The light-activation plays an important role in reducing the cytotoxicity of Fuji Lining LC. Following the manufacturer' recommendation regarding the light-curing regime may prevent toxic effect to the pulp cells. © 2005 Academy of Dental Materials.

Application of a serum/protein-free medium for the growth of mammalian cell lines and high level production of classical, variant and very virulent strain of infectious bursal disease virus (IBDV)

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

Evaluation of tissue reaction, cell viability and cytokine production induced by Sealapex Plus

The aim of this study was to investigate the effects of mineral trioxide aggregate (MTA), Sealapex, and a combination of Sealapex and MTA (Sealapex Plus) on the reaction of subcutaneous connective tissue of rats, and on cell viability and cytokine production in mouse fibroblasts. The tissue reaction was carried out with dentin tubes containing the materials implanted in the dorsal connective tissue of rats. The histological analysis was performed after 7 and 30 days. Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts to evaluate the cell viability by MTT assay. ELISA assays were also performed after 24 h of exposure of the mouse fibroblasts to set material disks. Histopathologic examination showed Von Kossa-positive granules that were birefringent to polarized light for all the studied materials at the tube openings. No material inhibited the cell viability in the in vitro test. It was detected IL-6 production in all root-end filling materials. MTA and Sealapex Plus induced a slight raise of mean levels of IL-1β. The results suggest that Sealapex Plus is biocompatible and stimulates the mineralization of the tissue.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research. © 2013 Spandidos Publications Ltd. All rights reserved.