Kisspeptin (Kp-10) inhibits in vitro osteogenic differentiation of multipotent mesenchymal stromal cells extracted from the bone marrow of adult rats.

Kisspeptin (Kp-10) is a neuropeptide that binds to GPR54 receptors, exerting several functions mainly in the nervous and reproductive systems of the body. However, its effects and mechanisms of action on the skeletal system remain poorly understood. This study evaluated the effects of different concentrations of Kp-10 on in vitro osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) extracted from the bone marrow (BM) of adult Wistar rats. Two-month-old female rats were euthanized to extract BM from long bones to obtain MSCs. Four experimental groups were established in vitro: a control and Kp-10 at concentrations of 0.01, 0.05 and, 0.1 µg/mL. After induction of osteogenic differentiation, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase activity, collagen synthesis, percentage of area covered by MSCs/field and mineralized nodules/field, and immunocytochemistry of the GPR54 receptor tests. Furthermore, evaluation of gene transcripts for type I collagen, Runx-2, Bmp-2, bone sialoprotein, osteocalcin and osteopontin was performed using real-time RT-qPCR. It was observed that MSCs expressed GPR54 receptor to which Kp-10 binds during osteogenic differentiation, promoting a negative effect on osteogenic differentiation. This effect was observed at all the Kp-10 concentrations in a concentration-dependent manner, characterized by a decrease in the activity of alkaline phosphatase, collagen synthesis, mineralized nodules, and decreased expression of gene transcripts for type I collagen, osteocalcin, osteopontin, and Runx-2. Thus, Kp-10 inhibits in vitro osteogenic differentiation of MSCs extracted from the BM of adult Wistar rats.

Effect of the addition of different concentrations of Solanum glaucophyllum desf. extract on chondrocyte cultures from the growth cartilage of newborn rats.

Solanum glaucophyllum Desf. is a calcinogenic plant responsible for enzootic calcinosis that affects ruminants and causes alterations in bone and cartilaginous tissues, among others. It is believed that changes in cartilage tissue, with reduced bone growth, are due to hypercalcitoninism, caused by excess vitamin D. However, we hypothesized that S. glaucophyllum Desf. can act directly on chondrocytes and therefore, chondrocyte cultures from the epiphysis of the long bones of newborn rats were used as a model to elucidate the direct effects of S. glaucophyllum Desf. on bone growth. Plant samples were collected from Cañuelas, Argentina. An aliquot of the plant extract was used to quantify vitamin D (1,25(OH)2D3). The effects of the three concentrations of the plant extract were tested in cultures of chondrocytes extracted from the epiphyses of the long bones of 32 three-day-old Wistar rats. A control group (without extract), and three groups treated with different concentrations of plant extract were formed: group 1 (100 µL/L); group 2 (1 mL/L), and group 3 (5 mL/L), containing respectively 1 × 10-9 M, 1 × 10-8 M, and 5 × 10-8 M of 1,25(OH)2D3. After 7, 14, and 21 days of culture, MTT assay for cell viability, alkaline phosphatase activity, and quantification of the percentage of areas with glycosaminoglycans (GAG) stained with periodic acid-Schiff (PAS) were performed. On day 7, all chondrocytes in group 3, that is, those with the highest concentration of plant extract, died. On days 14 and 21, groups 1 and 2 showed a significant reduction in chondrocyte viability compared to the control. At 7, 14, and 21 days, groups 1 and 2 showed significantly lower alkaline phosphatase activity than the control. On day 21, group 2 showed a significant reduction in areas with PAS + GAGs. There were no significant differences between the groups in the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan. The S. glaucophyllum Desf. extract directly affected growing rat chondrocytes by reducing viability, alkaline phosphatase activity, and GAG synthesis without altering the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan, which may be one of the mechanisms by which there is a reduction in bone growth in animals intoxicated by the plant.

Qualidade do leite cru refrigerado avaliado por diferentes laboratórios credenciados pelo MAPA / Quality of refrigerated raw milk evaluated by different laboratories accredited by MAPA

O objetivo deste trabalho foi analisar e correlacionar os resultados de qualidade do leite cru refrigerado de dez propriedades rurais de Viçosa (MG) obtidos por métodos de referências do Ministério da Agricultura, Pecuária e Abastecimento e por espectrofotômetros de luz infravermelha em três laboratórios (A, B e C) credenciados pelo mesmo órgão de fiscalização. As amostras de leite foram analisadas quanto à contagem bacteriana, contagem de células somáticas (CCS) e teores de gordura, proteína e extrato seco desengordurado (ESD). Não houve discordância de amostras de leite quanto aos teores de proteína e gordura. Porcentagens diferentes de discordância foram observadas entre os resultados dos laboratórios e método de referência quanto à CCS, contagem bacteriana e teores de ESD. Os teores médios de proteína dos laboratórios A e B e os teores médios de ESD de todos laboratórios não foram estatisticamente iguais (p<0,05) aos teores obtidos pelo método de referência. Os teores de gordura obtidos no laboratório B não tiveram correlação estatística (p>0,05) com os teores obtidos pelo método de referência. Observou-se que as CCS e contagens bacterianas médias de todos os laboratórios foram estatisticamente iguais (p>0,05) e correlacionadas (p<0,05) às contagens médias obtidas pelos métodos de referência. Conclui-se que os teores de proteína em dois laboratórios e ESD em todos os laboratórios não são equivalentes com os métodos de referência do MAPA. É importante que os laboratórios revejam a calibração dos seus equipamentos quanto à quantificação dos teores de sólidos do leite.

The objective of this study was to analyze and to correlate the results of refrigerated raw milk quality in ten farms of Viçosa (MG). The milk was analyzed by the Ministério da Agricultura, Pecuária e Abastecimento standard methods and by infrared spectrophotometers in three laboratories (A, B and C) accredited by the same institution. The milk samples were analyzed for bacterial counts, somatic cell count (SCC) and fat, protein and solids not fat (SNF) tenors. There were no disagreement milk samples as the protein and fat tenors. Different percentages of disagreement were observed between the results of laboratory and standard method as the SCC, bacterial count and SNF tenor. The protein mean tenor of A and B laboratories and the SNF mean tenor of all laboratories were not statistically equal (p<0.05) the tenors obtained by standard method. The fat tenors obtained in B laboratory were not statistically correlated (p>0.05) with tenors obtained by the standard method. It was observed that the means of SCC and bacterial counts of all laboratories were statistically the same (p>0.05) and correlated (p<0.05) to the means counts obtained by standard methods. It is concluded that the protein tenors in two laboratories and SNF in all laboratories are not equivalent with the reference methods of MAPA. It is important that laboratories review the calibration of the equipment on the quantification of milk solids tenors.

Canine umbilical cord perivascular tissue: A source of stem cells for therapy and research.

There are numerous sources of multipotent mesenchymal stromal cells (MSC) with therapeutic potential, and bone marrow is the main one. However, pain, lack of donors and comorbidities associated with harvesting stimulate the search for new sources of MSCs. The aim of this work is to obtain cells from umbilical cord (UC) perivascular tissue of dogs and characterize them as MSCs. For this, the UC was obtained from therapeutic cesarean sections and submitted to enzymatic digestion. The obtained cells were subjected to growth and proliferation tests, as well as the analysis of surface markers, differentiation test in three mesenchymal lineages and analysis of differentiation markers expression. From all the UC used in this study an adherent with fibroblastoid shape cell was obtained, with an initial number of 4.8 × 105 of cells. The growth curves showed a lag phase from 0 to 24 h, followed by a phase of growth of 24 to 168 h, and then phase of cell decay. The doubling time was kept around 15 h until the sixth passage, from which there were signs of cellular senescence. The differentiation assays demonstrated the ability of cells to differentiate into osteoblasts, adipocytes and chondrocytes when subjected to the induction mediums. The study of surface markers was positive for adhesion markers and negative for hematopoietic markers. Thus, cells obtained from canine UC perivascular tissue by enzymatic digestion are multipotent MSC and the protocol developed ensures the perivascular origin of these cells.