Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Brossi P.M., Baccarin R.Y.A. & Massoco C.O. 2012 Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro? Pesquisa Veterinaria Brasileira 32(12):1355-1360. Departamento de Clinica Medica, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Butanta, Sao Paulo, SP 5508-210, Brazil. E-mail: baccarin@ usp.br Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)(4) - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P<0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium

Viability of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction after freezing and thawing process

Cinco cavalos adultos foram submetidos à coleta de medula óssea do esterno e de tecido adiposo da região glútea. As amostras foram processadas para obtenção da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo, o número de células obtidas e a viabilidade celular foram determinados. Em seguida, realizou-se o congelamento das amostras em solução contendo 20% de soro fetal bovino e 10% de dimetilsulfóxido. Depois de um mês, realizou-se o descongelamento das amostras e a viabilidade celular foi novamente mensurada. Os resultados revelaram que as técnicas utilizadas tanto para coleta de medula óssea quanto de tecido adiposo em equinos são simples, rápidas e seguras. As metodologias adotadas para o processamento das amostras foram eficientes, obtendo-se aproximadamente 95% de viabilidade celular. Após o descongelamento, a viabilidade média das amostras de células mononucleares da medula óssea foi de 86% e da fração vascular estromal do tecido adiposo de 64%. Frente à importância da terapia celular na clínica médica de equinos, concluiu-se que é necessária a realização de mais estudos, visando padronizar uma técnica de criopreservação que mantenha a integridade das células da fração mononuclear da medula óssea e da fração vascular estromal do tecido adiposo de equinos

Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment

Abstract\ud \ud Background\ud Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank.\ud \ud \ud Methods\ud Expression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-Cnu/nu mice to verify the safety of the MSCs from these sources.\ud \ud \ud Results\ud Cultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects.\ud \ud \ud Conclusions\ud SF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity.This research was supported by Fundação Coordenação de Aperfeiçoamento\ud de Pessoal de Nível Superior (CAPES), Brasília, SP, Brazil, and by Fundação de\ud Amparo à Pesquisa do Estado de São Paulo (FAPESP), São Paulo, SP, Brazil.\ud These sponsors did not have any influence on the study design, on the\ud collection, analysis and interpretation of data, or on the writing of the\ud manuscript and decision to submit for publication

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro? Os componentes do sangue afetam a produção de (ERO) pelas células sinoviais de equinos in vitro?

Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)¹ - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (PProdutos derivados do sangue são comumente usados, tanto no homem como em cavalos, para tratar diversas doenças musculoesqueléticas, principalmente devido a seus efeitos antioxidantes e anti-inflamatórios. Contudo, efeitos antioxidantes nunca foram demonstrados em células de líquido sinovial in vitro. Caso esses efeitos sejam efetivamente demonstrados, uma nova perspectiva para justificar a aplicação clínica desses produtos poderia surgir. Sendo assim, o objetivo do presente estudo foi investigar os efeitos antioxidantes de dois produtos derivados de sangue - plasma (produto de sangue não condicionado - UBP) e uma preparação comercial de sangue (produto de sangue condicionado - CBP)¹ - sobre células estimuladas extraídas do líquido sinovial de equinos. Articulações tibiotársicas saudáveis (60) foram puncionadas para a obtenção de células de líquido sinovial; essas células foram concentradas, processadas, estimuladas com lipopolissacarídeos (LPS) ou forbol 12-miristato 13-acetato (PMA), e avaliadas por citometria de fluxo quanto à produção de espécies reativas de oxigênio (ROS). Após a adição de ambos os produtos derivados do sangue - UBP ou CBP - ocorreu uma significativa queda no burst oxidativo em células do líquido sinovial (P<0,05). Não houve diferença entre os efeitos de UBP e CBP. Em conclusão, tratamento de células estimuladas do líquido sinovial de equinos com UBP ou CBP restaurou eficientemente o equilíbrio redox das células

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.O objetivo deste estudo foi avaliar o cultivo de células da fração mononuclear da medula óssea e da fração vascular estromal do tecido adiposo de equinos em dois diferentes meios. Cinco cavalos foram submetidos à aspiração da medula óssea do esterno e à coleta de tecido adiposo da região glútea, próxima à inserção da cauda. A fração mononuclear e a fração vascular estromal foram obtidas das amostras e cultivadas em meio DMEM suplementado com soro fetal bovino a 10% ou em meio AIM-V. As culturas foram observadas uma vez por semana com um microscópio de luz invertida, com o intuito de se realizar uma análise qualitativa das características morfológicas das células, bem como do aspecto geral do cultivo celular. As unidades formadoras de colônia (CFU) foram contadas nos dias 5, 15 e 25 do cultivo celular. Durante a primeira semana, foram observadas diferenças entre amostras obtidas de mesma origem mantidas em diferentes meios. O número de colônias foi significativamente maior nas amostras de medula óssea em relação às amostras de tecido adiposo

Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules.

There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development