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Primary cell cultures are essential tools for elucidating the physiopathological mechanisms of the cardiovascular system. Therefore, a primary culture growth protocol of cardiovascular smooth muscle cells (VSMCs) obtained from human abdominal aortas was standardized. Ten abdominal aorta samples were obtained from patients diagnosed with brain death who were organ and tissue donors with family consent. After surgical ablation to capture the aorta, the aortic tissue was removed, immersed in a Custodiol® solution, and kept between 2 and 8 °C. In the laboratory, in a sterile environment, the tissue was fragmented and incubated in culture plates containing an enriched culture medium (DMEM/G/10% fetal bovine serum, L-glutamine, antibiotics and antifungals) and kept in an oven at 37 °C and 5% CO2. The aorta was removed after 24 h of incubation, and the culture medium was changed every six days for twenty days. Cell growth was confirmed through morphological analysis using an inverted optical microscope (Nikon®) and immunofluorescence for smooth muscle alpha-actin and nuclei. The development of the VSMCs was observed, and from the twelfth day, differentiation, long cytoplasmic projections, and adjacent cell connections occurred. On the twentieth day, the morphology of the VSMCs was confirmed by actin fiber immunofluorescence, which is a typical characteristic of VSMCs. The standardization allowed VSMC growth and the replicability of the in vitro test, providing a protocol that mimics natural physiological environments for a better understanding of the cardiovascular system. Its use is intended for investigation, tissue bioengineering, and pharmacological treatments.
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Aorta Abdominal , Doenças Vasculares , Humanos , Morte Encefálica/metabolismo , Morte Encefálica/patologia , Músculo Liso Vascular/metabolismo , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Modelos Teóricos , Miócitos de Músculo Liso , Encéfalo , Células CultivadasRESUMO
Cell volume regulation is an essential strategy for the maintenance of life under unfavorable osmotic conditions. Mechanisms aimed at minimizing the physiological challenges caused by environmental changes are crucial in anisosmotic environments. However, aquatic ecosystems experience multiple stressors, including variations in salinity and heavy metal pollution. The accumulation of heavy metals in aquatic ecosystems has a significant effect on the biota, leading to impaired function. The aim of this study was to investigate the capacity of volume regulation in isolated cells of the sea anemone Bunodosoma cangicum exposed to nominal copper (Cu) concentrations of 5 and 50 µg L-1, associated or not with hypoosmotic (15) or hyperosmotic (45) shock for 15 min. In the absence of the metal, our results showed volume maintenance in all osmotic conditions. Our results showed that cell volume was maintained under all osmotic conditions in the absence of Cu. Similarly, no significant differences were observed in cell volumes under isosmotic and hyperosmotic conditions in the presence of both Cu concentrations. A similar homeostatic response was observed under the hypoosmotic condition with 5 µg L-1 Cu. Our results showed an increase in cell volume with exposure of the cells to the hypoosmotic condition and 50 µg L-1 Cu. The response could be associated with the increased bioavailability of Cu, reduced ability to resist multixenobiotics and their efflux pathways, and the impairment of water efflux in specialized transmembrane proteins. Therefore, B. cangicum pedal disk cells can tolerate osmotic variations in aquatic ecosystems. However, the capacity to regulate cell volume under hypoosmotic conditions can be affected by the presence of a metal contaminant (50 µg L-1 Cu), which could be due to the inhibition of water channels.
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Metais Pesados , Anêmonas-do-Mar , Poluentes Químicos da Água , Animais , Cobre/metabolismo , Anêmonas-do-Mar/metabolismo , Ecossistema , Metais Pesados/metabolismo , Tamanho Celular , Poluentes Químicos da Água/metabolismoRESUMO
ABSTRACT Objective This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. Methods Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. Results Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. Conclusion The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.
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Bovine gammaherpesvirus type 4 (BoHV-4) shows tropism for the endometrium, in which it causes the death of epithelial and stroma cells. Despite having anti-apoptotic genes in its genome, experiments based on immortalized cell lines have shown that BoHV-4 induces cell death by apoptosis. In the present study, we evaluated BoHV-4 replication, pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) mitochondrial genes expression and chromatin condensation in bovine endometrium primary culture cells (BEC) and in the Madin Darby bovine kidney (MDBK) cell line. Results showed that BoHV-4 has a preference for replication in BEC cells over the MDBK cell line, demonstrated by the high viral titer that is consistent with the tropism of the virus. In BEC cells, chromatin condensation was consistent with the values of viral kinetics at the late stage of infection, accompanied with a balance in the mRNA levels of apoptotic mitochondrial proteins. As a consequence, in those cells viral transmission would be enhanced by inhibiting apoptosis in the early stage of virus proliferation, allowing the complete production of viral progeny, and then, the induction of apoptosis in late stages would allow neighboring cells infection. In MDBK cells replication kinetics was coincident with the up-regulation of Bcl-2, which suggests that the productive infection in MDBK is associated with a lytic phase of the virus or another cell death pathway (probably autophagy mechanism) at the late stage of infection. The results agree with the study of nuclear morphology, where a constant chromatin condensation was observed over time. It is clear that the documented BoHV-4 apoptotic responses observed in the cell lines studied above are not valid in cells from primary cultures. The data presented in this study suggest that BoHV-4 could induce apoptosis in BEC cells without a leading role of the mitochondria pathway. Further studies will be necessary to characterize in detail the programmed cell death pathways involved in BoHV-4 infection in the primary cell cultures evaluated.
Assuntos
Herpesvirus Bovino 4 , Animais , Apoptose , Bovinos , Linhagem Celular , Cromatina , Feminino , Herpesvirus Bovino 4/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Replicação ViralRESUMO
Echinococcus multilocularis is the etiological agent of alveolar echinococcosis (AE), a serious parasitic disease in the Northern Hemisphere. The E. multilocularis primary cell cultivation system, together with E. multilocularis genome data and a range of pioneering molecular-based tools have advanced the research on this and other cestodes. RNA interference (RNAi) and microRNA knock-down have recently contributed to the study of the cellular and molecular basis of tapeworm development and host-parasite interaction. These, as well as other techniques, normally involve an electroporation step for the delivery of RNA, DNA, peptides, and small molecules into cells. Using transcriptome data and bioinformatic analyses, we herein report a genome-wide comparison between primary cells of E. multilocularis and primary cells under electroporated conditions after 48 h of culture. We observed that ~ 15% of genes showed a significant variation in expression level, including highly upregulated genes in electroporated cells, putatively involved in detoxification and membrane remodeling. Furthermore, we found genes related to carbohydrate metabolism, proteolysis, calcium ion binding and microtubule processing significantly altered, which could explain the cellular dispersion and the reduced formation of cellular aggregates observed during the first 48 h after electroporation.
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Cestoides , Infecções por Cestoides , Equinococose , Echinococcus multilocularis , Animais , Equinococose/parasitologia , Echinococcus multilocularis/genética , Eletroporação , Cultura Primária de CélulasRESUMO
Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.
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Animal models represent a crucial tool for biological research, so the establishment of new cultures is fundamental for the discovery of new therapies and the understanding of mechanisms of cell development in the most diverse animals. Here, we report the successful establishment of two new primary cell cultures derived from a South American bat (Artibeus planirostris). The establishment of a new bat culture can help in the investigation of new zoonoses since bats have been proposed as carriers of these diseases. We evaluated the chromosomal stability of cells from different passages. Primary cultures were collected from ear tissues and bone marrow of A. planirostris. Cultures were expanded, and osteogenic and adipogenic inductions were conducted for 21 days. For osteogenic differentiation, the medium was supplemented with 0.1 µM dexamethasone, 3 mM ß-glycerophosphate, and 10 µM L-ascorbic acid 2-phosphate. For adipogenic differentiation, the medium was supplemented with 5 µM rosiglitazone, 0.4 µM insulin, 0.1 mM indomethacin, and 0.1 µM dexamethasone. After the induction period, the cells were stained with Alizarin Red to assess osteogenic differentiation and Oil Red O to assess adipogenic differentiation. We observed the appearance of lipid droplets in adipocytes and the extracellular deposition of calcium matrix by osteocytes, indicating that bone marrow-derived cells and skin-derived cells of A. planirostris could successfully differentiate into these lineages. Also, the number of chromosomes remained stable for both primary cultures during passages 2, 4, 6, and 8.
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Técnicas de Cultura de Células , Separação Celular , Quirópteros/metabolismo , Células-Tronco Mesenquimais , Pele , Animais , Células Cultivadas , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Pele/citologia , Pele/metabolismoRESUMO
RESUMEN Las células madre humanas nacen con la creación de la vida misma y algunas de estas permanecen durante toda la vida. Por consiguiente, se pueden hallar en tejidos adultos y utilizarlas para investigaciones a nivel básico y aplicado. Actualmente, en nuestro país existe un creciente interés en el estudio y aplicación de células madre; sin embargo, existe poco conocimiento acerca del procedimiento para su identificación. Es por ello que este artículo tiene como objetivo dar a conocer, desde un punto de vista práctico, un procedimiento para el cultivo e identificación de células madre/estromales obtenidas de lipoaspirado humano (Adipose Stem Cells) con fines de investigación, el cual incluye la caracterización a nivel de inmunofenotipo, el potencial de diferenciación celular, la expresión génica y el control de calidad del cultivo celular, que sirva de apoyo para los profesionales de la comunidad científica peruana que deseen desarrollar esta línea de investigación.
ABSTRACT Human stem cells are born with the creation of life itself and some of them remain throughout life. Therefore, they can be found in adult tissues and used for basic and applied research. Currently, in our country there is a growing interest in the study and application of stem cells; however, little is known about the identification procedure. For this reason, this study aims to present, from a practical point of view, a procedure for the culture and identification of stem/stromal cells obtained from human lipoaspirate (Adipose Stem Cells), for research purposes. This procedure includes the immunophenotype characterization, cell differentiation potential, gene expression and cell culture quality control; and will serve as support for Peruvian scientific community professionals who wish to develop this line of research.
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Células-Tronco , Técnicas de Cultura de Células , Pesquisa , Separação Celular , Tecido Adiposo , Inquéritos e Questionários , Medicina Regenerativa , Cultura Primária de Células , Terapia Baseada em Transplante de Células e TecidosRESUMO
Some medical applications of magnetic nanoparticles require direct contact with healthy tissues and blood. If nanoparticles are not designed properly, they can cause several problems, such as cytotoxicity or hemolysis. A strategy for improvement the biological proprieties of magnetic nanoparticles is their functionalization with biocompatible polymers and nonionic surfactants. In this study we compared bare magnetite nanoparticles against magnetite nanoparticles coated with a combination of polyethylene glycol 3350 (PEG 3350) and polysorbate 80 (Tween 80). Physical characteristics of nanoparticles were evaluated. A primary culture of sheep adipose mesenchymal stem cells was developed to measure nanoparticle cytotoxicity. A sample of erythrocytes from a healthy donor was used for the hemolysis assay. Results showed the successful obtention of magnetite nanoparticles coated with PEG 3350-Tween 80, with a spherical shape, average size of 119.2 nm and a zeta potential of +5.61 mV. Interaction with mesenchymal stem cells showed a non-cytotoxic propriety at doses lower than 1000 µg/mL. Interaction with erythrocytes showed a non-hemolytic propriety at doses lower than 100 µg/mL. In vitro information obtained from this work concludes that the use of magnetite nanoparticles coated with PEG 3350-Tween 80 is safe for a biological system at low doses.
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Abstract INTRODUCTION: Biomphalaria snails may display varying levels of susceptibility to Schistosoma mansoni infection. We have been developing an in vitro model to study the interaction between the snail and the parasite, using tissue-derived cell cultures from Biomphalaria. METHODS: The digestive gland- and kidney-derived cells from primary cultures of resistant (B. tenagophila Taim) and susceptible (B. tenagophila HM and B. glabrata BH) strains of Biomphalaria were exposed to S. mansoni sporocysts. RESULTS: S. mansoni sporocysts were surrounded and encapsulated exclusively by cells derived from the digestive gland (DG) of B. tenagophila Taim. The process was followed by a marked decrease in the number of free sporocysts in the culture medium. The morphological characteristics of DG-derived cells in culture have been described. CONCLUSIONS: Cells derived from DG (but not SK) primary cultures of B. tenagophila Taim may participate in S. mansoni sporocyst control.
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Animais , Biomphalaria , Esquistossomose mansoni , Schistosoma mansoni , Oocistos , Interações Hospedeiro-ParasitaRESUMO
SUMMARY: Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. Gingival fibroblasts present structural function, being able to modulate their metabolic capacity, which is reflected in the tissue morphology. The possibility of culturing fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal surgical clinic. The objective of the present article is to present a method of obtaining and cultivating viable human gingival fibroblasts for in vitro research. Explants derived from periodontal surgical discards were used, grown in 25 cm2 bottles to obtain a primary cell culture. After observing the proliferation and growth of the fibroblasts that interconnected and formed a monolayer network, involving the periphery of the explants, it was possible to remove the explants, to make the passage and the new subcultures were obtained in a ratio of 1:1. After 7 days, the amount of viable cells was analyzed in triplicate, using the Neubauer chamber technique, in cell culture bottles of 25 mm2 (T25) and 75 mm2 (T75). Fibroblasts were described and subclassified morphologically. The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were subclassified into reticular and fusiform, being predominant those with fusiform morphology. In conclusion, culture of explant of human gingival connective tissue is a viable method for obtaining gingival connective tissue cells suitable for laboratory tests in cell culture, aiming at obtaining constructs for gingival tissue engineering.
RESUMEN: El cultivo celular es una herramienta importante en los laboratorios de investigación médica, odontológica y biológica, que apoyan las terapias celulares y las estrategias de bioingeniería de tejidos. Los fibroblastos gingivales presentan una función estructural, pudiendo modular su capacidad metabólica, que se refleja en la morfología tisular. La posibilidad de cultivar fibroblastos in vitro, en monocapa o en andamios tridimensionales, para trasplantes posteriores in vivo abre perspectivas importantes para la clínica de cirugía periodontal. El objetivo del presente artículo es presentar un método para obtener y cultivar fibroblastos gingivales humanos viables para investigación in vitro. Se utilizaron explantes derivados de los descartes quirúrgicos periodontales, crecidos en frascos de 25 cm2 para obtener un cultivo de células primarias. Después de observar la proliferación y el crecimiento de los fibroblastos que se interconectaron y formaron una red de monocapa, que involucraba la periferia de los explantes, fue posible eliminar los explantes, hacer el pasaje y los nuevos subcultivos se obtuvieron en una proporción de 1:1. Después de 7 días, la cantidad de células viables se analizó por triplicado, utilizando la técnica de cámara de Neubauer, en botellas de cultivo celular de 25 mm2 (T25) y 75 mm2 (T75). Los fibroblastos fueron descritos y sub-clasificados morfológicamente. Los resultados mostraron un patrón de crecimiento en ambas botellas, pero con un número mayor en botellas de 75 cm2. Las células con morfología fibroblástica se subclasificaron en reticulares y fusiformes, predominando aquellas con morfología fusiforme. En conclusión, el cultivo de explante de tejido conectivo gingival humano es un método viable para obtener células de tejido conectivo gingival adecuadas para pruebas de laboratorio en cultivos celulares, con el objetivo de obtener construcciones para la ingeniería del tejido gingival.
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Humanos , Células do Tecido Conjuntivo , Técnicas de Cultura de Células/métodos , Bioengenharia/métodos , Gengiva/citologia , Biologia Celular , FibroblastosRESUMO
BACKGROUND: Ovarian cancer is the most lethal of all gynecologic malignancies. The relationship between sexual steroids receptors and ovarian cancer progression has been largely evaluated. The presence of progesterone receptors has been associated with an increase of a disease-free period and overall survival in patients with ovarian carcinoma. In the present study, primary cultures of ovarian carcinoma obtained from 35 patients diagnosed with epithelial ovarian cancer were evaluated for cell survival after treatment with 10- 8 M of 17ß-estradiol, progesterone, testosterone and dihydrotestosterone. RESULTS: The results were analyzed considering histological subtypes: low grade serous, high grade serous, endometrioid and mucinous carcinoma; clear cell carcinoma was not included due to failure in obtaining successful cultures of this subtype. A significant reduction of cell survival was observed after progesterone treatment in endometrioid ovarian carcinoma. Changes were not observed in low grade serous, high grade serous and mucinous carcinoma. The effect of progesterone was related to the presence of progesterone receptor (PR), a 43% reduction in the cell number was observed in PR (+) endometrioid ovarian carcinoma. CONCLUSIONS: This study supports the importance of progesterone and the presence of progesterone receptor in the reduction of ovarian cancer progression in the endometrioid ovarian carcinoma.
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Antineoplásicos Hormonais/farmacologia , Carcinoma Endometrioide/patologia , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Progesterona/farmacologia , Adulto , Carcinoma Endometrioide/metabolismo , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Estudos Prospectivos , Receptores de Progesterona/metabolismo , Receptores de Esteroides/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Characteristics of melanoma cells have been deciphered by studies carried out in two dimensional cell cultures growing as adherent monolayers on the bottom of plastic flasks. Melanoma cells can be cultured with a considerable degree of success, and, depending on the further use of the cells obtained in the culture, methodologies have to be adjusted to obtain reliable results. Although there are many melanoma continuous cell lines, in vitro 2D and 3D melanoma primary cell culture may be a more useful model to investigate interactions between cancer cells and immune system, as well as the effect of cytotoxic treatments and personalized medicine in environments more similar to the physiological conditions.Here, we described a protocol which employs many strategies to obtain primary 2D and 3D melanoma cultures as a model to study cell-cell and cell-microenvironment interactions that must be considered to properly design personalized cancer treatments, as well as for testing novel anticancer drugs and drug delivery vehicles.
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Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Melanoma/patologia , Cultura Primária de Células/métodos , Neoplasias Cutâneas/patologia , Antineoplásicos/uso terapêutico , Biópsia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Humanos , Melanoma/sangue , Melanoma/tratamento farmacológico , Melanoma/imunologia , Cultura Primária de Células/instrumentação , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/imunologia , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Esferoides Celulares , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
O objetivo deste estudo foi avaliar um novo método de texturização por PEO com incorporação de Ca e P na superfície do Ti-6Al-4V em ossos de baixa densidade, por meio de avaliação in vitro, ex-in vivo e in vivo, em função de parâmetros topográficos e reparacionais. 57 ratas Wistar (Rattus novergicus), sendo 38 ratas com 6 meses de idade (Grupos OXV - submetidas à ovariectomia e SHAM - cirurgia fictícia) e 19 ratas senis (18 meses de idade: Grupo SENIL), foram divididas para realização do estudo ex-in vivo (n=9) e in vivo (n=48). Os grupos para análise ex-in vivo foram submetidos à eutanásia e os fêmures foram removidos e transportados em meio de cultura contendo meio essencial mínimo modificação alfa (α- MEM) suplementado com 500 µg/mL de gentamicina e 3 µg/mL de fungisona. As células-tronco mesenquimais de medula óssea (CTMsMO) dos fêmures, foram isoladas e cultivadas em meio de crescimento para manterem-se como CTMs. Após alcançar a subconfluência, as células foram cultivadas em 3 superfícies de discos de Ti-6Al-4V, grupo CONTROLE (superfície usinada) grupo AC (superfície tratada por Ataque Ácido e Jateamento) e grupo PEO (superfície tratada por Oxidação de Plasma Eletrolítico com associação de Cálcio e Fosforo). Para avaliação das respostas celulares foram realizados ensaios de viabilidade celular, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteina óssea (BSP) e osteopontina (OPN), atividade da fosfatase alcalina (ALP) e formação de matriz mineralizada. Os dados foram submetidos ao teste ANOVA 1 fator ou Kruskal-Wallis (p < 0,05). Nos experimentos in vivo, após 90 dias, foi instalado um implante em cada tíbia, sendo um implante pertencente ao grupo PEO e o outro implante do grupo AC. Após 42 dias da instalação dos implantes, 8 animais de cada grupo foram submetidos à eutanásia e suas tíbias passaram pela descalcificação, para a análise histológica e imunoistoquímica (OPG, RANKL, OC e TRAP). As demais ratas, após a eutanásia, tiveram suas tíbias coletadas e analisadas em microtomografia computadorizada (BV.TV, Tb.Th, Tb.N, Tb.Sp e Po(tot)) e, em seguida os implantes submetidos ao torque reverso em torquímetro digital (N.cm). A outra metade das tíbias foram processadas com inclusão em resina fotopolimerizadas para cortes calcificados e assim, a análise por microscopia confocal (calceina e alizarina) e em seguida, análise histométrica. Os dados foram submetidos ao teste ANOVA 1 fator ou Kruskal-Wallis, seguido de pós teste Tukey; p < 0,05). A análise da viabilidade celular mostrou que em todos os grupos testes de CTMs-MO para SHAM, OVX e SENIL apresentaram um crescimento progressivo nos diferentes tempos de 3, 7 e 10 dias. Avaliação da expressão gênica através dos genes Runx2, SP7/Osterix, ALP, BSP, OC e OPN e analise pela imunofluorecência apresentaram uma leve tendência de melhores respostas nas CTMs-MO SHAM para o grupo AC, CTMs-MO OVX para o grupo PEO e CTMsMO SENIS características semelhantes nos grupos AC e PEO. A atividade da fosfatase alcalina ocorreu maior expressão na superfície PEO no grupo SHAM, maior expressão na superfície CONTROLE no grupo OVX e no grupo SENIL houve um equilíbrio em todas as superfícies. A superfície PEO apresentou maior formação de nódulos de mineralização (21º dia) em todos os grupos. Nos experimentos in vivo, as análises histológicas mostrou maior neoformação óssea no grupo PEO quando comparado ao grupo AC nos grupos SHAM e OVX, e no grupo SENIL foram similares os resultados. A avaliação imunoistoquímica demonstrou um equilíbrio em todos os grupos na comparação de superfícies na proteína TRAP, para o processo de remodelação (OPG e RANKL) e mineralização (OC) nos grupos SHAM e SENIL (p>0,05), ocorrendo uma diminuição no grupo OVX, no qual os resultados do PEO foram mais favoráveis que AC nessa conformação. Na análise microtomográfica, BV.TV os resultados foram semelhantes em ambos os grupos, porém em OVX AC mostrou menor porcentagem de volume ósseo e uma maior porosidade (Po(tot)). A análise biomecânica por torque-reverso (N.cm) mostrou que os maiores valores pertenciam ao grupo PEO. A dinâmica do tecido ósseo representada pelo turnover ósseo, observado através dos fluorocromos (Calceína e Alizarina) mostrou-se similares nos grupos experimentais. As superficies PEO E AC nesse estudo demonstram que possuem uma grande capacidade de promoção da formação óssea independente dos tipos ósseos experimentais (SHAM, OVX e SENIL), tanto na área de contato osso e implante (ELCOI), quanto para a área de osso neoformado (AON). Diante daslimitações do estudo in vitro e in vivo, os resultados foram esclarecedores para acreditar que o método de texturização aqui testado, por meio da Oxidação por Plasma Eletrolítico (PEO), favoreceu à formação óssea, principalmente nos ossos mais críticos (OVX), inclusive evidenciando maior maturação óssea nos períodos mais tardios aqui analisados(AU)
The objective of this study was to evaluate a new PEO texturing method with Ca and P incorporation on the Ti-6Al-4V surface in low bone density, by means of in vitro, ex vivo and in vivo evaluation through topographic and repairment parameters. 57 Wistar rats (Rattus novergicus), being 38 at 6 months of age (OXV Groups - submitted to ovariectomy and SHAM surgery) and 19 senile rats (18 months of age: SENIL Group) were divided into three subgroups: ex-in vivo (n = 9) and in vivo (n = 48). The Groups for ex-in vivo analysis were euthanized and femurs were removed and transported in culture medium containing minimal alpha modification (α- MEM) medium supplemented with 500 µg / ml gentamicin and 3 µg / ml fungizone. The mesenchymal stem cells from bone marrow (MSC-M) of the femur were isolated and cultured in growth medium to remain as MSCs. After reaching the subconfluence, the cells were grown on 3 surfaces of Ti-6Al-4V discs, CONTROL group (machined surface) group AC (surface treated by etched-acid) and PEO group (surface treated by Electrolytic Plasma Oxidation with the association of Calcium and Phosphorus). Cell viability assays, gene expression of osteoblastic markers, bone sialoprotein (BSP) and osteopontin (OPN), alkaline phosphatase (ALP) activity, and mineralized matrix formation were performed to evaluate cellular responses. Data were submitted to ANOVA 1 factor test or Kruskal-Wallis test (P <0.05). In the groups for the in vivo study, after 90 days, an implant was installed on each tibia, one implant to the PEO group and another implant of the AC group. After 42 days of implant implantation, eight animals from each group underwent euthanasia and their tibiae underwent decalcification for histological and immunohistochemical analysis (OPG, RANKL, OC, and TRAP). The other rats, after euthanasia, had their tibiae collected and analyzed in computerized microtomography (BV.TV, Tb.Th, Tb.N, Tb.Sp and Po (tot)), and then implants submitted to reverse torque in Digital torque wrench (N.cm). Another half of the tibiae were processed with inclusion in photopolymerized resin for calcified cuts and thus the analysis by confocal microscopy (calcein and alizarin) and then histometric analysis. Data were submitted to 1-factor ANOVA or Kruskal-Wallis test, followed by Tukey test; p <0.05). The cell viability analysis showed that in all groups, MOH tests for SHAM, OVX, and SENIL showed a progressive growth in the different times of 3, 7 and 10 days. Evaluation of gene expression through the Runx2, SP7 / Osterix, ALP, BSP, OC and OPN genes and immunofluorescence analysis showed a slight tendency for better responses in the CTMs- MO SHAM for the AC group, CTMs-MO OVX for the PEO group and CTMs-MO SENIS features similar in AC and PEO groups. The alkaline phosphatase activity was higher on the PEO surface in the SHAM group, the greater expression on the CONTROL surface in the OVX group and the SENIL group showed a balance on all surfaces. The PEO surface presented a higher formation of mineralization nodules in all groups (21st day). For the in vivo analyzes, the histological analysis showed greater bone neoformation in the PEO group when compared to the AC group in the SHAM and OVX groups, and in the SENIL group, the results were similar. The immunohistochemical evaluation showed a balance in all groups in the comparison of surfaces in the TRAP protein, for the remodeling process (OPG and RANKL) and mineralization (OC) in the SHAM and SENIL groups (p> 0.05). OVX group, in which PEO results were more favorable than AC in this confirmation. In the microtomographic analysis, BV.TV the results were similar in both groups, but in OVX AC showed a lower percentage of bone volume and a higher porosity (Po (tot)). Biomechanical analysis by torque-reverse (N.cm) showed that the highest values belonged to the PEO group. The dynamics of the bone tissue represented by the bone turnover observed through the fluorochromes (Calcein and Alizarin) were similar in the experimental groups. The PEO and AC surfaces in this study demonstrate that they have a great ability to promote bone formation independent of experimental bone types (SHAM, OVX, and SENIL), both in the area of bone and implant contact (ELCOI) and in the area of newly formed bone (AON). Considering the limitations of the in vitro and in vivo study, the results were enlightening to believe that the texturing method tested here, through Electrolytic Plasma Oxidation (PEO), favored bone formation, mainly in the most critical bones (OVX), including evidence of increased bone maturation in the later periods analyzed here(AU)
Assuntos
Animais , Ratos , Osteoporose , Implantes Dentários , Cultura Primária de Células , Ratos Wistar , Oxidação , Células-Tronco MesenquimaisRESUMO
OBJECTIVES: Corneal endothelial cell (CEC) isolation and harvest aim to produce engineered grafts to solve donor corneal tissue shortage. To yield high amounts of CEC maintaining morphological and molecular characteristics, several isolation and culture conditions are reported. Here, we combined direct explant culture, with three different coating conditions and a two-step media approach to compare confluence efficiency, morphology, and specific molecular markers expression. DATA DESCRIPTION: Confluence was reached after 2 weeks in the three coating conditions (Matrigel, collagen I, and in uncoated plates) using a two-step approach (proliferative medium without pituitary extract, followed by stabilizer basal medium). Na/K-ATPase and GPC4 markers were detected by immunocytochemistry while GPC4, CD200, and TJP1 by RT-PCR in the three CEC coating culture conditions. CEC in proliferative medium showed spindle morphology in the three conditions. Polygonal morphology was seen in CEC cultures using basal medium under uncoated and collagen I coated plates. CEC cultured in Matrigel-coated plates remained with spindle morphology in basal medium.
Assuntos
Técnicas de Cultura de Células/métodos , Colágeno Tipo I/metabolismo , Células Endoteliais/citologia , Endotélio Corneano/citologia , Antígenos CD/genética , Proliferação de Células , Células Cultivadas , Colágeno , Combinação de Medicamentos , Células Endoteliais/metabolismo , Expressão Gênica , Glipicanas/genética , Glipicanas/metabolismo , Humanos , Imuno-Histoquímica , Laminina , Proteoglicanas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
The genetics background underlying the aggressiveness of chondrosarcoma (CS) is poorly understood. One possible cause of malignant transformation is chromosomal instability, which involves an error in mitotic segregation due to numerical and/or functional abnormalities of centrosomes. The present study aimed to evaluate centrosome amplification in cryopreserved samples of tumor tissue from patients with CS. An analysis was performed on 3 primary cultures of tumors from patients who underwent surgery between January 2012 and December 2012 at the Department of Orthopedics at the Barretos Cancer Hospital (Barretos, Brazil). Additionally, cryopreserved tumor specimens were analyzed from 10 patients. The data were assessed using immunocytochemistry and immunohistochemistry staining techniques with monoclonal antibody anti-γ-tubulin. A total of 4 samples of CS cultured cells were obtained from 3 patients. A recurrence of a histological grade III tumor was detected in a female patient with Ollier's syndrome. The other 2 cases were grade I and III. The incidence of centrosome amplification in the primary cultures ranged from 15-64% of the cells. Whereas control cultured fibroblasts showed baseline levels of 4% amplified cells. For the cryopreserved specimens, two independent observers analyzed each sample and counted the cells stained with γ-tubulin, verifying the percentage of affected cells to be a mean of 14%, with the number of clusters ranging between 0-6 per slide. In conclusion, centrosome amplification was found to be a consistent biological feature of CS and may underlie chromosomal instability in this tumor.
RESUMO
A fotobioestimulação por laser e LED é uma tendência terapêutica inovadora e não invasiva. Os efeitos fotofísicos e fotoquímicos dessa terapia geram imunomodulação, aceleram a cicatrização e angiogênese, bem como reduzem a dor. Dessa forma tem se buscado o emprego desses estímulos no tecido ósseo, porém ainda inexistem padrões definidos para obter a melhor fotobioestimulação nas células ósseas. O objetivo desse trabalho foi avaliar a capacidade da fotobioestimulação na viabilidade celular e mineralização de células da granulação óssea de ratos (rGO). Células rGO na 6ª passagem foram plaqueadas em placas de 96 poços para os ensaios de viabilidade celular (1x10³) e em placas de 24 poços para os ensaios de cicatrização de feridas in vitro (1x104), mineralização e atividade da fosfatase alcalina (FALC) (4x104). As células receberam DMEM (10% SFB) e irradiações com laser (AlGaAs- 660nm e AlGaInP-810nm) e LED (637±15nm). Os grupos experimentais foram: laser vermelho (3 e 5 J/cm²), laser infravermelho (3 e 5 J/cm²) e LED (3 e 5s), além dos grupos controles, positivo (C+) e negativo (C-, 1%SFB). Para os ensaios de mineralização e atividade de fosfatase alcalina, além do meio convencional, grupos com meio osteogênico e os mesmos tratamentos luminosos foram acrescentados. A viabilidade celular foi avaliada pelos testes do MTT e cristal violeta nos períodos de 24, 48, 72 e 96h. O ensaio de cicatrização de feridas in vitro foi avaliado por meio da porcentagem da área de fechamento da ferida nos períodos de 12, 24, 36, 48h. O teste de mineralização foi feito por meio do teste com vermelho de alizarina nos períodos de 14, 21 e 28 dias enquanto que a atividade da FALC foi medida em 7, 14 e 21 dias. A análise estatística foi realizada através dos testes ANOVA complementados por Tukey (p<0,05). Os resultados mostraram que as terapias com luz de maneira geral aumentaram a viabilidade o fechamento da ferida in vitro, principalmente os grupos laser vermelho e LED5s (p<0,05). Pode-se observar um bom desempenho do grupo LED5s no ensaio de mineralização, onde nos grupos que receberam meio osteogênico houve um efeito somatório com a ação da fotobioestimulação promovendo maior produção de nódulos in vitro. Também, as terapias com luz, estimularam a produção de nódulos mineralizados nos grupos que receberam meio convencional de forma a superar o C+ osteogênico (p<0,05), denotando uma ação de indução osteogênica a partir da fotobioestimulação. A fosfatase alcalina foi estimulada pelos tratamentos com luz no período de 7 dias (p<0,05). Em conclusão, as terapias com laser e LED foram capazes de estimular a viabilidade e migração celular e eventos de mineralização em osteoblastos, sendo que o laser vermelho e LED promoveram os melhores resultados.(AU)
Photobiomodulation by laser and LED is a new therapeutic non-invasive trend. Photophysical and photochemical effects occur in immunomodulation, acceleration of wound healing and angiogenesis and reduction of pain. These effects are desired in bone tissue but there are no defined parameters for light irradiation and no consensus for the best effect on osseous cells. The aim of this study was to evaluate photobiomodulation effects on cell viability and mineralization events of rat osseous granulation cells (rGO). Cells in 6th passage were plated in 96-well plates for viability tests (1x10³ cells), and 24-well plates for in vitro wound healing test (1x104 cells), mineralization and alkaline phosphatases (AF) activity (4x104 cells). Cells were cultured in DMEM (10% bovine fetal serum) and irradiation with lasers (AlGaAs-660nm e AlGaInP-810nm) and LED (637±15nm). Experimental groups were red laser (3 and 5 J/cm²), infrared laser (3 and 5 J/cm²), LED (3 and 5s), positive(C+) and negative controls (C-, 1% bovine fetal serum). For mineralization and AF assays, other groups with osteogenic medium and same light treatments were added. Cell viability was evaluated by MTT and crystal violet tests at 24, 48, 72 and 96h. In vitro wound healing test evaluated the percentage of wound closure area by cells migration at 12, 24, 36, 48h. Mineralization test was done by alizarin red at 14, 21 and 28 days. AF activity was measured at 7, 14 and 21 days. Statistical analysis was performed by ANOVA complemented by Tukeys test (p<0,05). Results showed that light therapies in general increased viability and wound healing closure, mostly red laser and LED5s (p<0,05). Best results in mineralization stimulation were observed for LED5s. In groups with osteogenic medium, a synergistic effect of photobiomodulation resulted in higher numbers of mineral nodules. Light groups stimulated higher mineral nodule formation than positive control (p>0.05) even in groups with regular medium, showing an osteogenic induction by light. Increased AF activity was observed at 7 days in light treatment groups (p<0,05). In conclusion, laser and LED photobiostimulation increased viability, cell migration and mineralization events in osteoblasts with best results for red laser and LED.(AU)
Assuntos
Animais , Ratos , Luz , Terapia com Luz de Baixa Intensidade/métodos , Osteoblastos/efeitos da radiação , Fosfatase Alcalina/análise , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Doses de Radiação , Reprodutibilidade dos Testes , Fatores de Tempo , Cicatrização/efeitos da radiaçãoRESUMO
BACKGROUND: Periodontal ligament (PDL) has been reported to be a source of mesenchymal stem cells (MSCs).New vascular networks from undifferentiated cells are essential for repair/regeneration of specialized tissues, including PDL. The current study aims to determine potential of CD105(+)-enriched cell subsets of periodontal ligament cells (PDLSCs) to differentiate into endothelial cell (EC)-like cells and to give insights into the mechanism involved. METHODS: CD105(+)-enriched PDLSCs were induced to EC differentiation by endothelial growth medium 2 (EGM-2) for 3, 7, 14, and 21 days, with mRNA/protein levels and functional activity assessed by: 1) real-time polymerase chain reaction; 2) Western blotting; 3) fluorescence-activated cell sorting; 4) immunohistochemistry; 5) immunofluorescence; 6) matrigel; and 7) small interfering RNA assays. RESULTS: Data analyses demonstrated that EGM-2 treated PDLSCs presented increased expression of EC markers, including: 1) CD105; 2) kinase domain-containing receptor; and 3) Ulex europaeus agglutinin 1, and were able to form cord/tube-like structures. Gene and protein expression analysis showed that neuropilin 2 (NRP2), a key factor for vascular development, was significantly downregulated during EC differentiation. NRP2 was constitutively expressed in mouse PDL tissues by immunohistochemistry analysis, and NRP2 knockdown in CD105(+)-enriched PDLSCs resulted in increased cord/tube-like structures in a matrigel assay. CONCLUSION: These findings demonstrated the potential of CD105(+)-enriched PDLSCs to support angiogenesis, and NRP2 as a pivotal factor regulating this process.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Neuropilinas/fisiologia , Ligamento Periodontal , Animais , Citometria de Fluxo , CamundongosRESUMO
The histopathological hallmarks present in Alzheimer's disease (AD) brain are plaques of Aß peptide, neurofibrillary tangles of hyperphosphorylated tau protein, and a reduction in nicotinic acetylcholine receptor (nAChR) levels. The role of nAChRs in AD is particularly controversial. Tau protein function is regulated by phosphorylation, and its hyperphosphorylated forms are significantly more abundant in AD brain. Little is known about the relationship between nAChR and phospho-tau degradation machinery. Activation of nAChRs has been reported to increase and decrease tau phosphorylation levels, and the mechanisms responsible for this discrepancy are not presently understood. The co-chaperone BAG2 is capable of regulating phospho-tau levels via protein degradation. In SH-SY5Y cell line and rat primary hippocampal cell culture low endogenous BAG2 levels constitute an intracellular environment conducive to nicotine-induced accumulation of phosphorylated tau protein. Further, nicotine treatment inhibited endogenous expression of BAG2, resulting in increased levels of phosphorylated tau indistinguishable from those induced by BAG2 knockdown. Conversely, overexpression of BAG2 is conducive to a nicotine-induced reduction in cellular levels of phosphorylated tau protein. In both cases the effect of nicotine was p38MAPK-dependent, while the α7 antagonist MLA was synthetic to nicotine treatment, either increasing levels of phospho-Tau in the absence of BAG2, or further decreasing the levels of phospho-Tau in the presence of BAG2. Taken together, these findings reconcile the apparently contradictory effects of nicotine on tau phosphorylation by suggesting a role for BAG2 as an important regulator of p38-dependent tau kinase activity and phospho-tau degradation in response to nicotinic receptor stimulation. Thus, we report that BAG2 expression dictates a functional intracellular switch between the p38-dependent functions of nicotine on tau phosphorylation levels via the α7 nicotinic receptor.
Assuntos
Chaperonas Moleculares/metabolismo , Nicotina/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas tau/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
Fibroblasts are cells widely used in cell culture, both for transient primary cell culture or permanent as transformed cell lines. Lately, fibroblasts become cell sources for use in disease modeling after cell reprogramming because it is easily accessible in the body. Fibroblasts in patients will maintain all genetic background during reprogramming into induced pluripotent stem cells. In spite of their large use, fibroblasts are obtained after an invasive procedure, a superficial punch skin biopsy, collected under patient's local anesthesia. Taking into consideration the minimum patient's discomfort during and after the biopsy procedure, as well as the aesthetics aspect, it is essential to reflect on the best site of the body for the biopsy procedure combined with the success of getting robust fibroblast cultures in the lab. For this purpose, we compared the efficiency of four biopsy sites of the body (skin from eyelid, back of the ear, abdominal cesarean scar and groin). Cell proliferation assays and viability after cryopreservation were measured. Our results revealed that scar tissue provided fibroblasts with higher proliferative rates. Also, fibroblasts from scar tissues presented a higher viability after the thawing process.