Alteraciones hematológicas en sangre periférica de pacientes con COVID-19 / Molecular study of leukemias: essential tool for the hematologist

Introducción: Los primeros casos con neumonía atípica de etiología desconocida fueron reportados en Wuhan, China en diciembre de 2019. En enero 2020 se describió como agente causal un nuevo tipo de virus de la familia Coronaviridae, denominado SARS-CoV-2. Objetivo: Evaluar la significación clínica de los cambios hematológicos y morfológicos en la sangre periférica de pacientes con COVID-19. Métodos: Se realizó un estudio descriptivo, observacional, transversal que incluyó a los pacientes con COVID-19 que ingresaron en el Hospital Clínico Quirúrgico Docente Freyre de Andrade desde el 1ro de junio hasta 31 de septiembre de 2021. Los pacientes fueron asignados a dos grupos según fueron admitidos, en las unidades de vigilancia intensiva o en la unidad de cuidados intensivos. Se les realizó hemograma completo y lámina periférica el día del ingreso para evaluar la significación clínica de estas variables en la evolución de estos pacientes. Resultados: El sexo femenino predominó en los pacientes ingresados en unidades de vigilancia intensiva (67,36 %) y el masculino en los ingresados en unidades de cuidados intensivos (63,26 %). La media de edad fue mayor en el grupo de pacientes en cuidados intensivos (65,83 años). La leucocitosis y el menor recuento de plaquetas predominaron en los pacientes ingresados en cuidados intensivos, seguido de linfopenia. Las macroplaquetas, las vacuolas citoplasmáticas y las granulaciones tóxicas fueron más frecuentes en el grupo de cuidados intensivos. Conclusiones: El hemograma y el frotis de sangre periférica son útiles para diagnosticar y predecir la evolución de los pacientes y permiten un mejor manejo de la infección.

Introduction: The first cases of atypical pneumonia of unknown etiology were reported in Wuhan, China in December 2019. In January 2020 a new virus from Coronaviridae family was described as causal agent and was named SARS-COV-2. Objectives: To evaluate the clinical significance of numerical values of complete blood count (CBC) and morphologic changes on peripheral blood on patients with COVID-19. Methods: A descriptive, observational, transversal study included patients with diagnosis of COVID-19 admitted in Freyre de Andrade Hospital in Havana, between June 1st and September 31st of 2022 was carried out. Patients were assigned to two groups according to their admission in intensive vigilance ward or intensive care unit. CBC test and peripheral blood smear were performed on admission day to evaluate the significance on clinical evolution. Results: Female sex predominated on intensive vigilance group (67,36 %) and male in intensive care group (63,26 %). Media of age was superior in intensive care group (67,83 years). Leukocytosis and low level of platelets count were significantly more common in more severe group followed by lymphopenia. The presence of big platelets, cytoplasmic vacuoles and toxic granules were more common in intensive care unit group. Conclusions: The CBC and peripheral blood smear are useful tools to diagnose and predict clinical evolution and allow a better management of infection.

Cytocentrifugation as an additional method to study echinoderm coelomocytes: a comparative approach combining living cells, stained preparations, and energy-dispersive x-ray spectroscopy

Introduction: Echinoderm coelomocytes have traditionally been investigated through a morphological approach using light microscopy, which relies on the idea of constant cell shape as a stable character. However, this can be affected by biotic or abiotic conditions. Objective: To analyze if the consistency in cell morphology offered by the cytocentrifugation method, might be used as a convenient tool to study echinoderm coelomocytes. Methods: Cells of Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus, and Echinometra lucunter (Echinoidea) were spread on microscope slides by cytocentrifugation, stained, and analyzed through light microscopy. Additionally, fluorescence microscopy, scanning electron microscopy, and energy-dispersive x-ray spectroscopy were applied to cytospin preparations, to complement the analysis of granular and colorless spherulocytes of Eucidaris tribuloides. Results: Altogether, 11 cell types, including phagocytes, spherulocytes, vibratile cells, and progenitor cells were identified in the samples analyzed. The granular spherulocyte, a newly-described cell type, was observed in all Echinoidea and was very similar to the acidophilic spherulocytes of Holothuria (Holothuria) tubulosa. Conclusions: Cytocentrifugation proved to be versatile, either as the main method of investigation in stained preparations, or as a framework on which other procedures may be performed. Its ability to maintain a constant morphology allowed accurate correspondence between live and fixed/stained cells, differentiation among similar spherulocytes as well as comparisons between similar cells of Holothuroidea and Echinoidea.

Introducción: Los celomocitos de equinodermos se han investigado tradicionalmente a través de un enfoque morfológico utilizando microscopía óptica, que se basa en la idea de la forma celular constante como un carácter estable. Sin embargo, esto puede verse afectado por condiciones bióticas o abióticas. Objetivo: Analizar si la consistencia en la morfología celular que ofrece el método de citocentrifugación podría utilizarse como una herramienta conveniente para estudiar los celomocitos de equinodermos. Métodos: Células de Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus y Echinometra lucunter (Echinoidea) se esparcieron en portaobjetos de microscopio por citocentrifugación, se tiñeron y analizaron mediante microscopía óptica. Adicionalmente, se aplicó microscopía de fluorescencia, microscopía electrónica de barrido y espectroscopía de rayos X con dispersión de energía a las preparaciones de citoespina, para complementar el análisis de los esferulocitos granulares e incoloros de Eucidaris tribuloides. Resultados: En total, se identificaron en las muestras analizadas 11 tipos de células, incluidos fagocitos, esferulocitos, células vibrátiles y células progenitoras. El esferulocito granular, un tipo de célula recién descrito, se observó en todos los Echinoidea y fue muy similar a los esferulocitos acidófilos de Holothuria (Holothuria) tubulosa. Conclusiones: La citocentrifugación demostró ser un método bastante versátil, ya sea como el método principal de investigación en preparaciones teñidas o como un marco en el que se pueden realizar otros procedimientos. Su capacidad para mantener una morfología constante permitió una correspondencia precisa entre las células vivas y las células fijas/teñidas, la diferenciación entre esferulocitos similares, así como comparaciones entre células similares de Holothuroidea y Echinoidea.

Peritoneal fluid collection in healthy cattle and evaluation of changes in cell morphology during storage in refrigerated and non-refrigerated samples / Colheita do fluido peritoneal de bovinos sadios e verificação das alterações na morfologia celular quando mantido refrigerado ou à temperatura ambiente

This study aimed to evaluate the appropriate sites of abdominocentesis for peritoneal fluid collection in cattle and to investigate the time of cell viability in vitro, comparing three methods of sample conservation. Twenty-one healthy cattle (19 females and 2 males) were subjected to a laparocentesis procedure to obtain peritoneal fluid, with punctures in three defined sites: left cranial, right cranial, and right caudal. The total peritoneal fluid collected was divided into three aliquots and maintained under three preservation conditions: room temperature (26°C), refrigeration (4°C), and room temperature (26°C) with the addition of 1µL of 10% formaldehyde per 1mL of peritoneal fluid. The peritoneal fluid analysis performed immediately after collection consisted of: physical examination (color, appearance, volume, and specific gravity), biochemical measures (pH, total protein, fibrinogen, creatinine, and glucose), and cellularity (total and differential counts). The determination of proteins and the examination of cells were repeated in each separate aliquot at two, four, six, and eight hours after harvest. Data were analyzed through repeated measures ANOVA or Friedman test. The harvest was productive in 67% of cattle. The left cranial and the right cranial puncture sites were the most appropriate. Peritoneal fluid analyzed after collection, the total protein concentration ranged from 1.4 to 3.6g/dL, and number of leukocytes ranged from 54 to 1,322 cells/µL; 60 to 95% of leukocytes were lymphocytes. The protein concentration decreased, but the absolute values of leukocytes, lymphocytes, and segmented neutrophils did not change up to eight hours after collection, independent of the maintenance method. Cell lysis was delayed by cooling, and the addition of formaldehyde did not help preserve the integrity of cellular morphology. Laparocentesis is a safe and secure procedure in cattle and maybe more productive when performed in specific sites on the left or right sides of the cranial abdominal wall. Peritoneal fluid samples may be analyzed with reliable results for up to eight hours after collection when kept refrigerated and for up to six hours when kept at room temperature.(AU)

O estudo teve como objetivo avaliar os locais adequados de laparocentese para a colheita de fluido peritoneal de bovinos e estabelecer o tempo de viabilidade celular in vitro, comparando três métodos de conservação. Vinte e um bovinos hígidos (19 fêmeas e 2 machos) foram submetidos ao procedimento de laparocentese para obtenção de fluido peritoneal, com punção em três pontos definidos: cranial esquerdo, cranial direito e caudal direito. O volume total do líquido peritoneal foi dividido em três alíquotas mantidas sob três métodos de conservação: temperatura ambiente (26°C); refrigeração (4°C); e temperatura ambiente (26°C) com adição de 1µL de formol 10% para cada 1mL de líquido peritonial. A análise do líquido peritoneal realizada imediatamente após sua obtenção consistiu em: exames físico (cor, aspecto, volume e densidade); bioquímicos (pH, proteína total, fibrinogênio, creatinina e glicose); e da celularidade (contagens total e diferencial). A determinação de proteínas e o exame da celularidade foram repetidos, em cada alíquota separada, as duas, quatro, seis e oito horas após a colheita. Análise de variâncias de medidas repetidas ou teste de Friedman foram empregados para avaliação ao longo do tempo. A colheita foi produtiva em 67% dos bovinos e os locais de punção craniais esquerdo e direito foram os mais adequados. A concentração de proteína total variou de 1,4 a 3,6g/dL e o número de leucócitos de 54 a 1.322 células/µL, com predomínio de linfócitos (60 a 95% das células) no fluido peritoneal analisado logo após a colheita. A concentração de proteínas diminuiu, mas os valores absolutos de leucócitos, de linfócitos e de neutrófilos segmentados não se modificaram até oito horas após a colheita, independente do método de manutenção das amostras. A lise celular foi retardada pela refrigeração e a adição de formol não contribuiu para preservar a integridade da morfologia celular. A laparocentese é um procedimento seguro e de execução fácil em bovinos sendo mais produtiva quando realizada em locais específicos à esquerda ou à direita craniais da parede abdominal. Amostras de fluido peritoneal podem ser analisadas com resultados confiáveis quando mantidas refrigeradas por até oito horas após a colheita e quando mantidas à temperatura ambiente por até seis horas.(AU)

MLO-Y4 osteocytic cell clones express distinct gene expression patterns characteristic of different stages of osteocyte differentiation / Clones de las células osteocíticas MLO-Y4 tienen diferentes patrones de expresión génica característicos de diferentes estadíos de diferenciación osteocítica

Osteocytes are the most abundant bone cell and are formed when osteoblasts become embedded in the bone matrix. Through changes in gene expression and paracrine effects, osteocytes regulate the number of osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, which are needed to maintain bone mass. MLO-Y4 is the better characterized osteocytic cell line; however, lacks expression of sclerostin, the product of the SOST gene, which is fundamental for osteocyte function and blocks bone formation. With the objective to isolate MLO-Y4 clones with different gene expression profiles, we performed cultures at very low density of MLO-Y4 cells stably transfected with nuclear green fluorescent protein (MLOnGFP). Cell morphology was visualized under a fluorescence microscope. Once the cells reached 80% confluency, RNA was extracted and quantitative real time PCR was performed. Clones exhibit different sizes and morphology, with some cells showing a spindle-like shape and others with abundant projections and a star-like shape. Gene expression also differed among clones. However, none of the clones examined expressed SOST. We conclude that the MLO-nGFP clones constitute a useful tool to study osteocyte differentiation and the role of osteocytes in the control of bone formation and resorption in vitro. (AU)

Los osteocitos son las células más abundantes del hueso y se forman cuando los osteoblastos se encuentran rodeados de matriz ósea. A través de cambios en la expresión génica y efectos paracrinos, los osteocitos controlan el número de osteoblastos que forman el hueso, y osteoclastos que resorben el hueso, células necesarias para mantener la masa ósea. Las células MLO-Y4 son la línea celular osteocítica más investigada; sin embargo, no expresan esclerostina, el pro esclerostina, el producto del gen SOST que bloquea la formación ósea y es indispensable para la función de los osteocitos. Con el objetivo de aislar clones de las células MLO-Y4 con diferentes perfiles de expresión génica, realizamos cultivos a muy baja densidad de las células transfectadas en forma estable con proteína verde fluorescente nuclear (MLO-nGFP). La morfología celular fue evaluada utilizando un microscopio de fluorescencia. Una vez que las células alcanzaron el 80% de confluencia, el ARN fue extraído y analizado por PCR cuantitativa en tiempo real. Las células de los diferentes clones tienen diferentes tamaños y morfología, algunas células son fusiformes y otras con proyecciones citoplasmáticas abundantes y en forma de estrella. La expresión de los genes también varió en los distintos clones. Sin embargo, ninguno de ellos expresó SOST. En conclusión, los clones de las células MLO-nGFP constituyen una herramienta útil para estudiar la diferenciación de los osteocitos y el rol de estas células en el control de la formación y resorción ósea in vitro. (AU)

Giant Anteater (Myrmecophaga tridactyla Linnaeus, 1758) of the Brazilian cerrado: hematology and storage effect / Tamanduás-bandeiras (Myrmecophaga tridactyla Linnaeus, 1758) do cerrado brasileiro: hematologia e efeito de estocagem

Giant Anteater (Myrmecophaga tridactyla) is a vulnerable species because of progressive habitat destruction, mostly affected by wildfires and car accidents. The increasing number of animals that are attended by wildlife rescue centres reinforces the need of knowledge about haematological parameters, especially for those that inhabit Brazilian cerrado biome. For this purpose and in order to establish reference values for this species and also to compare them with previous studies, haematological analysis of captive giant anteaters from Brazilian cerrado were performed. Moreover, the alterations of blood samples after 24 and 48 hours of storage at refrigeration temperatures (4oC) and preserved with two different EDTA concentrations (5% and 10%) were studied. Means and standard deviations of haematological parameters analysed immediately after collection were: RBC: 2,07 x106/µL ± 0,40; hematocrit: 38,08%± 5,93; haemoglobin: 11.33g/dL±2.15; MCV:186.52 fL±21.72; MCHC: 29.68g/dL±2.56; MCH: 55.08pcg±5,94; total leucocytes: 8.142/µL±2.441; neutrophils: 5.913/µL±2.168; lymphocytes: 1.460/µL±740; eosinophil: 522/µL±385; monocytes: 247/µL±176; thrombocytes: 123.458/µL±31.362 and total plasma protein: 6.23g/dL±0.49. This data shows evidence of the existence of important differences between these values and others from other areas, either from Brazil or from other South American countries. Those variations might be connected to environment, genetic, nutritional and/or management factors. Regarding the storage effect analysis, it can be concluded that in giant anteaters, haematological analysis can be performed until 24h after collection without any significant alterations on the haematological parameters, except for thrombocytes. Concerning the different EDTA concentrations, it can be concluded that there are no quantitative differences in haematological variables. Nevertheless, relevant morphologic alterations in blood cells can be observed after a 24h storage period, being most noticeable in the leucocytes. Those alterations can lead to misinterpretation of the results, interfering diagnosis, prognosis and treatment.(AU)

O tamanduá-bandeira (Myrmecophaga tridactyla) é uma espécie vulnerável devido à destruição progressiva do seu habitat natural, sendo afetado por queimadas e atropelamentos. O aumento na casuística de atendimentos de animais silvestres reforça a necessidade de se ter conhecimento dos parâmetros hematológicos, em especial para os que vivem no bioma do cerrado. Por isso, este trabalho teve por objetivos realizar o hemograma de tamanduás-bandeiras de cativeiro localizados no cerrado brasileiro, a fim de estabelecer valores de referência para essa espécie e compará-los a estudos prévios. Além disso, verificar quais alterações podem ser encontradas em amostras armazenadas por 24 e 48 horas após a colheita, em temperatura de refrigeração (4oC) e tratadas com duas concentrações distintas de EDTA (5% e 10%). A média e o desvio padrão das variáveis hematológicas encontradas nas amostras processadas logo após a colheita foram: hemácias (2,07x106/µL±0,40); volume globular (38,08%± 5,93); hemoglobina (11,33g/dL±2,15); VCM (186,52 fL±21,72); CHCM (29,68g/dL±2,56); HCM (55,08pcg±5,94); leucócitos totais (8.142/µL±2.441); neutrófilos (5.913/µL±2.168); linfócitos (1.460/µL±740); eosinófilos (522/µL±385); monócitos (247/µL±176); plaquetas (123.458/µL±31.362) e proteínas plasmáticas totais (6,23g/dL±0,49). Tais dados permitem afirmar que existem importantes diferenças entre os valores hematológicos destes em relação aos animais provenientes de outras regiões, tanto do Brasil quanto de outros países da América do Sul. Provavelmente, tais divergências estão associadas a fatores ambientais, genéticos, nutricionais e/ou de manejo. Quanto à análise das amostras estocadas, conclui-se que, em tamanduás-bandeiras, as amostras para a realização de hemograma podem ser processadas até 24 horas após a colheita, sem alteração significativa das variáveis hematológicas, com exceção das plaquetas. Com relação às duas concentrações de EDTA, pode-se inferir que não há diferença quantitativa entre ambas para as variáveis hematológicas. Contudo, é possível constatar que o EDTA promove alterações morfológicas relevantes nas células sanguíneas após 24 horas de armazenamento, sendo os leucócitos os mais afetados. Tais alterações, quando relatadas, podem induzir interpretações equivocadas, interferindo no diagnóstico, prognóstico e tratamento.(AU)

Evaluación de la citotoxicidad de tres cementos selladores endodóncicos utilizados en cirugía periapical: estudio in vitro / Cytotoxicity assessment of three endodontic sealing cements used in periapical surgery. In vitro study

Introducción: Existen diversos materiales de retroobturación, pero poco se sabe de su toxicidad sobre fibroblastos gingivales. Objetivo: Evaluar la citotoxicidad de tres materiales de retroobturación sobre fibroblastos gingivales humanos y fibroblastos de la línea L929. Material y métodos: Los medios condicionados de los materiales de retroobturación EndoSequence® BC RRMTM (ERRM), trióxido mineral agregado MTA Angelus® blanco (MTA) y material de restauración intermedia (IRM®) se obtuvieron en fresco, al tiempo de fraguado, y después de 1, 24 y 72 horas del tiempo de fraguado. La morfología celular fue evaluada por microscopia de luz y la viabilidad celular fue evaluada a través de la actividad metabólica mitocondrial con 3-(4,5-dimetiltiazol-2-il)-2,5-difenil bromuro de tetrazolio (MTT). El análisis estadístico se realizó por ANOVA. Resultados: El material ERRM no mostró efectos citotóxicos sobre los fibroblastos. Sin embargo, el MTA y el IRM® mostraron citotoxicidad moderada y alta, respectivamente. Esto revela que el MTA y el IRM® no son completamente inocuos. Conclusión: Los materiales biocerámicos como el ERRM pueden ser considerados los materiales de retroobturación más biocompatibles.

Introduction: Presently there are many retrofilling materials in themarket, nevertheless, little is known about their toxicity on gingival fibroblasts. Objective: To assess cytotoxicity of three materials tohuman gingival fibroblasts and L929 mouse fibroblasts cell line. Material and methods: EndoSequence® BC RRMTM (ERRM; rootrepair material), white MTA Angelus® (MTA) and intermediaterestoration material (IRM®) conditioned media were obtained whenmaterials were freshly mixed, at setting time and after 1, 24 and72 hours of setting time. Cell morphology was assessed with lightmicroscopy and cell viability was assessed through mitochondrialmetabolic activity with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Statistical analysis was conducted withANOVA. Results: We found that ERRM material did not exhibitcytotoxic effects on used fibroblast, nevertheless, MTA and IRM® respectively exhibited moderate and severe cytotoxicity, thusindicating the materials were not fully harmless. Conclusion: Bioceramic cements like ERRM could be considered the mostcompatible retrofilling-materials.

Morphological analysis of peripheral blood cells of Chelonoidis carbonaria (Spix, 1824) / Análise morfológica das células do sangue periférico de Chelonoidis carbonaria (Spix, 1824)

This present work describe the peripheral blood cell morphology from Chelonoids carbonaria. To do this were used ten animal specimens clinically healthy, six female and four male, submitted to peripheral blood collect by jugular vein. Blood was collected to prepare blood smears, without the use of anticoagulants. The slides were stained and analyzed microscopically to describe the cell morphology. The mature erythrocytes had an ellipsoid shape and a grain-free nucleus; immature ones were circular. The leukocytes, granulocytes and agranulocytes were also circular. The heterophils had cytoplasmic granules with various elongated shapes, and the eosinophils had a uniform round shape. The basophils had highly dense basophilic granules, stained in blue; the granules were irregularly arranged and also inside the nucleus. The lymphocytes were circular with a large circular nucleus. The thrombocytes were small, with basophilic staining and a small cytoplasm (the nucleus occupied almost the entire cell). The morphological results found in this study are consistent with cell types of other chelonians.

O presente estudo descreve a morfologia dos constituintes celulares do sangue periférico de Chelonoides carbonaria. Para tanto, 10 espécimes adultos, sendo seis fêmeas e quatro machos, clinicamente saudáveis foram submetidos à coleta de sangue periférico através da veia jugular. O sangue foi recolhido para preparar esfregaços sanguíneos, sem a utilização de anticoagulantes. As lâminas foram coradas e analisadas microscopicamente para descrever a morfologia da célula. Os eritrócitos maduros têm a forma elipsóide e apresentam núcleo central sem granulações; nas fases imaturas mostram-se arredondados. Os leucócitos, granulócitos e agranulócitos, também são circulares. Os heterófilos possuem grânulos citoplasmáticos com várias formas alongadas e nos eosinófilos são uniformes e arredondados. Os basófilos possuem grânulos altamente densos e basofílicos, corados em azul; os grânulos são dispostos de forma irregular e também no interior do núcleo. Os linfócitos são circulares com um grande núcleo circular. Os trombócitos são pequenos, com coloração basofílica e citoplasma escasso (o núcleo ocupa quase toda a célula). Os resultados encontrados nesta pesquisa são compatíveis com a morfologia encontrada nesses tipos celulares em outros quelônios.

Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture / Morfologia e morfometria das células-tronco mesenquimais isoladas de medula óssea de gato

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x106) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.(AU)

As células tronco mesenquimais são utilizadas na terapia de várias doenças na medicina humana e veterinária. As células tronco foram isoladas da medula óssea de gato, entretanto, existem poucos dados referentes a morfologia e não existem informações sobre a morfometria das células tronco isoladas da medula óssea. Os objetivos do presente estudo foram o isolamento, avaliação do crescimento, potencial de diferenciação e caracterização morfológica e morfométrica das células mesenquimais de gato isoladas de medula óssea. A diferenciação in vitro foi realizada para confirmar a multipotencialidade das células mesenquimais de gato (diferenciação em osteoblastos, condrócitos, adipócitos). As células mesenquimais foram mantidas em cultivo para avaliações morfológica e morfométrica. As células foram coradas e observadas em microscopia ótica. As mensurações foram realizadas com 24, 48, 72 e 120h de cultura (primeira e terceira passagens). O teste não paramétrico ANOVA foi utilizado e as médias foram comparadas pelo teste de Tukey. O número médio de células mononucleares obtido foi de 12,29 (±6,05x106) células/mL de medula óssea. As células mesenquimais são longas e fusiformes, e escamosas com citoplasma abundante. No estudo morfométrico, observou-se aumento no comprimento médio das células durante a primeira passagem. As medidas de comprimento das células foram: 106,97±38,16µm e 177,91±71,61µm, respectivamente, na primeira e terceira passagens (24 horas). As medidas de largura das células foram: 30,79±16,75 µm e 40,18±20,46 µm, respectivamente, na primeira e terceira passagens (24 horas). O comprimento do núcleo na primeira passagem aumentou de 16,28µm (24h) para 21,29µm (120h) e na terceira passagem foi de 26,35µm (24h) para 25,22µm (120h). As informações são importantes para futuras aplicações e uso da célula mesenquimal de gato.(AU)

Isolamento e caracterização de células mesenquimais do tecido adiposo de cães / Isolation and characterization of canine adipose-derived mesenchymal stem cells

As células-tronco mesenquimais (CTMs) diferenciam-se em várias linhagens e têm potencial de utilização na medicina regenerativa. As CTMs podem ser isoladas de vários tecidos de animais adultos. O objetivo deste estudo foi o isolamento das CTMs do tecido adiposo de cães, seu cultivo e diferenciação. Foram coletadas amostras de tecido adiposo subcutâneo de cinco cães. As CTMs foram isoladas, obtendo-se 146.803 (±49.533) células/g, cultivadas e diferenciadas em osteoblastos, adipócitos e condrócitos. Avaliaram-se a cinética do crescimento, a morfologia e a viabilidade celular. A caracterização citoquímica comprovou a natureza mesenquimal das células isoladas. O cultivo foi iniciado com 20.000 células/mL, verificando-se crescimento rápido até 72 horas (220.000 células/mL), fase exponencial entre 72 e 192 horas (455.000 células/mL), seguida de platô por saturação da densidade com 240 horas (355.000 células/mL). A viabilidade celular variou entre 96 e 100%. As CTMs em cultivo são fibroblásticas, fusiformes, com citoplasma basofílico e núcleo esférico. O comprimento médio das células variou entre 80,85 e 98,36µm, a largura média entre 17,40 e 28,79µm e o diâmetro médio do núcleo entre 15,46 e 17,74µm.

The applications of mesenchymal stem cells (MSCs) are becoming increasingly more promising for regenerative medicine and tissue engineering fields. MSCs can be isolated from adult animals from a variety of tissues, such as the adipose. This study focused on the isolation, culture and differentiation of MSCs from canine adipose tissue. Samples of subcutaneous adipose tissue from five dogs were collected. These cells were isolated, cultured and differentiated into osteoblasts, adipocytes and chondrocytes. We obtained 146,803 (±49,533) cells/g. Growth kinetics and viability studies were conducted during cell culture and the evaluation of cell differentiation was successfully performed by cytochemistry. The cell cultures were initiated with 20,000 MSCs/ml. Rapid growth was observed at 72 hours (220,000 cells/ml), the exponential phase between 72 and 192 hours (455,000 cells/ml) and saturation at 240 hours (355,000 cells/ml). The cellular viability ranged from 96 to 100%. MSCs in culture are fibroblastic cells, fusiform with basophilic cytoplasm and spherical nucleus. The length and width means of the cells and nuclear diameter ranged from 80.85-98.36µm, 17.40-28.79µm and 15.46-17.74µm respectively.