Adipose and amnion-derived mesenchymal stem cells: Extracellular vesicles characterization and implication for reproductive biotechnology.

The stem cell-based research for reproductive biotechnology has been widely studied and shows promise for repairing defective tissue or degenerated cells to treat different diseases. The adipose tissue and amniotic membrane have awakened great interest in regenerative medicine and arises as a promising source of mesenchymal stem cells. Both types, adipose and amniotic derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages.. We aimed to evaluate the effect of basal supplementation of exosomes in cell cultures with canine amniotic mesenchymal stem cells (MSCs). Mesenchymal stem cells derived from canine amniotic and adipose tissue were isolated and cultured performing cell passages until 80-90% confluence was reached. The growth curve was determined and peak cell growth was observed in the second passage. The cells were then characterized and differentiated into adipogenic, chondrogenic and osteogenic lineages. Extracellular vesicles from amnion were isolated using an ultracentrifugation protocol and characterized by nanosight analysis. To evaluate their ability to improve cellular viability in naturally inefficient passages, exosomes were co-cultures to the MSC cells. The results showed a 15-20% increase in the expansion rate of cultures supplemented with vesicles extracted in the first and second passages when compared to the control group. Statistical analysis using the Dunnett test (p ≤ 0.05) corroborated this result, showing a positive correlation between supplementation and expansion rate. These results indicate not only the importance of exosomes in the cell communication process but also the feasibility of the culture supplementation protocol for therapeutic purposes. The potential of the AMSCs for reproductive biotechnology is undoubted, however, their application to repair reproductive disorders and the involved mechanisms remain elusive. The strategies to enable the Adipose Stem Cells and AMSCs application in reproductive biotechnology and optimize their use for tissue regeneration open new venues using exosomes interactions.

Secretion pattern of canine amniotic stem cells derived extracellular vesicles.

Extracellular vesicles (EVs) derived from stem cells (SCs) have regenerative potential and the possibility of being used in treating chronic diseases. EVs present lower risk of tumorigenicity and easily to isolation and storage. Therefore, this research aims to compare the morphological characteristics of the EVs (up to 150nm) derived from stem cells obtained from canine amniotic membranes in different passages during the in vitro culture. For this, cells from the amniotic membranes were isolated, cultured, and characterized. In order to answer our aim, the number of cells was normalized at each passage to generate conditioned media for EVs separation. The cells were differentiated into adipogenic, chondrogenic, and osteogenic tissue, to characterize these cells as mesenchymal stem cells (MSC). Moreover, flow cytometry analysis was performed and showed that the MSC were positive for CD90, CD105 and negative for CD34, CD45, mesenchymal and hematopoietic markers, respectively. For EVs analysis, MSC in different passages (P0-P2) were culture until 80% of confluence, then the medium was replaced by EVs depleted medium. After 48h, culture medium was collected and centrifuged to separate EVs, followed by nanoparticle tracking analysis. The EVs were also characterized by western blot and transmission electron microscopy (TEM). EVs were positive for Alix and negative for Cytochrome C as well as presented the traditional cup-shape by transmission electronic microscopy. Our results demonstrated that the concentration in the different passages was increased in P0 compared to P1 and P2 (p<0.05). No differences were found in EVs size (P0=132nm, P1=130nm and P2=120nm). Together, these results demonstrate that P0 of MSC is enriched of EVs when compared to later passages, suggesting that this passage would be the best to be applied in pre-clinical tests. Despite that, more studies are necessary to identify the EVs content and how the cells will respond to treatment with them.

Secretion pattern of canine amniotic stem cells derived extracellular vesicles

Extracellular vesicles (EVs) derived from stem cells (SCs) have regenerative potential and the possibility of being used in treating chronic diseases. EVs present lower risk of tumorigenicity and easily to isolation and storage. Therefore, this research aims to compare the morphological characteristics of the EVs (up to 150nm) derived from stem cells obtained from canine amniotic membranes in different passages during the in vitro culture. For this, cells from the amniotic membranes were isolated, cultured, and characterized. In order to answer our aim, the number of cells was normalized at each passage to generate conditioned media for EVs separation. The cells were differentiated into adipogenic, chondrogenic, and osteogenic tissue, to characterize these cells as mesenchymal stem cells (MSC). Moreover, flow cytometry analysis was performed and showed that the MSC were positive for CD90, CD105 and negative for CD34, CD45, mesenchymal and hematopoietic markers, respectively. For EVs analysis, MSC in different passages (P0-P2) were culture until 80% of confluence, then the medium was replaced by EVs depleted medium. After 48h, culture medium was collected and centrifuged to separate EVs, followed by nanoparticle tracking analysis. The EVs were also characterized by western blot and transmission electron microscopy (TEM). EVs were positive for Alix and negative for Cytochrome C as well as presented the traditional cup-shape by transmission electronic microscopy. Our results demonstrated that the concentration in the different passages was increased in P0 compared to P1 and P2 (p<0.05). No differences were found in EVs size (P0=132nm, P1=130nm and P2=120nm). Together, these results demonstrate that P0 of MSC is enriched of EVs when compared to later passages, suggesting that this passage would be the best to be applied in pre-clinical tests. Despite that, more studies are necessary to identify the EVs content and how the cells will respond to treatment with them.(AU)

Potential application of aminiotic stem cells in veterinary medicine.

In regenerative medicine stem cell biology has become one of the most interesting and more often studied subject. The amniotic membrane is the innermost layer of the fetal membranes and is considered a potential tool to treat many pathologies. It is used because it can be collected from discarded fetal material and is a rich source of stem cells with high proliferation and plasticity ratio capable of proliferating and differentiate in vitro. We propose to elucidate the characteristics and potencial clinical application of cells derived of amniotic membrane in veterinary medicine.

Morphology of male and female reproductive tract of the ocelot (Leopardus pardalis).

The Ocelot (Leopardus pardalis) is the largest species of this genus, despite having broad distribution in the Americas; it is included in the main list of endangered species. Their conservation is widely studied, but there is a lack of studies about their morphology. In order to contribute to the knowledge of its reproductive system, five male and female ocelots were examined macro- and microscopically by histological techniques. Macroscopic analysis of the male reproductive system revealed presence of prostate and bulbourethral gland located caudally to the urinary bladder and a penis with small spicules. Microscopically, the testes were encased by the tunica albuginea and divided it into lobules with 5-10 tubules per lobe. In females, macroscopic analysis demonstrated two ovaries position dorsally in the sublumbar region and caudal to the kidneys. The bicornuate uterus is composed by uterine horns (12 to 14 cm in length), which travels from the ovaries in a caudal direction to form a small uterine body (4 cm in length). The ovary analysis revealed, in longitudinal section, medullary region composed of loose connective tissue, a stroma rich in blood vessels, and an external parenchymal region surrounded by a tunica albuginea. The results of the study confirmed the similarity between ocelot's reproductive system as domestic cat's ones and showing for the first time the complete morphological tool to highlight these organs and tissue in this male and female endangered wild felid specie. The present study open venue for other researchers to consider morphological and preservationist features and aimed to help at long-term conservation of wild felines.

Morphology of male and female reproductive tract of the ocelot (Leopardus pardalis)

The Ocelot (Leopardus pardalis) is the largest species of this genus, despite having broad distribution in the Americas; it is included in the main list of endangered species. Their conservation is widely studied, but there is a lack of studies about their morphology. In order to contribute to the knowledge of its reproductive system, five male and female ocelots were examined macro- and microscopically by histological techniques. Macroscopic analysis of the male reproductive system revealed presence of prostate and bulbourethral gland located caudally to the urinary bladder and a penis with small spicules. Microscopically, the testes were encased by the tunica albuginea and divided it into lobules with 5-10 tubules per lobe. In females, macroscopic analysis demonstrated two ovaries position dorsally in the sublumbar region and caudal to the kidneys. The bicornuate uterus is composed by uterine horns (12 to 14 cm in length), which travels from the ovaries in a caudal direction to form a small uterine body (4 cm in length). The ovary analysis revealed, in longitudinal section, medullary region composed of loose connective tissue, a stroma rich in blood vessels, and an external parenchymal region surrounded by a tunica albuginea. The results of the study confirmed the similarity between ocelot's reproductive system as domestic cat's ones and showing for the first time the complete morphological tool to highlight these organs and tissue in this male and female endangered wild felid specie. The present study open venue for other researchers to consider morphological and preservationist features and aimed to help at long-term conservation of wild felines.(AU)

Morphology of male and female reproductive tract of the ocelot (Leopardus pardalis)

The Ocelot (Leopardus pardalis) is the largest species of this genus, despite having broad distribution in the Americas; it is included in the main list of endangered species. Their conservation is widely studied, but there is a lack of studies about their morphology. In order to contribute to the knowledge of its reproductive system, five male and female ocelots were examined macro- and microscopically by histological techniques. Macroscopic analysis of the male reproductive system revealed presence of prostate and bulbourethral gland located caudally to the urinary bladder and a penis with small spicules. Microscopically, the testes were encased by the tunica albuginea and divided it into lobules with 5-10 tubules per lobe. In females, macroscopic analysis demonstrated two ovaries position dorsally in the sublumbar region and caudal to the kidneys. The bicornuate uterus is composed by uterine horns (12 to 14 cm in length), which travels from the ovaries in a caudal direction to form a small uterine body (4 cm in length). The ovary analysis revealed, in longitudinal section, medullary region composed of loose connective tissue, a stroma rich in blood vessels, and an external parenchymal region surrounded by a tunica albuginea. The results of the study confirmed the similarity between ocelot's reproductive system as domestic cat's ones and showing for the first time the complete morphological tool to highlight these organs and tissue in this male and female endangered wild felid specie. The present study open venue for other researchers to consider morphological and preservationist features and aimed to help at long-term conservation of wild felines.

Canine fetus immune system at late development.

The immune system is mainly responsible for protecting the organism against agents that may interfere in its homeostasis. Thus, understand how this system develops and operates is very important, for create new therapies to assist this system in its operation, such as its failure. In domestic dogs, few studies show how actually occurs the development, maturation and functioning of the immune system. Therefore, this study demonstrates the development and possible activation of it on dog fetus from late gestational period by in situ and microscopic analyzes.

Canine fetus immune system at late development

The immune system is mainly responsible for protecting the organism against agents that may interfere in its homeostasis. Thus, understand how this system develops and operates is very important, for create new therapies to assist this system in its operation, such as its failure. In domestic dogs, few studies show how actually occurs the development, maturation and functioning of the immune system. Therefore, this study demonstrates the development and possible activation of it on dog fetus from late gestational period by in situ and microscopic analyzes.(AU)

Potential application of aminiotic stem cells in veterinary medicine

In regenerative medicine stem cell biology has become one of the most interesting and more often studied subject. The amniotic membrane is the innermost layer of the fetal membranes and is considered a potential tool to treat many pathologies. It is used because it can be collected from discarded fetal material and is a rich source of stem cells with high proliferation and plasticity ratio capable of proliferating and differentiate in vitro. We propose to elucidate the characteristics and potencial clinical application of cells derived of amniotic membrane in veterinary medicine.(AU)

Potential application of aminiotic stem cells in veterinary medicine

In regenerative medicine stem cell biology has become one of the most interesting and more often studied subject. The amniotic membrane is the innermost layer of the fetal membranes and is considered a potential tool to treat many pathologies. It is used because it can be collected from discarded fetal material and is a rich source of stem cells with high proliferation and plasticity ratio capable of proliferating and differentiate in vitro. We propose to elucidate the characteristics and potencial clinical application of cells derived of amniotic membrane in veterinary medicine.

Canine fetus immune system at late development

The immune system is mainly responsible for protecting the organism against agents that may interfere in its homeostasis. Thus, understand how this system develops and operates is very important, for create new therapies to assist this system in its operation, such as its failure. In domestic dogs, few studies show how actually occurs the development, maturation and functioning of the immune system. Therefore, this study demonstrates the development and possible activation of it on dog fetus from late gestational period by in situ and microscopic analyzes.

Felinos paraplégicos e perfil de seus tutores: estudo retrospectivo de 15 casos / Paraplegic felines and the profile of their owners: a retrospective study of 15 cases / Felinos parapléjicos y perfil de sus tutores: estudio retrospectivo de 15 casos

Traumas são a segunda maior causa de morte em gatos, perdendo apenas para envenenamentos. No presente trabalho, 15 tutores de gatos paraplégicos responderam um questionário online. A causa de maior prevalência da paraplegia foi maus tratos (40%). A média de idade foi de 1 ano e 7 meses. Em média, os animais fazem/fizeram tratamento durante 11 meses, sendo que 26,7% só utilizou medicação anti-inflamatória e analgésica, enquanto dentre os demais, 27,3% foram submetidos à cirurgia, 72,7% fizeram acupuntura, 45,5% fisioterapia, 9% terapia celular e 9% outros. Entretanto, 33,3% dos tutores não observaram melhora. As doenças concomitantes mais predominantes foram constipação e retenção urinária (53,5%), e cistites recorrentes (40%), seguidas de escaras (33,3%), incontinência fecal e urinária (26,7%), dermatite de contato na região da fralda (6,7%) e necrose de cauda (6,7%). 40% dos tutores cogitaram ou foram instruídos a realizar a eutanásia, sendo que 16,7% optaram pelo procedimento. Apesar da paraplegia, 93,3% dos tutores consideram que seus animais apresentam boa qualidade de vida.

Traumas are the second largest cause of death in cats, behind only for poisonings. In the present study, 15 owners of paraplegic cats answered an online questionnaire. The greatest prevalence of paraplegia was animal cruelty (40%). The mean age was 1 year and 7 months. On average, the animals treated for 11 months, with 26.7% only using anti-inflammatory and analgesic medication, while among the others, 27.3% underwent surgery, 72.7% acupuncture, 45.5 % physiotherapy, 9% cell therapy and 9% others. However, 33.3% of the owners did not notice improvement. The most frequent concomitant diseases were constipation and urinary retention (53.5%), and recurrent cystitis (40%), followed by pressure sores (33.3%), fecal and urinary incontinence (26.7%), diaper dermatitis (6.7%) and tail necrosis (6.7%). 40% of the owners considered or were instructed to perform euthanasia, and 16.7% opted for the procedure. Despite paraplegia, 93.3% of the owners considered that their animals had a good quality of life.

Traumatismos son la segunda causa de muerte en gatos, perdiendo sólo para envenenamientos. En el presente trabajo, 15 tutores de gatos parapléjicos respondieron un cuestionario en línea. La causa de la mayor prevalencia de la paraplejia fue maltrato (40%). EI promedio de edad fue de 1 ano y 7 meses. En promedio, los animales hacen/han hecho un tratamiento durante 11 meses, siendo que el 26,7% sólo utilizó medicación antiinflamatoria y analgésica, mientras que los demás, el 27,3% fueron sometidos a la cirugía, el 72,7% hicieron acupuntura, 45,5 % fisioterapia, 9% terapia celular y 9% otros. Sin embargo, el 33,3% de los pacientes no observaron mejoría. Las enfermedades concomitantes más predominantes fueron constipación y retención de orina (53,5%), y cistitis recurrentes (40%), seguidas de escaras (33,3%), incontinencia fecal y urinaria (26,7%), dermatitis de contacto región del panal (6,7%) y necrosis de cola (6,7%). EI 40% de los tutores pensó o fue instruido para realizar una eutanasia, y el 16,7% optó por el procedimiento. A pesar de la paraplejia, el 93,3% de los derechos de autor en sus animales presentan buena calidad de vida.

Felinos paraplégicos e perfil de seus tutores: estudo retrospectivo de 15 casos / Paraplegic felines and the profile of their owners: a retrospective study of 15 cases / Felinos parapléjicos y perfil de sus tutores: estudio retrospectivo de 15 casos

Traumas são a segunda maior causa de morte em gatos, perdendo apenas para envenenamentos. No presente trabalho, 15 tutores de gatos paraplégicos responderam um questionário online. A causa de maior prevalência da paraplegia foi maus tratos (40%). A média de idade foi de 1 ano e 7 meses. Em média, os animais fazem/fizeram tratamento durante 11 meses, sendo que 26,7% só utilizou medicação anti-inflamatória e analgésica, enquanto dentre os demais, 27,3% foram submetidos à cirurgia, 72,7% fizeram acupuntura, 45,5% fisioterapia, 9% terapia celular e 9% outros. Entretanto, 33,3% dos tutores não observaram melhora. As doenças concomitantes mais predominantes foram constipação e retenção urinária (53,5%), e cistites recorrentes (40%), seguidas de escaras (33,3%), incontinência fecal e urinária (26,7%), dermatite de contato na região da fralda (6,7%) e necrose de cauda (6,7%). 40% dos tutores cogitaram ou foram instruídos a realizar a eutanásia, sendo que 16,7% optaram pelo procedimento. Apesar da paraplegia, 93,3% dos tutores consideram que seus animais apresentam boa qualidade de vida.(AU)

Traumas are the second largest cause of death in cats, behind only for poisonings. In the present study, 15 owners of paraplegic cats answered an online questionnaire. The greatest prevalence of paraplegia was animal cruelty (40%). The mean age was 1 year and 7 months. On average, the animals treated for 11 months, with 26.7% only using anti-inflammatory and analgesic medication, while among the others, 27.3% underwent surgery, 72.7% acupuncture, 45.5 % physiotherapy, 9% cell therapy and 9% others. However, 33.3% of the owners did not notice improvement. The most frequent concomitant diseases were constipation and urinary retention (53.5%), and recurrent cystitis (40%), followed by pressure sores (33.3%), fecal and urinary incontinence (26.7%), diaper dermatitis (6.7%) and tail necrosis (6.7%). 40% of the owners considered or were instructed to perform euthanasia, and 16.7% opted for the procedure. Despite paraplegia, 93.3% of the owners considered that their animals had a good quality of life.(AU)

Traumatismos son la segunda causa de muerte en gatos, perdiendo sólo para envenenamientos. En el presente trabajo, 15 tutores de gatos parapléjicos respondieron un cuestionario en línea. La causa de la mayor prevalencia de la paraplejia fue maltrato (40%). EI promedio de edad fue de 1 ano y 7 meses. En promedio, los animales hacen/han hecho un tratamiento durante 11 meses, siendo que el 26,7% sólo utilizó medicación antiinflamatoria y analgésica, mientras que los demás, el 27,3% fueron sometidos a la cirugía, el 72,7% hicieron acupuntura, 45,5 % fisioterapia, 9% terapia celular y 9% otros. Sin embargo, el 33,3% de los pacientes no observaron mejoría. Las enfermedades concomitantes más predominantes fueron constipación y retención de orina (53,5%), y cistitis recurrentes (40%), seguidas de escaras (33,3%), incontinencia fecal y urinaria (26,7%), dermatitis de contacto región del panal (6,7%) y necrosis de cola (6,7%). EI 40% de los tutores pensó o fue instruido para realizar una eutanasia, y el 16,7% optó por el procedimiento. A pesar de la paraplejia, el 93,3% de los derechos de autor en sus animales presentan buena calidad de vida.(AU)

Uso da glicerina para a substituição do formaldeído na conservação de peças anatômicas / Gliceryn can replace formaldehyde for anatomic conservation

A formalização é a principal forma de conservação de corpos e peças anatômicas, devido principalmente ao seu baixo custo. A técnica baseia-se na utilização do formaldeído (5-20%) como fixador e conservador. Entretanto existem vários fatores negativos à sua utilização, como odor desagradável, escurecimento, aumento de peso e rigidez das peças e sérios problemas ambientais quando descartada de forma incorreta, além de ser um produto classificado pela Agência Internacional de Pesquisas em Câncer como altamente cancerígeno. Existem várias opções para substituir o formaldeído, a glicerinação é uma delas, cujo será tratada neste trabalho. A glicerina atua como antifúngico e bactericida, além de ter muitas vantagens em relação ao formol, tratando-se de odor, textura e coloração, além de não ser prejudicial a saúde. Porém o seu custo ainda é elevado e isso explica sua pouca utilização em laboratórios de anatomia. Este trabalho tem como objetivo mostrar as vantagens do uso da glicerina em relação ao formol e propor uma possível substituição deste produto cancerígeno para preservar a saúde dos alunos, funcionários e professores dentro do laboratório, além de permitir um ambiente mais agradável para o aprendizado.(AU)

The formalization is the main form of body and anatomic conservation, mainly due to its low cost. The technique is based on the use of formaldehyde (5-20%) as fixative and preservative. However there are several negative factors on their use, unpleasant smell, browning, weight gain and stiffness of parts and serious environmental problems if disposed of incorrectly, and is a product classified by the International Agency for Research on Cancer as highly carcinogenic. There are several options to replace formaldehyde and glicerinação is one of them, whose which will be treated in this work. Glycerin dehydrates the cell and acts as antifungal and antibacterial, and has many advantages compared to formaldehyde. But its cost is still high and this explains their limited use in the anatomy lab. This work aims to show the advantages of using glycerin in relation to formaldehyde and propose a possible replacement of this carcinogen to preserve the health of students, staff and teachers within the laboratory, and allows a more pleasant environment for learning.(AU)

Uso da glicerina para a substituição do formaldeído na conservação de peças anatômicas / Gliceryn can replace formaldehyde for anatomic conservation

A formalização é a principal forma de conservação de corpos e peças anatômicas, devido principalmente ao seu baixo custo. A técnica baseia-se na utilização do formaldeído (5-20%) como fixador e conservador. Entretanto existem vários fatores negativos à sua utilização, como odor desagradável, escurecimento, aumento de peso e rigidez das peças e sérios problemas ambientais quando descartada de forma incorreta, além de ser um produto classificado pela Agência Internacional de Pesquisas em Câncer como altamente cancerígeno. Existem várias opções para substituir o formaldeído, a glicerinação é uma delas, cujo será tratada neste trabalho. A glicerina atua como antifúngico e bactericida, além de ter muitas vantagens em relação ao formol, tratando-se de odor, textura e coloração, além de não ser prejudicial a saúde. Porém o seu custo ainda é elevado e isso explica sua pouca utilização em laboratórios de anatomia. Este trabalho tem como objetivo mostrar as vantagens do uso da glicerina em relação ao formol e propor uma possível substituição deste produto cancerígeno para preservar a saúde dos alunos, funcionários e professores dentro do laboratório, além de permitir um ambiente mais agradável para o aprendizado.(AU)

The formalization is the main form of body and anatomic conservation, mainly due to its low cost. The technique is based on the use of formaldehyde (5-20%) as fixative and preservative. However there are several negative factors on their use, unpleasant smell, browning, weight gain and stiffness of parts and serious environmental problems if disposed of incorrectly, and is a product classified by the International Agency for Research on Cancer as highly carcinogenic. There are several options to replace formaldehyde and glicerinação is one of them, whose which will be treated in this work. Glycerin dehydrates the cell and acts as antifungal and antibacterial, and has many advantages compared to formaldehyde. But its cost is still high and this explains their limited use in the anatomy lab. This work aims to show the advantages of using glycerin in relation to formaldehyde and propose a possible replacement of this carcinogen to preserve the health of students, staff and teachers within the laboratory, and allows a more pleasant environment for learning.(AU)