Experimental study of amniotic membrane or topical propolis on induced cutaneous wound healing in Wistar rats / Estudo experimental do uso da membrana amniótica e da própolis tópica na cicatrização de feridas cutâneas induzidas em ratos Wistar

The study of propolis and the amniotic membrane as alternatives to promote the acceleration of healing has grown considerably. These products have shown to be easy to use, have bactericidal or bacteriostatic action and are affordable. The objective of the present study was to evaluate the action of the amniotic membrane of bitches and propolis in the healing of surgical wounds. To evaluate the efficacy of these methods the time for conclusion of the healing process of both groups was compared with a control group. In addition, the inflammatory response of each group was also evaluated to identify which alternative method could induce a better response in the healing process compared to the control group. For this purpose, 27 animals, Rattus norvegicus, variety Wistar, were used. Rats were separated into 3 groups with 9 animals each. Group I represented the 25% alcoholic propolis solution; Group II, Amniotic membrane; Group III was Control group. Wounds were assessed macroscopically for the lesion area, and microscopically for the histological quality of each wound layer and the intensity of inflammation and collagen deposition. After the macroscopic analysis, it was observed that there were no statistical differences among the 3 groups (p> 0.05) relevant to accelerate the healing process in terms of presence of inflammatory cells, fibroblasts, collagen, neovascularization, granulation tissue and re-epithelialization.

O estudo da própolis e da membrana amniótica como alternativa para promover a aceleração da cicatrização vem crescendo bastante, numa tentativa de buscar métodos alternativos para cicatrização de feridas que apresentem fácil utilização, ação bactericida ou bacteriostática e tenha custo acessível. Com o presente estudo objetivou-se avaliar a ação da membrana amniótica de cadelas e da própolis na cicatrização de feridas cirúrgicas, comparando o tempo no processo de cicatrização com o grupo controle e a resposta inflamatória de cada um, de maneira a identificar qual método alternativo induzirá melhor res-posta no processo de cicatrização em comparação ao grupo controle. Para tanto, foram utilizados 27 animais, Rattus norve-gicus, variedade Wistar, separados em três grupos com nove animais cada, sendo Grupo I – solução alcoólica de própolis a 25%; Grupo II – Membrana amniótica de cadelas; Grupo III – Grupo controle. As feridas foram avaliadas macroscopica-mente quanto à área da lesão, e microscopicamente quanto à qualidade histológica de cada leito da ferida e à intensidade de inflamação e deposição de colágeno. Após a análise macroscópica, constatou-se que não houve diferenças estatísticas (p>0,05) relevantes para acelerar o processo cicatricial, quanto a presença de células inflamatórias, fibroblastos, colágeno, neovasculari-zação, tecido de granulação e reepitelização, perante os tratamentos alternativos e o grupo controle no processo de cicatrização.

Experimental study of amniotic membrane or topical propolis on induced cutaneous wound healing in Wistar rats / Estudo experimental do uso da membrana amniótica e da própolis tópica na cicatrização de feridas cutâneas induzidas em ratos Wistar

The study of propolis and the amniotic membrane as alternatives to promote the acceleration of healing has grown considerably. These products have shown to be easy to use, have bactericidal or bacteriostatic action and are affordable. The objective of the present study was to evaluate the action of the amniotic membrane of bitches and propolis in the healing of surgical wounds. To evaluate the efficacy of these methods the time for conclusion of the healing process of both groups was compared with a control group. In addition, the inflammatory response of each group was also evaluated to identify which alternative method could induce a better response in the healing process compared to the control group. For this purpose, 27 animals, Rattus norvegicus, variety Wistar, were used. Rats were separated into 3 groups with 9 animals each. Group I represented the 25% alcoholic propolis solution; Group II, Amniotic membrane; Group III was Control group. Wounds were assessed macroscopically for the lesion area, and microscopically for the histological quality of each wound layer and the intensity of inflammation and collagen deposition. After the macroscopic analysis, it was observed that there were no statistical differences among the 3 groups (p> 0.05) relevant to accelerate the healing process in terms of presence of inflammatory cells, fibroblasts, collagen, neovascularization, granulation tissue and re-epithelialization.(AU)

O estudo da própolis e da membrana amniótica como alternativa para promover a aceleração da cicatrização vem crescendo bastante, numa tentativa de buscar métodos alternativos para cicatrização de feridas que apresentem fácil utilização, ação bactericida ou bacteriostática e tenha custo acessível. Com o presente estudo objetivou-se avaliar a ação da membrana amniótica de cadelas e da própolis na cicatrização de feridas cirúrgicas, comparando o tempo no processo de cicatrização com o grupo controle e a resposta inflamatória de cada um, de maneira a identificar qual método alternativo induzirá melhor res-posta no processo de cicatrização em comparação ao grupo controle. Para tanto, foram utilizados 27 animais, Rattus norve-gicus, variedade Wistar, separados em três grupos com nove animais cada, sendo Grupo I solução alcoólica de própolis a 25%; Grupo II Membrana amniótica de cadelas; Grupo III Grupo controle. As feridas foram avaliadas macroscopica-mente quanto à área da lesão, e microscopicamente quanto à qualidade histológica de cada leito da ferida e à intensidade de inflamação e deposição de colágeno. Após a análise macroscópica, constatou-se que não houve diferenças estatísticas (p>0,05) relevantes para acelerar o processo cicatricial, quanto a presença de células inflamatórias, fibroblastos, colágeno, neovasculari-zação, tecido de granulação e reepitelização, perante os tratamentos alternativos e o grupo controle no processo de cicatrização.(AU)

Aspectos clínicos de implantes de membrana amniótica no reparo da córnea em cães com úlceras complicadas / Clinical aspects of amniotic membrane implants in corneal repair in dogs with complicated ulcers

Background: Among numerous disorders treated by veterinary ophthalmology, therapies employed to solve corneal ulcers stand out. Amniotic membrane transplantation is an effective surgical technique for the treatment of complicated corneal ulcers in the dog, with highly satisfactory visual and cosmetic outcomes. However, in veterinary ophthalmology, reports on the use of the amniotic membrane and its corneal reconstructive potential are scarce. The objective of this work was to evaluate, for 21 days, the clinical aspects and the effectiveness of transplantation of canine amniotic membrane for corneal healing in two cases of complicated ulcers in dogs. Cases: Two Shih-Tzu dogs were diagnosed with complicated corneal ulcer. They underwent surgical treatment with the use of a glycerin-preserved canine amniotic membrane implant, which was secured on the cornea or the limbus. A surgical microscope at a magnification of 16x was used. The devitalized tissues at the periphery of the ulcer were excised, and multiple layers of amniotic membranes were sutured onto the cornea and near the limbus with interrupted absorbable suture. Protection of the canine amniotic membrane implants was performed with a scarified third eyelid flap, which was attached to the upper eyelid, and maintained for 14 days. During the first 14 days after the procedure, topical treatment with antibiotic eye drops was administered; protease inhibitors were also used. Antibiotics were used systemically for 10 days. Between days 14 and 21 after the transplantation procedure, protease inhibitors, corticosteroids, and lubricant were administered topically. Healing and clinical aspects were evaluated on days 0, 14, and 21. The parameters evaluated were blepharospasm, dazzle and direct pupillary reflexes, and consensual and threat responses. Corneal vascularization, hypopyon, synechia, opacity, and fluorescein dye penetration into the...

Aspectos clínicos de implantes de membrana amniótica no reparo da córnea em cães com úlceras complicadas / Clinical aspects of amniotic membrane implants in corneal repair in dogs with complicated ulcers

Background: Among numerous disorders treated by veterinary ophthalmology, therapies employed to solve corneal ulcers stand out. Amniotic membrane transplantation is an effective surgical technique for the treatment of complicated corneal ulcers in the dog, with highly satisfactory visual and cosmetic outcomes. However, in veterinary ophthalmology, reports on the use of the amniotic membrane and its corneal reconstructive potential are scarce. The objective of this work was to evaluate, for 21 days, the clinical aspects and the effectiveness of transplantation of canine amniotic membrane for corneal healing in two cases of complicated ulcers in dogs. Cases: Two Shih-Tzu dogs were diagnosed with complicated corneal ulcer. They underwent surgical treatment with the use of a glycerin-preserved canine amniotic membrane implant, which was secured on the cornea or the limbus. A surgical microscope at a magnification of 16x was used. The devitalized tissues at the periphery of the ulcer were excised, and multiple layers of amniotic membranes were sutured onto the cornea and near the limbus with interrupted absorbable suture. Protection of the canine amniotic membrane implants was performed with a scarified third eyelid flap, which was attached to the upper eyelid, and maintained for 14 days. During the first 14 days after the procedure, topical treatment with antibiotic eye drops was administered; protease inhibitors were also used. Antibiotics were used systemically for 10 days. Between days 14 and 21 after the transplantation procedure, protease inhibitors, corticosteroids, and lubricant were administered topically. Healing and clinical aspects were evaluated on days 0, 14, and 21. The parameters evaluated were blepharospasm, dazzle and direct pupillary reflexes, and consensual and threat responses. Corneal vascularization, hypopyon, synechia, opacity, and fluorescein dye penetration into the...(AU)

Desenvolvimento embrionário/fetal, placentação e glicosaminoglicanos no aparelho reprodutor feminino e na placenta / Embryonic/fetal development, placentation and glycosaminoglycans in the female reproductive tract and placenta

Background: Fetal attachments, placentation and embryonic development have been widely discussed in rodents such as agoutisand cavies, as well as research on glycosaminoglycans (GAGs) in rats and rabbits. Moreover, studies on buffalo, cattle and sheep aredescribed in ruminants, and work has also been reported in sheep with GAGs in placentoma. However, further studies are needed inthis regard, since there are reports of economic losses associated with reproductive failures described for cattle such as changes in thechorion and allantois, and in sheep in which changes between the transition from vitelline to allantois circulation have been discussed.Review: In relation to embryonic development, detailed studies have been described in rodents such as rats (12 days old), desertmouse (15 days old) and agoutis at 30 days. Macroscopic structures such as the cephalic region, nose, optic vesicle, cervical curvature, thoracic and pelvic limbs were observed, as well as microscopic structures such as the pituitary, lung, heart, brain cavity, liver,retina, and ossification regions. There are reports of buffalo and cattle studies in ruminants describing early embryonic development.However, the research in the case of sheep is limited, meaning there is only the ultrasound examination, such as gestational diagnosisand morphometric measurement of the embryonic vesicle. Still, studies with umbilical funicular and placental development of sheepwith different gestational ages can be highlighted. Regarding extraembryonic annexes, four important structures which contributeto embryonic maintenance have been reported. These are called the chorion, amnion, allantois and yolk sac, respectively, and areresponsible for originating the placenta, embryonic protection, collecting metabolic waste and early embryonic nutrition. In...

Desenvolvimento embrionário/fetal, placentação e glicosaminoglicanos no aparelho reprodutor feminino e na placenta / Embryonic/fetal development, placentation and glycosaminoglycans in the female reproductive tract and placenta

Background: Fetal attachments, placentation and embryonic development have been widely discussed in rodents such as agoutisand cavies, as well as research on glycosaminoglycans (GAGs) in rats and rabbits. Moreover, studies on buffalo, cattle and sheep aredescribed in ruminants, and work has also been reported in sheep with GAGs in placentoma. However, further studies are needed inthis regard, since there are reports of economic losses associated with reproductive failures described for cattle such as changes in thechorion and allantois, and in sheep in which changes between the transition from vitelline to allantois circulation have been discussed.Review: In relation to embryonic development, detailed studies have been described in rodents such as rats (12 days old), desertmouse (15 days old) and agoutis at 30 days. Macroscopic structures such as the cephalic region, nose, optic vesicle, cervical curvature, thoracic and pelvic limbs were observed, as well as microscopic structures such as the pituitary, lung, heart, brain cavity, liver,retina, and ossification regions. There are reports of buffalo and cattle studies in ruminants describing early embryonic development.However, the research in the case of sheep is limited, meaning there is only the ultrasound examination, such as gestational diagnosisand morphometric measurement of the embryonic vesicle. Still, studies with umbilical funicular and placental development of sheepwith different gestational ages can be highlighted. Regarding extraembryonic annexes, four important structures which contributeto embryonic maintenance have been reported. These are called the chorion, amnion, allantois and yolk sac, respectively, and areresponsible for originating the placenta, embryonic protection, collecting metabolic waste and early embryonic nutrition. In...(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.

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)

Effect of In Ovo Feeding of L-Glutamine to Chick Embryos

In ovo feeding (IOF), injecting nutrients into the amnion of the avian embryo may enhance hatchability, gastrointestinal development and serum metabolism changes. This hypothesis was evaluated with 5 IOF solutions containing L-glutamine. Were used 315 fertile Rhode Island Red eggs were used (breeders with 32-weeks). The experimental design was completely randomized with the treatments constituted by two controls and five solutions containing L-glutamine levels with 45 replicates each. Data collected were subjected to polynomial regression at 5% of significance. Differences (p>0.05) were observed in hatchability and intermediary mortality, with a gradual lower of decrease in hatchability from the IOF of L-glutamine. The yolk sac was higher (p 0.05) after the IOF (control and L-glutamine) in the in ovo fed embryos at 0.5% L-glutamine. IOF of L-glutamine alone enhanced the blood pH and reduced the other serum parameters (p 0.05), which may have fuelled more embryo development, mainly vital organs how as the heart and the liver due to the larger concentration of available nutrients for the embryo. The results of this study indicate that until 0.5% L-glutamine may be supplemented in-ovo to chick embryos without negative influence on chick weight and gastrointestinal tract development, acting as serum biochemical metabolism regulator and obtaining better hatchability.(AU)

Effect of In Ovo Feeding of L-Glutamine to Chick Embryos

In ovo feeding (IOF), injecting nutrients into the amnion of the avian embryo may enhance hatchability, gastrointestinal development and serum metabolism changes. This hypothesis was evaluated with 5 IOF solutions containing L-glutamine. Were used 315 fertile Rhode Island Red eggs were used (breeders with 32-weeks). The experimental design was completely randomized with the treatments constituted by two controls and five solutions containing L-glutamine levels with 45 replicates each. Data collected were subjected to polynomial regression at 5% of significance. Differences (p>0.05) were observed in hatchability and intermediary mortality, with a gradual lower of decrease in hatchability from the IOF of L-glutamine. The yolk sac was higher (p 0.05) after the IOF (control and L-glutamine) in the in ovo fed embryos at 0.5% L-glutamine. IOF of L-glutamine alone enhanced the blood pH and reduced the other serum parameters (p 0.05), which may have fuelled more embryo development, mainly vital organs how as the heart and the liver due to the larger concentration of available nutrients for the embryo. The results of this study indicate that until 0.5% L-glutamine may be supplemented in-ovo to chick embryos without negative influence on chick weight and gastrointestinal tract development, acting as serum biochemical metabolism regulator and obtaining better hatchability.

Structural plasticity and isolation of umbilical cord progenitor cells of agouti (Dasyprocta prymnolopha) raised in captivity / Plasticidade estrutural e isolamento de células progenitoras do cordão umbilical de cutias (Dasyprocta prymnolopha) criadas em cativeiro

The agouti has been used as an experimental model in several studies focused on reproductive biology. The umbilical cord, an embryonic attachment that connects the foetus to the placenta, has been reported as an important anatomical site for obtaining stem cells. The objective of this study was to describe macro- and microscopically the umbilical cord of agoutis at different stages of gestation, to expand and cultivate in vitro the progenitor cells and to report their morphological characteristics. Seven cutias were submitted to caesarean section to collect the umbilical cords: five were destined for studies of cord structure in different stages of gestation (30, 35, 50, 75 and 100 days postcoital), and two were collected in the third stage of gestation for isolation and cell culture. The umbilical cord of cutias assumes a spiral arrangement, with veins and arteries on it starting 50 days after coitus. The arteries present an outer layer of smooth muscle fibres in a longitudinal and circular arrangement and a medium layer of smooth muscle fibres with only longitudinal and intimate orientation and coated by the endothelium. The veins consist of longitudinal smooth muscle fibres with an extract of smooth muscle cells, and the endothelium, in all analysed gestational phases, is a structure bounded by simple pavement epithelial tissue originating from the amnion, adhered to Whartons Jelly and forming the umbilical vessels and allantoid duct. The proposed protocol allowed the collection of a high cellular concentration of umbilical cord progenitor cells from viable cutias.(AU)

A cutia vem sendo utilizada como modelo experimental em diversos estudos voltados à biologia reprodutiva. O cordão umbilical, anexo embrionário que une o feto à placenta, tem sido relatado como um importante sítio anatômico para obtenção de células-tronco. O objetivo deste estudo foi descrever macro e microscopicamente o cordão umbilical de cutias, em fases diferentes da gestação, expandir e cultivar in vitro as células progenitoras e relatar suas características morfológicas. Foram utilizadas sete cutias submetidas à cesariana para a coleta dos cordões umbilicais, cinco foram destinadas aos estudos da estrutura do cordão, em diferentes estágios de gestação (30, 35, 50, 75 e 100 dias pós-coito), e duas, no terço final da gestação, para isolamento e cultivo celular. O cordão umbilical de cutia assume disposição espiralada, com veias e artérias sobre ele a partir dos 50 dias após o coito. As artérias apresentam camada externa de fibras musculares lisas, disposição longitudinal e circular, camada média de fibras musculares lisas, apenas com disposição longitudinal e íntima revestida pelo endotélio. As veias constituídas por fibras musculares lisas longitudinais com um extrato de células musculares lisas e pelo endotélio. Em todas as fases gestacionais analisadas é uma estrutura delimitada por tecido epitelial simples pavimentoso, proveniente do âmnio, aderido a Geleia de Wharton e com formação de vasos umbilicais e ducto alantóide. O protocolo proposto permitiu a coleta de células progenitoras do cordão umbilical de cutias, viáveis com elevada concentração celular.(AU)

Effects of the application of the amniotic membrane in the healing process of skin wounds in rats

Purpose: To evaluate the efficacy of the application of the human amniotic membrane (HAM) on the inflammatory process, fibroblast proliferation, formation of collagenand reduction of skin wound areas in rats.Methods: Thirty six rats were submitted to a surgical injury induction and divided into two groups (n = 18): group C (control) and T (treated with the HAM). The macroscopic evolution in the wound area and the histological characteristics of the skin samples were evaluated.Results: The regression of the wound area was greater in group T. The histological analysis revealed a significant reduction (p < 0.05) in the inflammatory infiltrate in group T at all experimental periods compared with that in the control group. Furthermore, the group T presented a significant increase in the proliferation of fibroblasts at 14 and 21 days compared with group C (p < 0.05). Regarding the deposition of mature collagen fibers, there was an increase in the replacement of type III collagen by type I collagen in group T (p < 0.05).Conclusion: Treatment with the HAM reduced the healing time as well as the inflammatory responses, increased the proliferation of fibroblasts, and induced a higher concentration of mature collagen fibers.(AU)

Epitelização de enxertos cutâneos em feridas recentes de coelhos tratados com membrana amniótica canina e/ou laserterapia / Skin graft epithelialization in rabbit fresh wounds treated with amniotic membrane and/or laser therapy

A enxertia cutânea é uma técnica cirúrgica simples e bastante útil para o reparo de feridas, principalmente aquelas onde existe dificuldade da aplicação do fechamento primário ou de outras técnicas reconstrutivas. Entretanto, para a sobrevivência do enxerto, é necessário que o leito da ferida esteja saudável e com presença de tecido de granulação exuberante. O objetivo do presente trabalho foi avaliar a aplicação da membrana amniótica e da laserterapia como potenciais estimulantes da cicatrização em enxertos aplicados em feridas sem tecido de granulação. Foram utilizados 42 coelhos, divididos em quatro grupos: grupo controle (GC), grupo membrana (GM), grupo laser (GL) e grupo membrana e laser (GML), submetidos a avaliações macro e microscópicas. Na avaliação macroscópica, foi possível notar que os pacientes dos grupos nos quais a membrana amniótica foi utilizada (GM e GML) apresentaram evidências associadas à acentuada reação inflamatória, à falha de integração do enxerto e à consequente necrose dele. Já os pacientes do GL apresentaram melhor aspecto do enxerto no último dia de avaliação. Na análise microscópica, observou-se intensa integração do enxerto à derme, reepitelização acentuada e escassas células inflamatórias no local do enxerto no GL. O contrário foi observado nos pacientes do GM e GML, nos quais aparentemente houve rejeição da membrana. A formação de colágeno não se correlacionou com outros fatores, como inflamação e necrose, em nenhum dos grupos de tratamento. Dessa forma, é possível afirmar que a laserterapia mostrou ser efetiva, contribuindo para o processo cicatricial e a integração do enxerto. Já a membrana amniótica canina não deve ser utilizada para esse fim, pois provoca intensa reação inflamatória, além de impedir a nutrição do enxerto.(AU)

Skin grafting is a simple surgical technique and useful to repair wounds, especially those where there is a difficulty to apply primary closure skin or other reconstructive techniques. However, for graft survival a healthy wound bed and the presence of an exuberant granulation tissue are necessary. This study aimed to evaluate the application of amniotic membrane and laser therapy as potential healing stimulants in grafts applied in wounds without granulation tissue. For this, we used 42 rabbits divided into four treatment groups, control group (CG), membrane group (MG), laser group (LG) and membrane and laser group (MLG), submitted to macroscopic and microscopic evaluation. The macroscopic examination showed that the patients of the groups where the amniotic membrane was used in order to stimulate re-epithelialization (MG and MLG) presented evidences associated with severe inflammatory reaction, graft integration failure and consequent necrosis. LG patients apparently had the best graph aspect in the last valuation date. Microscopic examination showed intense integration of the graft to the dermis, high re-epithelialization level, and scarce inflammatory cells in the graft site of LG patients. The opposite was observed in patients in the MG and MLG groups, where a rejection of the membrane was observed. Finally, collagen formation was not correlated with other factors such as inflammation and necrosis in any of the treatment groups. We can conclude that laser therapy was effective, contributing to the healing process and integration of the graft. Thus, canine amniotic membrane should not be used for this purpose because it causes intense inflammatory reaction besides avoiding graft nutrition.(AU)

Epitelização de enxertos cutâneos em feridas recentes de coelhos tratados com membrana amniótica canina e/ou laserterapia / Skin graft epithelialization in rabbit fresh wounds treated with amniotic membrane and/or laser therapy

A enxertia cutânea é uma técnica cirúrgica simples e bastante útil para o reparo de feridas, principalmente aquelas onde existe dificuldade da aplicação do fechamento primário ou de outras técnicas reconstrutivas. Entretanto, para a sobrevivência do enxerto, é necessário que o leito da ferida esteja saudável e com presença de tecido de granulação exuberante. O objetivo do presente trabalho foi avaliar a aplicação da membrana amniótica e da laserterapia como potenciais estimulantes da cicatrização em enxertos aplicados em feridas sem tecido de granulação. Foram utilizados 42 coelhos, divididos em quatro grupos: grupo controle (GC), grupo membrana (GM), grupo laser (GL) e grupo membrana e laser (GML), submetidos a avaliações macro e microscópicas. Na avaliação macroscópica, foi possível notar que os pacientes dos grupos nos quais a membrana amniótica foi utilizada (GM e GML) apresentaram evidências associadas à acentuada reação inflamatória, à falha de integração do enxerto e à consequente necrose dele. Já os pacientes do GL apresentaram melhor aspecto do enxerto no último dia de avaliação. Na análise microscópica, observou-se intensa integração do enxerto à derme, reepitelização acentuada e escassas células inflamatórias no local do enxerto no GL. O contrário foi observado nos pacientes do GM e GML, nos quais aparentemente houve rejeição da membrana. A formação de colágeno não se correlacionou com outros fatores, como inflamação e necrose, em nenhum dos grupos de tratamento. Dessa forma, é possível afirmar que a laserterapia mostrou ser efetiva, contribuindo para o processo cicatricial e a integração do enxerto. Já a membrana amniótica canina não deve ser utilizada para esse fim, pois provoca intensa reação inflamatória, além de impedir a nutrição do enxerto.(AU)

Skin grafting is a simple surgical technique and useful to repair wounds, especially those where there is a difficulty to apply primary closure skin or other reconstructive techniques. However, for graft survival a healthy wound bed and the presence of an exuberant granulation tissue are necessary. This study aimed to evaluate the application of amniotic membrane and laser therapy as potential healing stimulants in grafts applied in wounds without granulation tissue. For this, we used 42 rabbits divided into four treatment groups, control group (CG), membrane group (MG), laser group (LG) and membrane and laser group (MLG), submitted to macroscopic and microscopic evaluation. The macroscopic examination showed that the patients of the groups where the amniotic membrane was used in order to stimulate re-epithelialization (MG and MLG) presented evidences associated with severe inflammatory reaction, graft integration failure and consequent necrosis. LG patients apparently had the best graph aspect in the last valuation date. Microscopic examination showed intense integration of the graft to the dermis, high re-epithelialization level, and scarce inflammatory cells in the graft site of LG patients. The opposite was observed in patients in the MG and MLG groups, where a rejection of the membrane was observed. Finally, collagen formation was not correlated with other factors such as inflammation and necrosis in any of the treatment groups. We can conclude that laser therapy was effective, contributing to the healing process and integration of the graft. Thus, canine amniotic membrane should not be used for this purpose because it causes intense inflammatory reaction besides avoiding graft nutrition.(AU)

Amniotic membrane as an option for treatment of acute Achilles tendon injury in rats

Purpose: To evaluate the effect of human amniotic membrane (hAM) fragment on inflammatory response, proliferation of fibroblast and organization of collagen fibers in injured tendon. Methods: Sixty rats were divided into 3 groups: C - surgical procedures without tendon lesion and with simulation of hAM application; I - surgical procedures, tendon injury and simulation of hAM application; T - surgical procedures, tendon injury and hAM application. These groups were subdivided into four experimental times (3, 7, 14 and 28 days). The samples underwent histological analysis and ATR-FTIR spectroscopy. Results: Histological analysis at 14 days, the T group showed collagen fibers with better alignment. At 28 days, the I group presented the characteristics described for the T group at 14 days, while this group presented aspects of a mature connective tissue. FT-IR analysis showed a clear distinction among the three groups at all experimental times and groups T and I presented more similarities to each other than to group C. Conclusion: Acute injury of tendon treated with human amniotic membrane fragment showed a faster healing process, reduction in inflammatory response, intense proliferation of fibroblasts and organization of collagen fibers.(AU)

The evolution of fetal membranes and placentation in carnivores and ungulates (Ferungulata)

Molecular phylogenetics has made a substantialcontribution to our understanding of the relationships between mammalian orders and has generated trees that can be used to examine the evolution of anatomical and physiological traits. We here summarize findings on fetal membranes and placen tation in Ferungulata, a clade comprising carnivores, pangolins, and even and odd-toed ungulates. Their Early ontogeny shows several conserve traits such as superficial attachment of the blastocyst, amnion formation by folding, a large allantoic sac and a temporary yolk sac placenta. In contrast, Several characters of the chorioallantoic placenta are derived, including the diffuse and cotyledonary placental types in ungulate sand zonary placenta in carnivores specializations of the interhaemal barrier, the presence of areolae or haemophagous regions and lack of stromal decidual cells. Ungulates Produce large amounts of placental proteins including placental lactogens and pregnancy-associated glycoproteins. Evolutionary in novations of the placental system may contribute to the high diversity of lifestyles within Ferungulata And be linked to the evolution of highly precocial offspring in ungulates.

The evolution of fetal membranes and placentation in carnivores and ungulates (Ferungulata)

Molecular phylogenetics has made a substantialcontribution to our understanding of the relationships between mammalian orders and has generated trees that can be used to examine the evolution of anatomical and physiological traits. We here summarize findings on fetal membranes and placen tation in Ferungulata, a clade comprising carnivores, pangolins, and even and odd-toed ungulates. Their Early ontogeny shows several conserve traits such as superficial attachment of the blastocyst, amnion formation by folding, a large allantoic sac and a temporary yolk sac placenta. In contrast, Several characters of the chorioallantoic placenta are derived, including the diffuse and cotyledonary placental types in ungulate sand zonary placenta in carnivores specializations of the interhaemal barrier, the presence of areolae or haemophagous regions and lack of stromal decidual cells. Ungulates Produce large amounts of placental proteins including placental lactogens and pregnancy-associated glycoproteins. Evolutionary in novations of the placental system may contribute to the high diversity of lifestyles within Ferungulata And be linked to the evolution of highly precocial offspring in ungulates.(AU)

Isolation, culture, characterization and cryopreservation of stem cells derived from amniotic mesenchymal layer and umbilical cord tissue of bovine fetuses / Isolamento, cultura, caracterização e criopreservação de células tronco derivadas de camada amniótica mesenquimal e de tecido do cordão umbilical de fetuses bovinos

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)

Isolation, culture, characterization and cryopreservation of stem cells derived from amniotic mesenchymal layer and umbilical cord tissue of bovine fetuses / Isolamento, cultura, caracterização e criopreservação de células tronco derivadas de camada amniótica mesenquimal e de tecido do cordão umbilical de fetuses bovinos

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)

Membrana amniótica: fonte alternativa de células-tronco mesenquimais em diversas espécies animais / Amniotic membrane: alternative source of mesenchymal stromal cells in several animals species

O crescente interesse do uso de células-tronco (CTs) na medicina regenerativa, estão baseadas na sua capacidade de autorrenovação, reparação tecidual e imunomodulação. A terapia utilizando células-tronco é uma ciência em formação considerada uma alternativa terapêutica promissora no tratamento de diversas patologias degenerativas, hereditárias e inflamatórias. A busca por fontes alternativas de CTs que possuam características como fácil obtenção e isolamento, tem levado ao estudo dos mais variados tecidos, onde se inclui os anexos embrionários como a membrana amniótica. Porém, para sua efetiva aplicação terapêutica são necessários estudos de suas características "in vitro", além de testes pré-clínicos e clínicos. Com isso, o objetivo deste trabalho é realizar uma revisão bibliográfica sobre a caracterização e aplicação clínica das células-tronco da membrana amniótica em diversas espécies de animais domésticos.(AU)

The increasing interest for the use of stem cells in regenerative medicine, is based on their self-renewal capacity, tissue repair and immunomodulation. The therapy with stem cells is a science in formation considered a promising therapeutic alternative for the treatment of various degenerative, hereditary and inflammatory disorders. The search for alternative sources of stem cells of easy obtention and isolation has led to the study of various tissues, includng embryonic attachments such as the amniotic membrane. However for effective therapeutic application are needed studies of the characteristics in vitro and preclinical and clinical trials. Thus, the aim of this study was a literature review regarding the characterization and clinical application of stem cells from the amniotic membrane in several domestic animal species.(AU)