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)

Antimicrobial activity of essential oils against Pasteurella spp isolated from the oral cavity of domestic cats / Avaliação da atividade antimicrobiana de óleos essenciais frente a isolados de Pasteurella spp oriundos da cavidade bucal de gatos domésticos

Pasteurella spp. have been identified predominantly in the oral microbiota of domestic cats. However, Pasteurella spp. was significantly more prevalent in cats with inflammatory oral disease; and consequently, it was considered as part of the etiology in this disease. In addition, in animals, Pasteurella spp. have become increasingly resistant to a large number of antimicrobials. Natural products, especially essential oils, could contribute to minimizing this issue. This study determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of six essential oils against Pasteurella spp. isolates from the oral cavity of domestic cats. Our results showed that essential oils with better antimicrobial effectiveness against most of the Pasteurella isolates were lemongrass, tea tree and clove, with inhibition values between 50 to 800 µg mL-¹. All essential oils showed bacteriostatic activity against the species of Pasteurella isolated from the domestic cats. These results suggested that lemongrass, tea tree and clove oils have potential to be used in products for oral hygiene and treatment of oral infections in domestic cats.

O gênero Pasteurella spp., considerado um comensal da cavidade bucal de gatos domésticos, vem sendo, nos últimos anos, apontado como possível agente etiológico de quadros inflamatórios crônicos bucais em felinos. Ademais, em animais, as espécies de Pasteurella têm apresentado cada vez mais resistência a um grande número de antimicrobianos de uso rotineiro. Nesse contexto, os produtos naturais, como óleos essenciais com potencial antimicrobiano tem sido alvo de estudos e apontados como alternativa terapêutica. Neste estudo, objetivou-se determinar a Concentração Mínima Inibitória (CMI) e da Concentração Bactericida Mínima (CBM) de seis óleos essenciais frente a isolados de Pasteurella spp. oriundos da cavidade bucal de gatos domésticos. Dos óleos essenciais testados, o capim-limão, tea tree, cravo e a camomila romana apresentaram ação bacteriostática frente aos isolados de Pasteurella spp. Contudo, os óleos de capim-limão, tea-tree e cravo apresentaram os melhores resultados, com valores de inibitórios entre 50 a 800 µg mL-¹. Esses resultados sugerem que os óleos de capim-limão, tea tree e cravo têm potencial para serem utilizados como produtos para higiene bucal e para o tratamento de infecções da cavidade bucal de gatos domésticos.

Characterization of hematopoietic stem cells from the canine yolk sac

The characterization of hematopoietic stem cells (HSC) from the canine yolk sac (cYS) can contribute to future gene therapies because it is possible to obtain information about the beginning of the development of the circulatory system through the characterization. The cYS is a likely source of HSC, which is a source of blood cell development in mammals. Studies in this field have been conducted for decades; however, interest in cellular therapy is currently at its peak with greater visibility, and these cells are a promising therapeutic tool for the treatment of diseases related to animals and humans. The aim of this study was to isolate and characterize HSC from the cYS embryos at 30 to 45 days of gestational age. Our results showed that the cYS was macroscopically located in the ventral region with a central portion and extremities. The cells in culture presented a circular morphology and cell clusters. The average cell viability was 22.55% dead cells out of 6.5 × 104 total cells. The cells were also able to form colonies on methylcellulose. Flow cytometry analysis revealed the expression of CD34, CD117, and CD45. Our results suggest that the cYS can be used as a source of hematopoietic cells, and this study is very important to understand the mechanism and development of the hematopoietic system in dogs.(AU)

Integration of heterologous bone matrix associated with polymethylmethacrylate in induced tibial bone defects. An experimental study in rabbits

ABSTRACT Purpose To analyze and compare the reactions at the interface between the composite, composed of fragmented heterologous mineralized bone matrix (MOMHF) and polymethylmethacrylate (PMMA), and the rabbit's tibias, through macroscopic evaluation and scanning electron microscopy (SEM) in different periods. Methods In this study, 12 New Zealand adult rabbits were used (E1: n = 3, E2: n = 3, E3: n = 3 and E4: n = 3). They had the right tibial defects filled with composite and were evaluated immediately after surgery and at 30, 60, 90, and 120 days. Results The composites were incorporated and integrated into the recipient beds in 100% of the cases, defined by the MOMHF osseointegration and the PMMA fibrointegration, with no sign of infection, migration, or rejection. Conclusions The behavior of the composites in the recipient beds demonstrates that these biomaterials have the potential to be used in bone defect repairs, offering, thus, better quality of life to the orthopedic patient.

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.

Pluripotent stem cells proliferation is associated with placentation in dogs

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.

Pluripotent stem cells proliferation is associated with placentation in dogs

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.(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.(AU)

Região metafisária da tíbia de coelhos: uma opção para estudos dos substitutos ósseos / Metaphyseal region of the tibia of rabbits: an option for bone substitutes studies

Apresenta-se um modelo experimental de ostectomia de tíbia em coelhos para estudo de biomateriais em processos de reparação óssea. Foi realizada falha segmentar de 6mm de diâmetro na região metafisária medial proximal de tíbia de 72 coelhos as quais foram preenchidas com substituto ósseo e avaliadas clinicamente, por exame radiográfico e por meio de tomografia computadorizada feixe cônico, em diferentes tempos. Conclui-se que a metáfise medial proximal de tíbia de coelhos é adequada como modelo para estudos que avaliem o comportamento de enxertos e/ou biomateriais em falhas ósseas.

Is presented an experimental model of tibial ostectomy in rabbits to study biomaterials during bone repair process. Segmental failure of 6 mm diameter was performed in the medial proximal tibial metaphyseal region of 72 rabbits, which were filled with bone substitute and evaluated by clinical exam, X-ray, and cone beam computed tomography at different times. It is concluded that the medial proximal tibial metaphysis region of rabbits is suitable as a model for studies that evaluate the behavior of grafts and/or biomaterials on bone defects.

Região metafisária da tíbia de coelhos: uma opção para estudos dos substitutos ósseos / Metaphyseal region of the tibia of rabbits: an option for bone substitutes studies

Apresenta-se um modelo experimental de ostectomia de tíbia em coelhos para estudo de biomateriais em processos de reparação óssea. Foi realizada falha segmentar de 6mm de diâmetro na região metafisária medial proximal de tíbia de 72 coelhos as quais foram preenchidas com substituto ósseo e avaliadas clinicamente, por exame radiográfico e por meio de tomografia computadorizada feixe cônico, em diferentes tempos. Conclui-se que a metáfise medial proximal de tíbia de coelhos é adequada como modelo para estudos que avaliem o comportamento de enxertos e/ou biomateriais em falhas ósseas.(AU)

Is presented an experimental model of tibial ostectomy in rabbits to study biomaterials during bone repair process. Segmental failure of 6 mm diameter was performed in the medial proximal tibial metaphyseal region of 72 rabbits, which were filled with bone substitute and evaluated by clinical exam, X-ray, and cone beam computed tomography at different times. It is concluded that the medial proximal tibial metaphysis region of rabbits is suitable as a model for studies that evaluate the behavior of grafts and/or biomaterials on bone defects.(AU)

Região metafisária da tíbia de coelhos: uma opção para estudos dos substitutos ósseos / Metaphyseal region of the tibia of rabbits: an option for bone substitutes studies

Apresenta-se um modelo experimental de ostectomia de tíbia em coelhos para estudo de biomateriais em processos de reparação óssea. Foi realizada falha segmentar de 6mm de diâmetro na região metafisária medial proximal de tíbia de 72 coelhos as quais foram preenchidas com substituto ósseo e avaliadas clinicamente, por exame radiográfico e por meio de tomografia computadorizada feixe cônico, em diferentes tempos. Conclui-se que a metáfise medial proximal de tíbia de coelhos é adequada como modelo para estudos que avaliem o comportamento de enxertos e/ou biomateriais em falhas ósseas.

Is presented an experimental model of tibial ostectomy in rabbits to study biomaterials during bone repair process. Segmental failure of 6 mm diameter was performed in the medial proximal tibial metaphyseal region of 72 rabbits, which were filled with bone substitute and evaluated by clinical exam, X-ray, and cone beam computed tomography at different times. It is concluded that the medial proximal tibial metaphysis region of rabbits is suitable as a model for studies that evaluate the behavior of grafts and/or biomaterials on bone defects.

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.

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)

Mid-pregnancy ewe shearing and the effects on fetus liver and muscle glycoprotein deposits

The reason why shearing ewes in midpregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoproteinreactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm2 in SE and 34,830.73 µm2 in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm2 and 31,942.7 µm2 , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.(AU)

Mid-pregnancy ewe shearing and the effects on fetus liver and muscle glycoprotein deposits

The reason why shearing ewes in midpregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoproteinreactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm2 in SE and 34,830.73 µm2 in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm2 and 31,942.7 µm2 , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.

Evaluation of polyamide surgical mesh as an abdominal ventral implant in rabbits

Purpose: To evaluate abdominal ventral wound healing by using a specific biomaterial, a handmade polyamide surgical mesh. Methods: A surgical incisional defect was made in ten rabbits to simulate a hernia in the ventral abdominal musculature. A polyamide surgical mesh was used in hernioplasty. They were monitored for surgical wound healing, and macroscopically and histologically evaluated at the end of the experiment. The polyamide surgical mesh did not cause foreign body reaction, pain, edema, or infection in the surgical site. The manure production was not affected by intestinal tissue adherences to the mesh, consistent with the ultrasonography result where adherences were not observed and organized scarring tissue formed in the incisional defect. The polyamide mesh was fixed over the abdominal wall, and its external and internal sides were surrounded by a vascularized connective tissue. Results: None of the experimental animals developed adherences from internal organs to the polyamide mesh, except two rabbits where the omentum formed adherence to the internal scarring tissue without present herniation or compromise of the rabbits health. Conclusion: Polyamide surgical mesh for hernioplasty presents, in rabbits, excellent biocompatibility, with minimal body adverse reactions and low cost.(AU)

Glial scar-modulation as therapeutic tool in spinal cord injury in animal models

Purpose: Spinal Cord injury represents, in veterinary medicine, most of the neurological attendances and may result in permanent disability, death or euthanasia. Due to inflammation resulting from trauma, it originates the glial scar, which is a cell interaction complex system. Its function is to preserve the healthy circuits, however, it creates a physical and molecular barrier that prevents cell migration and restricts the neuroregeneration ability. Methods: This review aims to present innovations in the scene of treatment of spinal cord injury, approaching cell therapy, administration of enzyme, anti-inflammatory, and other active principles capable of modulating the inflammatory response, resulting in glial scar reduction and subsequent functional improvement of animals. Results: Some innovative therapies as cell therapy, administration of enzymes, immunosuppressant or other drugs cause the modulation of inflammatory response proved to be a promising tool for the reduction of gliosis. Conclusion: Those tools promise to reduce gliosis and promote locomotor recovery in animals with spinal cord injury.(AU)

Bone marrow stem cell applied in the canine veterinary clinics / Células tronco de medula óssea utilizadas na clínica veterinária canina

Cell therapy represents an old therapeutic practice initiated with the transfusion of whole blood in different clinical situations. There is now a breakthrough in the study of multipotent stem cell therapy because of its functionality in regeneration of tissues, which promotes attention of the scientific community. Bone marrow is one of the main sources of multipotent stem cells, composed by hematopoietic stem cells responsible for the renewal of the cellular components of the blood, and mesenchymal stem cells that aid in the regeneration of tissues. These cells have a strong potential for the treatment of several diseases, due their main characteristics such as high plasticity, capacity for self-renewal and immunomodulation. Although, there are many studies that show good results with the use of cell therapy as a form of treatment for several diseases, some studies still show inconclusive or unsatisfactory results. Therefore, the objective of this study was to review the application of bone marrow stem cells in the canine model since improvements on the knowledge of the technique are necessary to enable its applicability with safety and efficacy.(AU)

A terapia celular representa uma antiga prática terapêutica iniciada com a transfusão de sangue total em diferentes situações clínicas. Atualmente há um avanço no estudo da terapia com células-tronco mesenquimais por conta de sua funcionalidade na regeneração de tecidos, o que promove uma crescente atenção do meio científico. A medula óssea é uma das principais fontes de células-tronco multipotentes, no qual se encontram as células-tronco hematopoiéticas, responsável pela renovação dos componentes celulares do sangue, e as células-tronco mesenquimais que auxiliam na regeneração de tecidos. Essas células têm um forte potencial para o tratamento de diversas enfermidades, uma vez que possuem como principais características alta plasticidade, capacidade de auto renovação e imunomodulação. Apesar de haver muitos trabalhos que apresentam bons resultados com a utilização da terapia celular como forma de tratamento para diversas enfermidades, alguns estudos ainda demonstram resultados inconclusivos ou não satisfatórios, por isso, objetivou-se com este trabalho revisar a aplicação das células-tronco derivadas da medula óssea no modelo canino uma vez que é necessário melhorias sobre o conhecimento da técnica para que possibilite a sua aplicabilidade com segurança e eficácia.(AU)