RESUMO

This prospective, comparative, randomized, horizontal, and double-blind clinical study investigated the clinical efficacy of leucocyte-poor platelet-rich plasma (PRP, n=8) or allogeneic adipose-derived stem cells (ADSC, n=8) in dogs with bilateral degenerative hip joint disease (DHJD). Sixteen dogs were treated with two intra-articular injections of PRP or ADSCs, within a 30-day interval. The Canine Brief Pain Inventory (CBPI), the Helsinki Chronic Pain Index (HCPI), and Visual Analogue Scales for pain (VAS-pain) and locomotion (VAS-loc) were assessed by the dog owners. Analysis-of-gait using a force plate, response to palpation (VAS-palp), and the descriptive numerical scale for pain (DNS) were measured by a veterinarian. The assessments were performed before (baseline), 30 and 60 days after the first treatment. Data were analyzed using the unpaired t test, paired Wilcoxon test, Fisher's exact test, and Mann-Whitney and Friedman tests (P<0.05). Compared with baseline HCPI, CBPI, VAS-pain, and VAS-palp scores reduced 41%, 52%, 51%, and 48% (P=0.0001-0.03) at 60 days in the ADSC group. In PRP-treated dogs, CBPI, VAS-loc, and DNS scores decreased by 43%, 43%, and 33% at 60 days, respectively (P=0.0003-0.011). Based on CBPI data, the rate of success at 60 days was 75% and 25% in the ADSC and PRP groups (P=0.13), respectively. Both therapies were apparently safe and effective to reduce chronic pain in dogs with bilateral DHJD during a 60-day period. However, a trend towards greater improvement was provided by the ADSC treatment.

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Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.

Assuntos

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Cell culture is an excellent alternative for the maintenance of cell lines and to obtain quality chromosome preparations of fishes. However, this methodology is still little employed, mainly because of the difficulty of standardization of cell cultures. In this study, we describe a methodology for the rapid acquisition of cell lineages and mitotic chromosomes for cytogenetic studies of fish species from muscle tissue cells. Our methodology is based on the use of a gelatin film, which provides better adhesion of a large number of cells and appropriate conditions for multiplication. The cells of Astyanax altiparanae, used as an experimental model, with fibroblast-like morphology, showed rapid cellular proliferation, resulting in a great number of cells. Chromosomal preparations of cultured cells showed the diploid number of the species, 2n = 50 chromosomes, in 80% of the cells examined, with chromosomes intact and distended. Cell populations were cryopreserved and after being recovered, these cells maintained their proliferative capacity. The development of this methodology represents an innovation for the fish cytogenetics area and it may bring a significant contribution to the conservation and study of several groups due to the difficulty of obtaining good-quality chromosome preparations.

RESUMO

BACKGROUND: The injection of mesenchymal stem cells (MSCs) directly into the bone of osteoporotic (OP) patients for rapid recovery has been studied worldwide. Scaffolds associated with MSCs are used to maintain and avoid cell loss after application. A unique heterologous fibrin sealant (HFS) derived from snake venom was evaluated for the cytotoxicity of its main components and as a three-dimensional biological scaffold for MSCs to repair a critical femur defect in osteoporotic rats. METHODS: The cytotoxicity of HFS was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay and transmission electron microscopy. The cells were cultured, characterized by flow cytometry and differentiated into the osteogenic lineage. Two-month-old rats underwent ovariectomy to induce OP. After 3 months, a 5 mm critical bone defect was made in the distal end of the rat femurs and filled with HFS; HFS + MSCs; and HFS + MSCs D (differentiated into the osteogenic lineage) to evaluate the effects. An injury control group (injury and no treatment) and blank control group (no injury and no treatment) were also included. The animals were observed at days 14 and 28 by microtomographic (micro-CT) analyses, histologic and biochemical analysis, as well as scanning electron microscopy. RESULTS: The results revealed that one of the compounds of HFS, the thrombin-like enzyme extracted from snake venom, had no cytotoxic effects on the MSCs. OP was successfully induced, as demonstrated by the significant differences in the levels of 17ß-estradiol, Micro-CT analyses and alkaline phosphatase between the ovariectomized (OVX) and non-ovariectomized (NOVX) groups. The histological data revealed that at 14 days after surgery in both the OVX and NOVX animals, the HFS + CTMs and HFS + CTMsD showed a higher formation of bone cells at the site in relation to the control group (without treatment). Collagen formation was evidenced through bone neoformation in all treated and control groups. No morphological differences in the femurs of the NOVX and OVX animals were observed after the surgical procedure. Scanning electron microscopy (SEM) confirmed the histological analysis. CONCLUSIONS: The new HFS composed of two non-toxic components for MSCs showed capacity to promote the recovery of the bone lesions in OVX and NOVX animals at 14 days after surgery. In addition, the HFS enabled the differentiation of MSCs into MSCs D in the group treated with HFS + MSCs. Using the MSCs and/or MSCs D together with this biopharmaceutical could potentially enable significant advances in the treatment of osteoporotic fractures. Future clinical trials will be necessary to confirm these results.

Assuntos

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Este estudo visou avaliar os efeitos do cloprostenol sódico na involução uterina, dosagem de progesterona (P4) e 13,14-dihydro-15-keto prostaglandina F2α (PGFM) e atividade folicular em vacas Nelore multíparas tratadas ou não com cloprostenol sódico nos dias 1 e 4 pós-parto (D0= nascimento), bem como testar a hipótese que o tratamento promove uma involução uterina mais rápida e retorno a atividade cíclica normal no puerpério. Para isso, foi feito ultrassom, palpação transretal, exame vaginoscópicoe determinação da concentração de P4 e PGFM nos dias 1, 7, 14, 28, 35 e 42 pós-parto (D0= nascimento). A vaginoscopia avaliou o óstio cervical (Escore 1 a 3) e descarga cervical (escore 1 a 5), a palpação transretal avaliou a involução uterina (escore 1 a 3) e o ultrassom avaliou o diâmetro dos folículos, tamanho dos cornos uterino e acumulo de fluido intrauterino (escore 0 a 3). O tratamento influenciou na descarga cervical (P= 0.034), e nas outras variáveis foi observada a influência do momento (P>0,05). Baseado nestes resultados, nós acreditamos que o uso desse protocolo nos animais com boa seleção genética, manejo e saudáveis pode ser desnecessário e promove gastos adicionais.(AU)

This study aimed to evaluate the effects of cloprostenol sodium at uterine involution and hormonal dosage of progesterone (P4) and 13,14-dihydro-15-keto prostaglandin F2α (PGFM) and follicular activity on multiparous Nelore cows treated or not with cloprostenol sodium on days 1 and 4 postpartum (D0= birth), as well as test the hypothesis that treatment promotes a faster uterine involution and return to normal cyclical activity at puerperium. For this, it was done ultrasound, transretal palpation, vaginoscopy exams and determination of concentration of P4 and PGFM on days 1, 7, 14, 28, 35 and 42 postpartum (D0= birth). Vaginoscopy evaluated cervical ostium (score 1 to 3) and cervical discharge (score 1 to 5), transrectal palpation evaluated uterine involution (score 1 to 3) and ultrasound evaluated the diameter of follicles, size of uterine horn and intrauterine fluid accumulation (score 0 to 3). Treatment influenced at cervical discharge (P= 0.034), and on the other variables it was observed influence of moment (P>0.05). Based on the results, we believe that the use of this protocol inanimals with good selection genetic, management and health can be unnecessary and promote additional spending.(AU)

El objetivo del estudio fue evaluar el efecto del cloprostenol sódico en la involución uterina y la concentración sérica de progesterona (P4) y 13,14-dihidro-15-keto prostaglandina F2α (PGFM) y actividad folicular en vacas multíparas Nelore tratadas o no, con cloprostenol sódico en los días 1 y 4 posparto (D0= día del nacimiento), de igual forma, comprobar la hipótesis de que el tratamiento promueve una involución uterina más rápida y un retorno de la actividad cíclica normal en el puerperio. Fue realizada ecografía, palpación transrectal, vaginoscopia y determinación de la concentración sérica de P4 y PGFM en los días 1, 7, 14, 28, 35, y 42 posparto (D0= día del nacimiento). La vaginoscopia evaluó el ostium cervical (escala de 1-3) y descarga cervical (escala de 1-5), la palpación transrectal determinó la involución uterina (escala de 1-3) y la ecografía evaluó el diámetro folicular, tamaño de los cuernos uterinos y la acumulación intrauterina de fluidos (escala de 0-3). El tratamiento influyó en la descarga cervical (P= 0.034), para las demás variables fue observado efecto del momento (P>0.05). Basados en los resultados, creemos que el uso de este protocolo en animales con buen manejo, salud y selección genética es innecesario y demanda gastos adicionales.(AU)

Assuntos

RESUMO

Buffaloes are dairy, meat and work farm animals very adapted to tropical regions, with large productivity and economic potential in several countries, including Brazil. In some states, the lack of technology in buffalo farming has increased reproductive and sanitary problems, turning imminent the extinction of these animals. In this contest, studies related to stem cell biotechnology have resulted in other possible utilization of buffaloes as experimental model, allowing the expansion of knowledge in this specie in particular cell characteristics. Stem cells have been the subject of many studies in several species, nevertheless buffaloes studies are scarce. Thus, this review aims to assemble and connect buffaloes and stem cells biotechnology studies to demonstrate the importance of research to increase the advancement of these two fields.(AU)

A Bubalinocultura abrange animais de tripla aptidão muito adaptados aos climas tropicais e com grande potencial produtivo e econômico em vários países, inclusive no Brasil. Em algumas regiões, a falta de tecnificação na produção de bubalinos tem agravado problemas reprodutivos e sanitários, tornando iminente a extinção desses animais. Nesse contexto, estudos relacionados à biotecnologia de células-tronco têm indicado resultados que viabilizam a utilização de búfalos como modelo experimental, além de permitir a ampliação dos conhecimentos acerca da espécie e de seu comportamento celular característico. As células-tronco têm sido alvo de muitas pesquisas em várias espécies, porém os estudos com bubalinos ainda são escassos. Desta forma, o objetivo dessa revisão foi reunir estudos relacionados à bubalinocultura e biotecnologia de células-tronco e demonstrar a importância das pesquisas que contribuem para o avanço dessas duas áreas.(AU)

La Bubalinocultura abarca animales de triple aptitud muy adaptados a los climas tropicales y con gran potencial productivo y económico en varios países, incluso en Brasil. En algunas regiones, la falta de tecnificación en la producción de bufalinos ha agravado problemas reproductivos y sanitarios, haciendo inminente la extinción de estos animales. En ese contexto, Estudios relacionados con la biotecnología de células madre han indicado resultados que viabilizan la utilización de búfalos como modelo experimental, además de permitir la ampliación de los conocimientos sobre la especie y su comportamiento celular característico. Las células madre han sido objeto de muchas investigaciones en várias espécies, pero los estudios con bubalinos todavía son escasos. De esta forma, el objetivo de esta revisión fue reunir estudios relacionados con la bubalinocultura y biotecnología de células madre y demostrar la importancia de las investigaciones que contribuyen al avance de estas dos áreas.(AU)

Assuntos

RESUMO

Buffaloes are dairy, meat and work farm animals very adapted to tropical regions, with large productivity and economic potential in several countries, including Brazil. In some states, the lack of technology in buffalo farming has increased reproductive and sanitary problems, turning imminent the extinction of these animals. In this contest, studies related to stem cell biotechnology have resulted in other possible utilization of buffaloes as experimental model, allowing the expansion of knowledge in this specie in particular cell characteristics. Stem cells have been the subject of many studies in several species, nevertheless buffaloes studies are scarce. Thus, this review aims to assemble and connect buffaloes and stem cells biotechnology studies to demonstrate the importance of research to increase the advancement of these two fields.

A Bubalinocultura abrange animais de tripla aptidão muito adaptados aos climas tropicais e com grande potencial produtivo e econômico em vários países, inclusive no Brasil. Em algumas regiões, a falta de tecnificação na produção de bubalinos tem agravado problemas reprodutivos e sanitários, tornando iminente a extinção desses animais. Nesse contexto, estudos relacionados à biotecnologia de células-tronco têm indicado resultados que viabilizam a utilização de búfalos como modelo experimental, além de permitir a ampliação dos conhecimentos acerca da espécie e de seu comportamento celular característico. As células-tronco têm sido alvo de muitas pesquisas em várias espécies, porém os estudos com bubalinos ainda são escassos. Desta forma, o objetivo dessa revisão foi reunir estudos relacionados à bubalinocultura e biotecnologia de células-tronco e demonstrar a importância das pesquisas que contribuem para o avanço dessas duas áreas.

La Bubalinocultura abarca animales de triple aptitud muy adaptados a los climas tropicales y con gran potencial productivo y económico en varios países, incluso en Brasil. En algunas regiones, la falta de tecnificación en la producción de bufalinos ha agravado problemas reproductivos y sanitarios, haciendo inminente la extinción de estos animales. En ese contexto, Estudios relacionados con la biotecnología de células madre han indicado resultados que viabilizan la utilización de búfalos como modelo experimental, además de permitir la ampliación de los conocimientos sobre la especie y su comportamiento celular característico. Las células madre han sido objeto de muchas investigaciones en várias espécies, pero los estudios con bubalinos todavía son escasos. De esta forma, el objetivo de esta revisión fue reunir estudios relacionados con la bubalinocultura y biotecnología de células madre y demostrar la importancia de las investigaciones que contribuyen al avance de estas dos áreas.

Assuntos

RESUMO

Este estudo visou avaliar os efeitos do cloprostenol sódico na involução uterina, dosagem de progesterona (P4) e 13,14-dihydro-15-keto prostaglandina F2α (PGFM) e atividade folicular em vacas Nelore multíparas tratadas ou não com cloprostenol sódico nos dias 1 e 4 pós-parto (D0= nascimento), bem como testar a hipótese que o tratamento promove uma involução uterina mais rápida e retorno a atividade cíclica normal no puerpério. Para isso, foi feito ultrassom, palpação transretal, exame vaginoscópicoe determinação da concentração de P4 e PGFM nos dias 1, 7, 14, 28, 35 e 42 pós-parto (D0= nascimento). A vaginoscopia avaliou o óstio cervical (Escore 1 a 3) e descarga cervical (escore 1 a 5), a palpação transretal avaliou a involução uterina (escore 1 a 3) e o ultrassom avaliou o diâmetro dos folículos, tamanho dos cornos uterino e acumulo de fluido intrauterino (escore 0 a 3). O tratamento influenciou na descarga cervical (P= 0.034), e nas outras variáveis foi observada a influência do momento (P>0,05). Baseado nestes resultados, nós acreditamos que o uso desse protocolo nos animais com boa seleção genética, manejo e saudáveis pode ser desnecessário e promove gastos adicionais.

This study aimed to evaluate the effects of cloprostenol sodium at uterine involution and hormonal dosage of progesterone (P4) and 13,14-dihydro-15-keto prostaglandin F2α (PGFM) and follicular activity on multiparous Nelore cows treated or not with cloprostenol sodium on days 1 and 4 postpartum (D0= birth), as well as test the hypothesis that treatment promotes a faster uterine involution and return to normal cyclical activity at puerperium. For this, it was done ultrasound, transretal palpation, vaginoscopy exams and determination of concentration of P4 and PGFM on days 1, 7, 14, 28, 35 and 42 postpartum (D0= birth). Vaginoscopy evaluated cervical ostium (score 1 to 3) and cervical discharge (score 1 to 5), transrectal palpation evaluated uterine involution (score 1 to 3) and ultrasound evaluated the diameter of follicles, size of uterine horn and intrauterine fluid accumulation (score 0 to 3). Treatment influenced at cervical discharge (P= 0.034), and on the other variables it was observed influence of moment (P>0.05). Based on the results, we believe that the use of this protocol inanimals with good selection genetic, management and health can be unnecessary and promote additional spending.

El objetivo del estudio fue evaluar el efecto del cloprostenol sódico en la involución uterina y la concentración sérica de progesterona (P4) y 13,14-dihidro-15-keto prostaglandina F2α (PGFM) y actividad folicular en vacas multíparas Nelore tratadas o no, con cloprostenol sódico en los días 1 y 4 posparto (D0= día del nacimiento), de igual forma, comprobar la hipótesis de que el tratamiento promueve una involución uterina más rápida y un retorno de la actividad cíclica normal en el puerperio. Fue realizada ecografía, palpación transrectal, vaginoscopia y determinación de la concentración sérica de P4 y PGFM en los días 1, 7, 14, 28, 35, y 42 posparto (D0= día del nacimiento). La vaginoscopia evaluó el ostium cervical (escala de 1-3) y descarga cervical (escala de 1-5), la palpación transrectal determinó la involución uterina (escala de 1-3) y la ecografía evaluó el diámetro folicular, tamaño de los cuernos uterinos y la acumulación intrauterina de fluidos (escala de 0-3). El tratamiento influyó en la descarga cervical (P= 0.034), para las demás variables fue observado efecto del momento (P>0.05). Basados en los resultados, creemos que el uso de este protocolo en animales con buen manejo, salud y selección genética es innecesario y demanda gastos adicionales.

Assuntos

RESUMO

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap+icaA+icaD+ isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay.

Assuntos

RESUMO

Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell banking.(AU)

As células tronco mesenquimais (CTMs) estão presentes na maioria dos tecidos adultos e possuem grande capacidade de multiplicação. Quando cultivadas in vitro são capazes de se auto renovar e dar origem a novos tipos celulares. As células tronco mais utilizadas, tanto na medicina humana como na medicina veterinária são as células tronco mesenquimais derivadas da medula óssea e do tecido adiposo. Atualmente, uma grande tendência para a utilização de CTMs obtidas de tecidos fetais, como a membrana amniótica (MA), matriz extravascular do cordão umbilical (TCU) e sangue do cordão umbilical (SCU) pode ser observada, já que estas fontes podem ser colhidas no momento do parto por uma técnica não invasiva. Dessa forma, o objetivo deste estudo foi isolar, caracterizar, diferenciar e criopreservar CTMs obtidas de MA e TCU de fetos bovinos colhidos no momento do parto e de fetos do terço inicial da gestação em abatedouro-frigorífico. As células foram recuperadas por meio de digestão enzimática tecidual, realizada com solução de colagenase 0,1%. Foram colhidas amostras de MA e TCU no momento do parto (n=6) e de MA e TCU no terço inicial de gestação (n=6), as quais foram submetidas às análises morfológicas, imunofenotípica por imunocitoquímica e citometria de fluxo, diferenciações in vitro nas linhagens osteogênica, adipogênica e condrogênica e ainda, avaliação da viabilidade após a criopreservação por citometria de fluxo. Todas as amostras dos diferentes grupos demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico todas as amostras foram imunomarcadas para CD44, NANOG e Oct-4, com ausência de marcação para MHC II. Na análise imunofenotipica por citometria de fluxo, todas as amostras apresentaram marcação para CD44, ausência de marcação para ou baixíssima expressão de CD34 (MA: TI-0,3%a, PA-3.4%b; TCU: TI-0,4%, PA-1.4%) e nula ou baixa expressão de MHC II (MA: TI-1.5%a, PA-9.7%b; UCT: TI-0.7%a, PA-5.7%b. Apresentaram também capacidade de diferenciação in vitro nas três linhagens mesodermais e quando analisadas pós criopreservação por citometria de fluxo, todas as amostras apresentaram viabilidade de 80%. Estes resultados indicam que MA e TCU, obtidos tanto no momento de parto como em abatedouro, de fetos bovinos podem ser utilizados como fonte não invasiva e indolor de CTMs e possibilitam a formação de bancos de armazenamento de células.(AU)

Assuntos

RESUMO

Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, [...](AU)

As células tronco mesenquimais (CTMs) estão presentes na maioria dos tecidos adultos e possuem grande capacidade de multiplicação. Quando cultivadas in vitro são capazes de se auto renovar e dar origem a novos tipos celulares. As células tronco mais utilizadas, tanto na medicina humana como na medicina veterinária são as células tronco mesenquimais derivadas da medula óssea e do tecido adiposo. Atualmente, uma grande tendência para a utilização de CTMs obtidas de tecidos fetais, como a membrana amniótica (MA), matriz extravascular do cordão umbilical (TCU) e sangue do cordão umbilical (SCU) pode ser observada, já que estas fontes podem ser colhidas no momento do parto por uma técnica não invasiva. Dessa forma, o objetivo deste estudo foi isolar, caracterizar, diferenciar e criopreservar CTMs obtidas de MA e TCU de fetos bovinos colhidos no momento do parto e de fetos do terço inicial da gestação em abatedouro-frigorífico. As células foram recuperadas por meio de digestão enzimática tecidual, realizada com solução de colagenase 0,1%. Foram colhidas amostras de MA e TCU no momento do parto (n=6) e de MA e TCU no terço inicial de gestação (n=6), as quais foram submetidas às análises morfológicas, imunofenotípica por imunocitoquímica e citometria de fluxo, diferenciações in vitro nas linhagens osteogênica, adipogênica e condrogênica e ainda, avaliação da viabilidade após a criopreservação por citometria de fluxo. Todas as amostras dos diferentes grupos demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico todas as amostras foram imunomarcadas para CD44, NANOG e Oct-4, com ausência de marcação para MHC II. Na análise imunofenotipica por citometria de fluxo, todas as amostras apresentaram marcação para CD44, ausência de marcação para ou baixíssima expressão de CD34 (MA: TI-0,3%a, PA-3.4%b; TCU: TI-0,4%, PA-1.4%) e nula ou baixa expressão de MHC II (MA: TI-1.5%a, PA-9.7%b; UCT: TI-0.7%a, PA-5.7%b. [...](AU)

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Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis/necrosis rates, immunophenotypic profile (IP), chromosomal stability, clonicity, and differentiation potential of UCIM-MSCs cryopreserved with four different mediums (with FBS: M1, M3, M4 and without FBS: M2). After 3 months of cryopreservation, samples were thawed and analyzed. The potential of differentiation in the mesodermal lineages, clonicity, and the chromosomal stability were maintained after cryopreservation of UCIM-MSCs with medium containing FBS. Changes (P < 0.05) at IP for some markers were observed at cells cryopreserved with medium M1-M3. Only the UCIM-MSCs cryopreserved with the CM (M4) had similar viability post-thaw (P = 0.23) when compared with fresh cells. We proved the hypothesis that the medium of cryopreservation containing CM supports the cryopreservation of UCIM-MSCs, at the experimental conditions, being the medium that better maintains the biological characteristics observed at fresh cells. Thus, future studies of UCIM-MSCs secretome should be conducted to better understand the beneficial and protective effects of the CM during the freezing process.

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Endometrial mesenchymal stem/progenitor cells (eMSCs) are multipotent cells known to modulate the immune system and have clinical application for human and animal health. This makes these bovines cells attractive for dual use as cellular therapy and experimental model. The aim of this study was to isolate, evaluate the differentiation potential, immunophenotypic and immunocytochemistry characteristics, chromosomal stability, cloning efficiency, and cryopreservation response of bovine eMSCs collected in two phases of the estrous cycle. For this, cells were isolated and submitted to differentiation for adipogenic and osteogenic lineage. The cells were then characterized by flow cytometer (FC) (vimentin, CD29, CD44, MHC-II, CD34) and immunocytochemistry (vimentin, pan-cytokeratin, CD44) and submitted to cytogenetic and cloning efficiency assay. The cells were also cryopreserved using two different medium of cryopreservation and analyzed by FC for viability, necrosis, late-apoptosis + necrosis, and initial apoptosis rates before and after cryopreservation. We obtained homogeneous cell populations which have fibroblastic morphology and adherence to plastic. These cells expressed high levels of markers CD29, CD44, and vimentin, low expression levels for CD34 and no MHC-II. The cells were chromosomally stable (2n = 60) with high cloning efficiency and no difference (P > 0.05) between medium of cryopreservation or phase was observed after thawing. We showed the presence and differentiation potential of bovine eMSCs, with chromosomal stability and great response to cryopreservation with both medium, which has implications for build biobanks or development of new therapeutic approaches to combat uterine diseases or to study.

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BACKGROUND AND OBJECTIVES: Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. METHODS AND RESULTS: For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs diluted in Hank's Balanced Solution (HBSS) was performed on right and left sides of the rump muscle. As a control, HBSS injections were performed caudally in the same muscle. Muscle biopsies were obtained as a control 30 days before transplantation (D-30). The biopsies were collected again on day 2 (left side) and day 7 (right side) post transplantation and examined histologically. All procedures were preceded by ultrasound examination and blood sampling. Hematologic evaluation remained within normal limits and no differences were observed between time points (p>0.05). Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups (control and treated). At histological evaluation it was found only discrete inflammation signals between D-30×D2 (p<0.05) in the treated group, without differences (p> 0.05) between the groups at different time points. CONCLUSIONS: Equine UC-MSCs under the experimental conditions did not promote severe inflammation that causes tissue damage or lead to its rejection by the host organism and therefore has a good potential for clinical use.

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BACKGROUND: Recent studies have demonstrated numerous biological properties of mesenchymal stem cells and their potential application in treating complex diseases or injuries to tissues that have difficulty regenerating, such as those affecting the central and peripheral nervous system. Thus, therapies that use mesenchymal stem cells are promising because of their high capacity for self-regeneration, their low immunogenicity, and their paracrine, anti-inflammatory, immunomodulatory, anti-apoptotic and neuroprotective effects. In this context, the purpose of this study was to evaluate the feasibility and safety of intrathecal transplantation of bone marrow-derived mesenchymal stem cells in horses, for future application in the treatment of neurological diseases. RESULTS: During the neurological evaluations, no clinical signs were observed that were related to brain and/or spinal cord injury of the animals from the control group or the treated group. The hematological and cerebrospinal fluid results from day 1 and day 6 showed no significant differences (P > 0.05) between the treated group and the control group. Additionally, analysis of the expression of matrix metalloproteinase (MMP) -2 and -9 in the cerebrospinal fluid revealed only the presence of pro-MMP-2 (latent), with no significant difference (P > 0.05) between the studied groups. CONCLUSIONS: The results of the present study support the hypothesis of the feasibility and safety of intrathecal transplantation of autologous bone marrow-derived mesenchymal stem cells, indicating that it is a promising pathway for cell delivery for the treatment of neurological disorders in horses.

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O puerpério é conhecido como o período que se inicia após o parto e persiste até o restabelecimento da condição de um animal não gestante. Compreendem na liberação dos restos placentários, involução uterina e mecanismos tais como contração uterina e digestão enzimática contribuem para a correta execução desses processos. Sua duração é variável e dependente de vários fatores tais como escore de condição corporal (ECC), produção leiteira, nutrição, condição uterina, entre outros. Tem-se também que para o estabelecimento de nova prenhez, é necessário que ocorra o retorno à ciclicidade por meio do crescimento, maturação e ovulação de um folículo e oócito sadios. Tal fato pode ser afetado pela presença do bezerro, amamentação e ECC em bovinos de corte e balanço energético, escore corporal, parição, estação do ano e doenças em bovinos de leite. Adicionalmente, é de extrema importância a característica do útero para o restabelecimento do estado reprodutivo fisiológico. Doenças uterinas podem afetar na liberação de substâncias que podem ser responsáveis pelo encurtamento ou prolongamento dos ciclos ovarianos, o que pode ocasionar perdas econômicas pelo fato de não atingir-se o desejável de produção de 1 bezerro/vaca/ano.

RESUMO

O puerpério é conhecido como o período que se inicia após o parto e persiste até o restabelecimento da condição de um animal não gestante. Compreendem na liberação dos restos placentários, involução uterina e mecanismos tais como contração uterina e digestão enzimática contribuem para a correta execução desses processos. Sua duração é variável e dependente de vários fatores tais como escore de condição corporal (ECC), produção leiteira, nutrição, condição uterina, entre outros. Tem-se também que para o estabelecimento de nova prenhez, é necessário que ocorra o retorno à ciclicidade por meio do crescimento, maturação e ovulação de um folículo e oócito sadios. Tal fato pode ser afetado pela presença do bezerro, amamentação e ECC em bovinos de corte e balanço energético, escore corporal, parição, estação do ano e doenças em bovinos de leite. Adicionalmente, é de extrema importância a característica do útero para o restabelecimento do estado reprodutivo fisiológico. Doenças uterinas podem afetar na liberação de substâncias que podem ser responsáveis pelo encurtamento ou prolongamento dos ciclos ovarianos, o que pode ocasionar perdas econômicas pelo fato de não atingir-se o desejável de produção de um bezerro/vaca/ano.

The postpartum period is known as the period that begins after birth and persists until the recovery of the condition of an animal that is not pregnant. They include the release of placental membranes, the uterine involution and mechanisms such as uterine contraction and enzymatic digestion contribute to the proper execution of these processes. Its duration is variable and dependent on several factors such as body condition score (BCS), milk production, nutrition, uterine condition, among others. It has also for the establishment of new pregnancy, it is necessary the return to cyclicity through growth, maturation and ovulation of a healthy follicle and oocyte. This can be affected by the presence of the calf, breast-feeding and BCS in beef cattle and energy balance, body condition score, parity, season and disease in dairy cattle. Additionally, it is extremely important the characteristic of the uterus for restoration, reproductive physiology. Uterine diseases can affect the release of substances that may be responsible for shortening or lengthening of ovarian cycles, which can cause economic losses because of not achieving is desirable to produce a calf / cow / year.

El puerperio es conocido como el periodo que se inicia después del parto y persiste hasta el restablecimiento de la condición de un animal no gestante. Comprende desde la liberación de los restos placentarios, involución uterina y mecanismos tales como contracción uterina y digestión enzimática, que contribuyen para la correcta ejecución de estos procesos. Su duración es variable y depende de varios factores, como condición corporal (CC), producción lechera, nutrición, estado del útero, entre otros. Para el restablecimiento de una nueva preñez, es necesario que ocurra el retorno del ciclo estral por medio del crecimiento, maduración y ovulación de un folículo y oocito sanos. Esto puede ser afectado por la presencia del ternero, amamantamiento y condición corporal en bovinos de corte y balance energético, condición corporal, parto, estación del año y enfermedades en bovinos de leche. Adicionalmente, es de extrema importancia la característica del útero para el restablecimiento del estado reproductivo fisiológico. Enfermedades uterinas pueden afectar la liberación de sustancias que pueden ser responsables del acortamiento o prolongación de los ciclos ovarianos, lo que puede ocasionar perdidas económicas por el hecho de no alcanzar lo deseado de producción de 1 ternero/vaca/año.

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The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

O objetivo deste estudo foi isolar, cultivar e caracterizar as células mesenquimais multipotentes estromais derivadas do sangue periférico (SpCTMs) equino. O sangue periférico foi coletado, seguido do isolamento das células mononucleadas utilizando o reagente de gradiente de densidade e o cultivo das células aderentes. Os anticorpos monoclonais mouse anti-horse CD13, mouse anti-horse CD44 e mouse anti-rat CD90 foram utilizados para a caracterização imunofenotípica da superfície das SpCTMs. Estas células também foram cultivadas utilizando meio de cultura específico para a diferenciação adipogênica e condrogênica. Não houve expressão do marcador CD13, mas os marcadores CD44 e CD90 foram expressos em todas as passagens testadas. Após 14 dias da diferenciação das células em adipócitos, gotículas de lipídeos foram observados através da coloração com Oil Red O. Vinte e um dias após a diferenciação condrogênica, as células foram coradas com o Alcian Blue. Embora a técnica de isolamento destas células necessite ser otimizada, o presente estudo demonstra a caracterização parcial das SpCTMs, classificando-as como um tipo de células progenitoras promissoras para o uso na terapia celular em equinos.

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The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.(AU)

O objetivo deste estudo foi isolar, cultivar e caracterizar as células mesenquimais multipotentes estromais derivadas do sangue periférico (SpCTMs) equino. O sangue periférico foi coletado, seguido do isolamento das células mononucleadas utilizando o reagente de gradiente de densidade e o cultivo das células aderentes. Os anticorpos monoclonais mouse anti-horse CD13, mouse anti-horse CD44 e mouse anti-rat CD90 foram utilizados para a caracterização imunofenotípica da superfície das SpCTMs. Estas células também foram cultivadas utilizando meio de cultura específico para a diferenciação adipogênica e condrogênica. Não houve expressão do marcador CD13, mas os marcadores CD44 e CD90 foram expressos em todas as passagens testadas. Após 14 dias da diferenciação das células em adipócitos, gotículas de lipídeos foram observados através da coloração com Oil Red O. Vinte e um dias após a diferenciação condrogênica, as células foram coradas com o Alcian Blue. Embora a técnica de isolamento destas células necessite ser otimizada, o presente estudo demonstra a caracterização parcial das SpCTMs, classificando-as como um tipo de células progenitoras promissoras para o uso na terapia celular em equinos.(AU)

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O interesse nas pesquisas com células-tronco derivadas de anexos fetais de diversas espécies cresceu exponencialmente nas últimas décadas em virtude de serem fontes de células-tronco adultas com potencial de diferenciação em diversas linhagens celulares que apresentam pouca ou nenhuma imunogenicidade, apresentando-se assim como alternativa de grande importância para a formação de bancos celulares. Apesar do crescente interesse, os estudos para espécie equina ainda são escassos. O objetivo deste trabalho foi isolar, caracterizar e diferenciar células-tronco mesenquimais (CTMs) derivadas do líquido amniótico equino obtidas do terço inicial, médio e final da gestação (LA-CTMs), comparando suas características. Foram colhidas 23 amostras de líquido amniótico as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e às diferenciações osteogênica, adipogênica e condrogênica in vitro. Todas as amostras demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico as células de todos os grupos foram imunomarcadas para CD44, PCNA e vimentina com ausência de marcação para citoqueratina e Oct-4. Na citometria de fluxo observou-se a expressão de CD44 e CD90 e ausência de expressão de CD34, sendo que os marcadores CD44 e CD90 mostraram padrão de expressão decrescente em relação ao desenvolvimento gestacional. As amostras obtidas de todas as fases da gestação foram capazes de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Portanto, as células obtidas do líquido amniótico apresentaram características morfológicas, imunofenotípicas e potencial de diferenciação típicos das CTMs, demonstrando que a colheita pode ser realizada em qualquer fase gestacional. No entanto, mais pesquisas devem ser realizadas principalmente quanto à expressão de marcadores de pluripotencialidade (como o Oct-4) e ao seu potencial de diferenciação em linhagens extra mesodermais já relatados na literatura.

The interest in stem cells derived from fetal annexes of many species has exponentially increased during the last decades, because they are adult stem cell sources with potential of differentiation in several cell lineages; which present little or no immunogenicity and are an alternative with great importance for storage cell banks. Despite the rising interest, studies for the equine species are still rare. The aim of this study was to isolate, characterize and differentiate mesenchymal stem cells derived from equine amniotic fluid obtained from initial, middle and late third of gestation (AF-MSCs), and compare their results. Twenty three samples from equine amniotic fluid were evaluated by morphological, immunocytochemical and immunophenotypical (Flow cytometer) assays and osteogenic, adipogenic and chondrogenic in vitro differentiation. All samples demonstrated plastic adhesion and fibroblastoid morphology. The immunocytochemical assay demonstrated cells from all the studied groups were positive for CD44, PCNA and vimentin and negative for cytokeratin and Oct-4. Flow cytometry demonstrated expression of CD44 and CD90 and no expression of CD34, where CD44 and CD90 markers presented decreasing pattern of expression in relation to the gestational development. All samples collected from all gestational phases were capable to differentiate in osteogenic, chondrogenic and adipogenic lineages. Thus, cells obtained from equine amniotic fluid presented morphological and immunophenotypical characteristics and potential of differentiation typical of MSCs showing that the collection can be performed at any stage of pregnancy. However, more studies should be performed about the expression of pluripotent markers as Oct-4 and the differentiation potential for extra mesodermal lineages prior demonstrated in the literature.

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