Preclinical study of experimental burns treated with photobiomodulation and Human Amniotic Membrane, both isolated and associated / Estudo pré-clínico de queimaduras experimentais tratadas com fotobiomodulação e membrana amniótica humana, isoladas e associadas / Estudio preclínico de quemaduras experimentales tratadas con fotobiomodulación y membrana amniótica humana, solas y combinadas

Abstract Objective: to evaluate the effect of photobiomodulation with low-level 660 nm laser alone or associated with Human Amniotic Membrane in the repair of partial-thickness burns in rats. Method: an experimental study conducted with 48 male Wistar rats, randomized into four groups: Control, Human Amniotic Membrane, Low-Level Laser Therapy, and Low-Level Laser Therapy associated with Human Amniotic Membrane. The histopathological characteristics of the skin samples were analyzed 7 and 14 days after the burn. The data obtained were submitted to the Kolmogorov-Smirnov and Mann-Whitney tests. Results: the histological analysis of the burn injuries showed a decrease in inflammation (p<0.0001) and an increase in proliferation of fibroblasts (p<0.0001) mainly at 7 days in all treatments related to the control group. At 14 days, the greater effectiveness in accelerating the healing process was significant (p<0.0001) in the Low-Level Laser Therapy group associated with the Human Amniotic Membrane. Conclusion: the association of photobiomodulation therapies with the Human Amniotic Membrane allowed verifying a reduction in the healing process time of the experimental lesions, stimulating its proposal as a treatment protocol in partial-thickness burns.

Resumo Objetivo: avaliar o efeito da fotobiomodulação com laser de baixa intensidade 660 nm isoladamente ou associada à membrana amniótica humana no reparo de queimaduras de espessura parcial em ratos. Método: estudo experimental com 48 ratos Wistar machos, randomizados em quatro grupos: Controle, Membrana Amniótica Humana, Terapia a Laser de Baixa Intensidade e Terapia a Laser de Baixa Intensidade associado à Membrana Amniótica Humana. As características histopatológicas das amostras de pele foram analisadas aos 7 e 14 dias após a queimadura. Os dados obtidos foram submetidos aos testes de Kolmogorov-Smirnov e Mann Whitney. Resultados: a análise histológica das lesões por queimadura mostrou a diminuição da inflamação (p <0,0001) e aumento da proliferação de fibroblastos (p <0,0001), principalmente nos 7 dias em todos os tratamentos relacionados ao grupo controle. Aos 14 dias, a maior efetividade na aceleração do processo cicatricial foi significativa (p<0,0001) no grupo Terapia a Laser de Baixa Intensidade associado à Membrana Amniótica Humana. Conclusão: a associação das terapias de fotobiomodulação à membrana amniótica humana permitiu comprovar redução no tempo do processo cicatricial das lesões experimentais, estimulando sua proposição como protocolo de tratamento em queimaduras de espessura parcial.

Resumen Objetivo: evaluar el efecto de la fotobiomodulación con láser de baja intensidad 660 nm de sola o combinada con la membrana amniótica humana en la reparación de quemaduras de espesor parcial en ratas. Método: estudio experimental con 48 ratas Wistar macho, aleatorizadas en cuatro grupos: Control, Membrana Amniótica Humana, Terapia con Láser de Baja Intensidad y Terapia con Láser de Baja Intensidad combinada con la Membrana Amniótica Humana. Las características histopatológicas de las muestras de piel fueron analizadas a los 7 y 14 días después de la quemadura. Los datos obtenidos fueron sometidos a las pruebas de Kolmogorov-Smirnov y Mann-Whitney. Resultados: el análisis histológico de las lesiones por quemadura mostró una disminución de la inflamación (p <0,0001) y un aumento de la proliferación de fibroblastos (p <0,0001) principalmente a los 7 días en todos los tratamientos en comparación con el grupo control; a los 14 días, en el grupo de Terapia con Láser de Baja Intensidad combinada con la Membrana Amniótica Humana la mayor efectividad en la aceleración del proceso de cicatrización fue significativa (p<0,0001). Conclusión: la asociación de terapias de fotobiomodulación con la membrana amniótica humana permitió comprobar que hubo una reducción en el tiempo del proceso de cicatrización de lesiones experimentales, lo cual favorece que se proponga como protocolo de tratamiento en quemaduras de espesor parcial.

Ruptura prematura de membranas ovulares / Premature rupture of ovular membranes

La ruptura prematura de las membranas ovulares se define como la pérdida de la integridad del amnios y corion antes del inicio del trabajo de parto, afecta el 3 % de los embarazos, causa un tercio de los partos pretérminos, los cuales ocupan el 10,49 % de los nacimientos y es el origen de altos índices de morbimortalidad perinatal. En la actualidad, el manejo de esta patología se orienta principalmente en evitar los factores de riesgo, hacer un diagnóstico adecuado, determinar la edad gestacional en que ocurre, realizar el monitoreo exhaustivo del bienestar materno-fetal y en decidir el momento idóneo de finalización de la gestación para minimizar sus complicaciones. Debido a la compleja y lábil estructura histológica de las membranas ovulares, se ha dejado a un lado el tratamiento directo de la entidad el cual sería sellar o reparar el defecto en sí. En los últimos años, numerosos estudios y protocolos clínicos de prestigiosos centros asistenciales han servido como guía para el manejo de esta entidad, pero en muy pocos se observa una terapia destinada a la reparación de dichas membranas o en sellar tal defecto. Las evidencias científicas demuestran que la regeneración y reparación de las membranas es lenta y compleja y los tratamientos propuestos para reparar o sellar su defecto no han gozado de la aceptación científica para su aprobación, sin embargo, el uso del parche hemático transvaginal endocervical autólogo luce como una alternativa terapéutica prometedora(AU)

The premature rupture of the ovular membranes is defined as the loss of the integrity of the amnion and chorion before the on set of labor, affects 3% of pregnancies, causes athird of preterm births which occupy 10,49% of births and is the origin of high rates of perinatal morbidity and mortality. At present, the management of this pathology is mainly oriented towards avoiding risk factors, making an adequate diagnosis, determining the gestational age in which it occurs, carrying out exhaustive monitoring of maternal-fetal well-being and deciding the ideal moment to end the treatment. Pregnancy to minimizeits complications. Due to the complex and labile histological structure of the ovular membranes, the direct treatment of the entity has been set a side, which would be to seal or repairthe defect it self. In recent years, numerous studies and clinicalprotocols from prestigious health care centers have served as aguide for the management of this entity, but very few have observed a therapy aimed at repairing said membranes or sealing such a defect. Scientific evidence shows that the regeneration and repair of the membranes is slow and complex and the treatment sproposed to repair or seal their defect have not enjoyed scientific acceptance for their approval, how ever, the use of the autologous endocervical transvaginal blood patch looks like a promising therapeutic alternative(AU)

Uso de membrana amniótica como cobertura temporal en pacientes pediátricos con quemaduras / Use of amniotic membrane as temporary coverage in pediatric patients with burn

Introducción: Los niños con quemaduras tienen un manejo complejo por lo cual el uso de la membrana amniótica como una cobertura temporal es un método válido para su implementación. Objetivos: Demostrar el uso de la membrana amniótica como cobertura temporal en pacientes pediátricos con quemaduras de segundo grado superficiales y profundas internados en el Centro Nacional de Quemaduras y Cirugías Reconstructivas (CENQUER), en julio y diciembre del 2021. Materiales y métodos: Estudio descriptivo de tipo retrospectivo mediante la revisión de casos clínicos-quirúrgicos de los pacientes pediátricos que ingresaron por diagnóstico de quemadura de segundo grado superficial y profundo en el CENQUER, durante el período comprendido entre julio y diciembre del 2021, donde se aplicó́ membrana amniótica humana. Resultados: Población de 14 pacientes, 11 provenían del departamento central y 3 del interior del país, con un promedio de 13% (R: 3%-25,5%) de superficie corporal quemada (SCQ) todos con quemaduras en promedio 7% tipo AB y 6% tipo ABB, 7 ingresaron con una gravedad moderada, 4 graves y 3 leves; 7 del sexo masculino y 7 del femenino , con un rango de edades entre 8 meses y 11 años cuya media fue de 4 años y una mediana de 7 años, con respecto al peso un promedio de 18 kg al ingreso % (R: 9-35 kg), las causas de las lesiones fueron 12 por escaldadura por agua caliente (85 %) y 2 por escaldadura con comida; 10 pacientes presentaron internación en sala y en la Unidad de terapia intensiva Pediátrica (UTIP) 4 pacientes, el promedio de tiempo transcurrido desde la quemadura a la consulta fueron de 7 hs (R: 1-48 hs), las regiones anatómicas que se cubrieron fueron: 4 pacientes en miembros superiores y tórax anterior (28 %), 2 exclusivamente en tórax anterior (16 %), 4 solo en miembros superiores (28 %), 4 en miembros inferiores (28 %), a los que se les colocó amnios por única ocasión en 10 pacientes a las 24 hs de la internación y en 4 pacientes a las 48 hs de internación, se utilizó antobioticoterapia parenteral en 3 pacientes y 11 pacientes no necesitaron antibioticoterapia parenteral, no se presentaron complicaciones en ninguno de los pacientes y tuvieron un promedio de internación de 8 días (R: 3- 16 días). Conclusión: El uso de membrana amniótica humana en pacientes quemados son favorables para los pacientes pediátricos; como demuestra en el estudio el menor uso de antibioticoterapia parenteral por la cobertura rápida de las heridas causadas por las quemaduras y el menor número de días de internación.

Introduction: Children with burns have a complex management for which the use of the amniotic membrane as a temporary cover is a valid method for its implementation. Objectives: Demonstrate the use of the amniotic membrane as temporary coverage in pediatric patients with superficial and deep second-degree burns admitted to the National Center for Burns and Reconstructive Surgery (CENQUER), in July and December 2021. Materials and methods: Descriptive retrospective study through the review of clinical-surgical cases of pediatric patients who were admitted due to a diagnosis of superficial and deep second-degree burns in the CENQUER, during the period between July and December 2021, where human amniotic membrane was applied. Results: Population of 14 patients, 11 came from the central department and 3 from the interior of the country, with an average of 13% (R: 3%-25.5%) of burned body surface area (SCQ), all with average 7% AB type burns. and 6% type ABB, 7 admitted with moderate severity, 4 severe and 3 mild; 7 males and 7 females, with an age range between 8 months and 11 years, whose average was 4 years and a median of 7 years, with respect to weight an average of 18 kg at admission % (R: 9- 35 kg), the causes of the injuries were 12 due to scalding due to hot water (85%) and 2 due to scalding with food; 10 patients were hospitalized in the ward and in the Pediatric Intensive Care Unit (PICU) 4 patients, the average time elapsed from the burn to the consultation was 7 hours (R: 1-48 hours), the anatomical regions that were covered were: 4 patients in the upper limbs and anterior thorax (28%), 2 exclusively in the anterior thorax (16%), 4 only in the upper limbs (28%), 4 in the lower limbs (28%), who received amnios for a single occasion in 10 patients at 24 hours of hospitalization and in 4 patients at 48 hours of hospitalization, parenteral antibiotic therapy was used in 3 patients and 11 patients did not need parenteral antibiotic therapy, there were no complications in any of the patients and they had an average hospital stay of 8 days (R: 3-16 days). Conclusion: The use of human amniotic membrane in burn patients is favorable for pediatric patients; As shown in the study, the lower use of parenteral antibiotic therapy due to the rapid coverage of wounds caused by burns and the lower number of days of hospitalization.

Aislación y caracterización de las células epiteliales del amnios / Isolation and characterization of amniotic ephitelial cells

RESUMEN: Las células epiteliales del amnios (hAECs) son células madre pluripotenciales; tienen capacidad de diferenciarse en células de las tres capas embrionarias. Como tales, se utilizan en algunas terapias regenerativas en medicina. Este estudio tiene por objetivo describir un protocolo de aislación de las células epiteliales del amnios (hAECs) a partir de placentas humanas de partos por cesárea, así como su caracterización y comportamiento in vitro. Se aislaron hAECs de 20 placentas de partos por cesárea con un protocolo optimizado. Se caracterizaron las células mediante citometría de flujo, microscopia óptica y de fluorescencia, y se evaluó la proliferación de las células mediante MTT a los 1, 3, 5 y 7 días con y sin β-mercaptoetanol en el medio de cultivo. El análisis histológico del amnios mostró un desprendimiento prácticamente completo de las células después de la segunda digestión del amnios. El promedio de células obtenidas fue de 10.97 millones de células por gramo de amnios. Las hAECs mostraron una proliferación limitada, la cual no fue favorecida por la adición de β-mercaptoetanol en el cultivo. Se observó un cambio de morfología espontanea de epitelial a mesenquimal después del cuarto pasaje. Las células epiteliales del amnios pueden ser aisladas con un protocolo simple y efectivo, sin embargo, presentan escasa capacidad proliferativa. Bajo las condiciones de este estudio, la adición de β-mercaptoetanol no favorece la capacidad proliferativa de las células.

SUMMARY: human amnion epithelial cells (hAECs) are pluripotent stem cells; they have the ability to differentiate into cells of the three embryonic layers, and are used in various regenerative therapies in medicine. This study aims to describe a protocol for the isolation of amnion epithelial cells (hAECs) from human placentas from cesarean delivery, as well as their characterization and culture conditions in vitro. hAECs were isolated from 20 cesarean delivery placentas with an optimized protocol. The cells were characterized by flow cytometry, light and fluorescence microscopy, and the proliferation of the cells was evaluated by MTT at 1, 3, 5 and 7 days with and without β-mercaptoethanol in the culture medium. Histological analysis of the amnion showed a practically complete detachment of the cells of the underlying membrane after the second digestion. The average number of cells obtained was 10.97 million cells per amnion. The hAECs perform a limited proliferation rate, which was not favored by the addition of β-mercaptoethanol in the culture. A spontaneous morphology change from epithelial to mesenchymal morphology is exhibited after the fourth passage. The epithelial cells of the amnion can be isolated with a simple and effective protocol, however, they present little proliferative capacity. Under the conditions of this study, the addition of β-mercaptoethanol does not favor the proliferation of the cells.

Curación de úlcera venosa crónica de la pierna con aloinjerto de membrana amniocoriónica humana fresca / Healing of a chronic ulcer of the lower limb of venous origin with fresh human amniochorionic membrane allograft

En su estado fresco, la membrana amniocoriónica contiene varias células multipotenciales, factores de crecimiento y proteínas de la matriz extracelular que contribuyen a la cicatrización de las úlceras vasculares crónicas. Para demostrar su efectividad, se recurrió a un aloinjerto de membrana placentaria humana fresca para tratar una úlcera venosa crónica, de 12 x 10 cm y con 40 años de evolución, en el zona maleolar interna e izquierda de una paciente de 89 años de edad. Transcurridos 60 días del injerto, la úlcera se encontraba cicatrizada en el 100 % de su superficie, observándose una cicatriz rosada clara en cuyos bordes se apreciaron intentos de pigmentación. El aloinjerto de membrana amniocoriónica humana fresca es una alternativa terapéutica para la curación de úlceras vasculares crónicas persistentes en las extremidades inferiores.

In its fresh state, the amniochorionic membrane contains various multipotential cells, growth factors, and extracellular matrix proteins that contribute to the healing of chronic vascular ulcers. To demonstrate its effectiveness, a fresh human placental membrane allograft was applied to a chronic venous ulcer in the lower limb of an 89-year-old female patient with a 12 x 10 cm ulcerated lesion of 40 years of evolution in the malleolar area of her left lower limb. Sixty days after the graft, the ulcer was healed in 100% of its surface and a light pink scar on the edges indicated possible pigmentation. Fresh human amniochorionic membrane allograft is a therapeutic alternative for the healing of refractory chronic vascular ulcers of the lower extremities.

Advances on human amniotic epithelial cells and its clinical application potential / 生理学报

Human amniotic epithelial cells (hAECs) are epithelial cells located on the placental amnion near the fetus. Different from other placental-derived stem cells, hAECs are derived from embryonic epiblast, and have been considered as seed cells for regenerative medicine. hAECs possess embryonic stem cell-like multi-differentiation capabilities and adult stem cell-like immunomodulatory properties. Compared with other types of stem cells, special properties of hAECs make them unique, including easy isolation, abundant cell numbers, non-tumorigenicity after transplantation, and the obviation of ethical debates. During the past two decades, the therapeutic potential of hAECs has been extensively investigated in various diseases. Accumulating evidence has demonstrated that hAECs contribute to repairing and remodeling the function of damaged tissues and organs through different molecular mechanisms. This article provides an in-depth review of the biological characteristics of hAECs, summarizes the research status of hAECs, and discusses the clinical application prospects of hAEC-based cell therapy.

Uso de membrana amniótica e injerto meek en un niño con quemaduras severas / Use of amniotic membrane and meek graft in a child with severe burns

El desbridamiento temprano seguido de cobertura con piel autóloga ha demostrado reducir la mortalidad en los pacientes quemados. En pacientes con quemaduras extensas, la carencia de zonas de piel donante es un factor limitante a la hora del tratamiento. El hecho de que no se pueda lograr un desbridamiento y cobertura completos en un primer tiempo favorece la infección de la quemadura y la septicemia, pudiendo tener consecuencias nefastas. Por tanto, es necesario disponer de otras opciones distintas a los injertos mallados de piel parcial, el tratamiento estándar hoy día. Presentamos el caso clínico de un niño con quemaduras severas y describimos los resultados obtenidos con el uso de la membrana amniótica como cobertura temporal y al injerto mallado tipo MEEK.

Early debridement followed by coverage with autologous skin has been shown to reduce mortality in burn patients. In patients with extensive burns, the lack of areas of donor skin is a limiting factor at the time of treatment. The fact that a complete debridement and coverage cannot be achieved in the first stage favors the infection of the burn and septicemia, which can have dire consequences. Therefore, it is necessary to have other options than partial skin mesh grafts, the standard treatment today. We present the clinical case of a child with severe burns and describe the results obtained with the use of the amniotic membrane as temporary covering and the MEEK-type mesh graft.

Amniotic membrane graft after excision of widespread conjunctival concretions / Enxerto de membrana amniótica após remoção de concreções conjuntivais

ABSTRACT Conjunctival concretions are single or clustered lesions frequently found on the palpebral conjunctiva. They are commonly present in older individuals, despite being rarely symptomatic. This case report describes an 83-year-old man with multiple conjunctival concretions, which were surgically treated. The patient was symptomatic on presentation and did not respond to conservative treatment. For this reason, a surgical approach was considered. After wide excision of the conjunctival lesions, a piece of amniotic membrane was fitted using fibrin glue. During follow-up, a markedly improvement in patient's symptoms was observed, along with nearly complete absence of conjunctival concretions. This is the first case report addressing extensive conjunctival concretions with a surgical approach using amniotic membrane. The authors conceived the technique described after noticing the limited clinical options in the literature. This technique was easily performed and achieved satisfactory results.

RESUMO As concreções conjuntivais representam lesões amareladas, simples ou múltiplas, frequentemente encontradas na conjuntiva palpebral. São mais prevalentes em idades avançadas e raramente sintomáticas. Este relato de caso descreve o quadro clínico de um paciente de 83 anos com múltiplas concreções conjuntivais, cirurgicamente tratadas. Por se tratar de um paciente sintomático com resposta insuficiente ao tratamento conservador, foi considerada a abordagem cirúrgica. Após remoção das lesões, foi aplicado um enxerto de membrana amniótica, adaptado com cola de fibrina. No acompanhamento pós-operatório, verificou-se melhoria significativa dos sintomas, com desaparecimento quase total das concreções conjuntivais. Este é o primeiro caso que descreve uma abordagem cirúrgica com utilização de membrana amniótica na resolução desse tipo de lesões. A técnica, de fácil execução e com resultados muito favoráveis, foi desenvolvida pelos autores após constatarem a escassez de alternativas na literatura.

Amniotic membrane transplantation in ocular surface diseases / Transplante de membrana amniótica em doenças da superfície ocular

Abstract Objective: To evaluate the clinical effectiveness of amniotic membrane transplantation for ocular surface reconstruction. Methods: Prospective study including 23 eyes of 21 patients who underwent amniotic membrane transplantation at Hospital de Clínicas da Universidade Federal do Paraná (HC-UFPR) and at Cirurgia e Diagnose em Oftalmologia do Paraná (CDOP) clinic, located in Curitiba, PR, Brazil, from may 2015 to july 2019. The amniotic membrane was collected from elective and term cesarean delivery, and conserved in preservation medium and glycerol 1:1, stored at -80° Celsius. The membrane was fixed on the ocular surface with 10-0 nylon, 8-0 vicryl, biological glue or a combination of these materials. Results: The ocular surface reconstruction was successful in 22 eyes (95.6%). Failure was observed only in 1 case (bullous keratopathy) in which the condition was maintained postoperatively. Patients' age ranged from 11-82 years, with a mean age of 37.4 years. There was a higher incidence in males (66.6%). A difference was perceived in the distribution of the affected eye (which was greater in the right eye - 65.2%). As for the previous ophthalmic surgery history, 12 of the 23 eyes had a positive history (52.2%). It was observed that all patients who had preoperative visual acuity assessed showed improvement or maintenance of corrected visual acuity. In the postoperative period, complications associated with the underlying disease were observed, although not particularly related to the amniotic membrane transplantation. There were not any cases of postoperative infection. Conclusions: There was an improvement in the general state of the ocular surface in almost all of the cases in which the transplant was performed. Therefore, the amniotic membrane can be considered a good alternative for reconstructing the ocular surface, as a single or supporting treatment.

Resumo Objetivo: Avaliar a eficácia clínica do transplante de membrana amniótica na reconstrução da superfície ocular. Métodos: Estudo prospectivo incluiu 23 olhos de 21 pacientes que realizaram transplante de membrana amniótica no Hospital de Clínicas da Universidade Federal do Paraná (UFPR) e na clínica de Cirurgia e Diagnose em Oftalmologia do Paraná (CDOP), localizados em Curitiba, PR, Brasil, no período de maio de 2015 a julho de 2019. A membrana amniótica foi captada a partir de parto cesárea eletivo e a termo, conservada em meio de preservação e glicerol 1:1 e armazenada a -80° Celsius. A membrana foi fixada na superfície ocular com fio nylon 10-0 ou vicryl 8-0 e/ou cola biológica. Resultados: A idade dos pacientes variou de 11-82 anos, com média de 37,4 anos. Houve maior incidência no sexo masculino (66,6%). Ocorreu diferença na distribuição do olho acometido (maior no olho direito - 65,2%). Quanto à história de cirurgia oftalmológica prévia, 12 dos 23 olhos tinham história positiva (52,2%). Observamos que nos pacientes em que foi possível a avaliação da acuidade visual pré-operatória, todos apresentaram melhora ou manutenção da acuidade visual. No pós-operatório foi observado complicações associadas à doença de base e não propriamente ao transplante de membrana amniótica. Não foram registrados casos de infecção pós-operatória. Conclusão: Houve melhora do estado geral da superfície ocular em quase totalidade dos casos em que o transplante foi realizado. Portanto, a membrana amniótica pode ser considerada uma boa alternativa para reconstrução da superfície ocular, como tratamento único ou coadjuvante.

Equine yolk sac: a stem cells source / Saco vitelino equino: una fuente de células madre

SUMMARY: Mesenchymal stem cells are characterized by in vitro high proliferation and multilineage potential maintenance. This study aimed to isolate and characterize equine YS mesenchymal stem cells and compare these with amniotic membranes. The yolk sac (YS) and amniotic membranes (AM) were obtained from 20 pregnant mares with gestational age around 30 days. Cells were cultured in α-MEM supplemented with 15 % FBS, 1 % antibiotic solution, 1 % L-glutamine and 1 % nonessential amino acids. To cell characterization we used cytogenetic analysis, fibroblast colony-forming unit assays, cell growth curves, immunophenotyping, flow cytometry, differentiation assays and teratoma formation. Results: Both cell sources presented fibroblastoid and epithelioid-like format. The YS cells have lower colony formation potential then AM ones, 3 versus 8 colonies per 103 plated cells. However, YS cells grew progressively while AM cells showed steady. Both, the YS and amnion cells immunolabeled for Oct-4, Nanog, SSEA-3, cytokeratin 18, PCNA, and vimentin. In addition, presented mesenchymal, hematopoietic, endothelial and pluripotency markers in flow cytometry. Discussion: Both cell sources presented high plasticity and differed into osteogenic, adipogenic, and chondrogenic lineages, and no tumor formation in nude mice was observed. The results suggest that horse YS may be useful for cell therapy such as amnion-derived cells.

RESUMEN: Las células madre mesenquimales se caracterizan por una alta proliferación in vitro y un mantenimiento potencial de múltiples líneas. Este estudio tuvo como objetivo aislar y caracterizar las células madre mesenquimales del saco vitelino equinas y compararlas con las membranas amnióticas. Se obtuvo el saco vitelino (SV) y las membranas amnióticas (MA) de 20 yeguas preñadas con edad gestacional de aproximadamente 30 días. Las células se cultivaron en α -MEM suplementado con 15 % de FBS, 1 % de solución antibiótica, 1 % de L-glutamina y 1 % de aminoácidos no esenciales. Para la caracterización celular utilizamos análisis citogenéticos, ensayos de unidades de colonias de fibroblastos, curvas de crecimiento celular, inmunofenotipaje, citometría de flujo, ensayos de diferenciación y formación de teratomas. Ambas fuentes celulares presentaron formato fibroblastoideo y epitelioide. Las células SV tienen un potencial de formación de colonias más bajo que las de MA, 3 versus 8 colonias por 103 células en placa. Sin embargo, las células SV crecieron progresivamente mientras que las células MA se mostraron estables. Tanto las células YS como las células amnios están inmunomarcadas para Oct-4, Nanog, SSEA-3, citoqueratina 18, PCNA y vimentina. Además, presentó marcadores mesenquimales, hematopoyéticos, endoteliales y pluripotenciales en citometría de flujo. Ambas fuentes celulares presentaron alta plasticidad y diferían en linajes osteogénicos, adipogénicos y condrogénicos, y no se observó formación de tumores en ratones. Los resultados sugieren que el SV de caballo puede ser útil para la terapia celular, como las células derivadas de amnios.

Characteristics of the equine placenta at first trimester / Características de la placenta equina en el primer trimestre

The equine placenta is a simple apposition of fetal and maternal tissues, becoming more complex with the formation of microcotyledons around days 75 and 100 of gestation. The present study aimed to describe the gross and microscopic morphology of early equine placenta. Embryonic/fetal membranes from thirty-seven mares were submitted to macroscopic description, light, scanning and transmission microscopy. Overall the gross characteristics of membranes were similar with already described for older stages. However, transmission electron microscopy evidenced high metabolic rate in chorion and allantois, and high secretion profile in amnion and even higher in yolk sac. Gene ontologies enrichment, using published data, pointed several common ontologies in allantoic and amniotic fluids, related to oxygen and iron transport, extracellular space and high-density lipoprotein receptor binding. Overall, the morphological and ontology enrichment could indicate allantois and amnion crosstalk.

La placenta equina es una simple aposición de tejidos fetales y maternos, que se vuelve más compleja con la formación de microcotiledones alrededor de los días 75 y 100 de gestación. El presente estudio tuvo como objetivo describir la morfología macroscópica y microscópica de la placenta equina temprana. Las membranas embrionarias / fetales de treinta y siete yeguas fueron sometidas a descripción macroscópica, luz, escaneo y microscopía de transmisión. En general, las características generales de las membranas fueron similares a las ya descritas para las etapas más antiguas. Sin embargo, la microscopía electrónica de transmisión mostró una alta tasa metabólica en corion y alantoides, y un alto perfil de secreción en amnios e incluso mayor en el saco vitelino. El enriquecimiento de ontologías génicas, utilizando datos publicados, señaló varias ontologías comunes en fluidos alantoideos y amnióticos, relacionados con el transporte de oxígeno y hierro, espacio extracelular y unión a receptores de lipoproteínas de alta densidad. En general, el enriquecimiento morfológico y ontológico podría indicar alantoides y diafonía de amnios.

Comparação dos meios de preparação e preservação de membrana amniótica humana para uso no tratamento de doenças da superfície ocular / Comparison of the preparation and preservation techniques of amniotic membrane used in the treatment of ocular surface diseases

Resumo Atualmente a membra amniótica (MA) tem obtido importância devido à comprovada capacidade de reduzir inflamação, auxiliar a cicatrização e epitelização, possuindo propriedades antimicrobianas e antivirais, além de baixa imunogenicidade. As indicações de seu uso na oftalmologia têm aumentado muito nas duas últimas décadas. Objetivo: Descrever a estrutura básica e as propriedades biológicas da MA em relação aos componentes da sua matriz extracelular e fatores de crescimento, as consequências de diferentes técnicas empregadas na sua preservação e esterilização, métodos para remoção do epitélio e a comparação dos custos dos diferentes meios de conservação atualmente empregados. Métodos: Pesquisa nas bases de dados do Portal da Biblioteca Virtual em Saúde (BVS), Pubmed, Cochrane, Scielo e Lilacs com as palavras-chave: membrana amniótica, transplante, reconstrução da córnea, doenças da conjuntiva. Resultados: A literatura é vasta na descrição dos efeitos de diversos agentes e técnicas na preparação da MA, dentre elas sua preservação, esterilização e desepitelização. A membrana desnuda tem sido a escolha para a reconstrução da superfície ocular, pois facilita a cicatrização. Em relação aos agentes conservantes, o glicerol é o meio mais utilizado mundialmente pelo baixo custo e facilidade de manuseio. Conclusão: A comparação das diversas técnicas nos guia na elaboração de protocolos de preparo da MA para uso oftalmológico. A membrana desnuda facilita a cicatrização em relação a com células epiteliais. O glicerol é o meio de conservação mais utilizado pelo baixo custo e facilidade de manuseio.

Abstract Currently, the amniotic membrane (AM) has obtained importance due to its ability to reduce inflammation, helping in the healing and epithelialization processes, having antimicrobial and antiviral properties and low immunogenicity. Its indications in ophthalmology have increased considerably in the past two decades. Objective: To describe the basic structure and biological properties of the AM, the components of the extracellular matrix and growth factors, the consequences of different techniques used in its preservation, and sterilization methods for the epithelium removal. To compare the costs of the different preservation solutions currently employed. Study design: literature review. Methods: Research in BVS databases, PubMed, Cochrane, Scielo and Lilacs with keywords: amniotic membrane transplantation, corneal reconstruction, conjunctival diseases. Results: The literature is vast in describing the effects of different agents and techniques used in the preparation of MA, including its preservation, sterilization and desepithelization. The naked membrane is the choice to reconstruct the ocular surface, as it facilitates the healing course. Regarding the preservatives, glycerol is the most used worldwide due its low cost and easy handling. Conclusion: Comparing different techniques guides us in developing a MA preparation protocol for ophthalmic use. The naked membrane facilitates the healing process compared with the presence of epithelial cells. The glycerol is the most used preservation method because of its low cost and easy handling.

Fresh Frozen Amniotic Membrane and Buccal Pad of Fat for Reconstruction of Oral Mucosal Defect After Surgical Excision of Leukoplakia

Objective: To compare the use of Fresh Frozen Amniotic Membrane (FFAM) and Buccal Pad of Fat (BPF) for reconstruction of oral mucosal defect after surgical excision of leukoplakia. Material and Methods: Twenty patients were randomly selected and divided into two groups. Group 1 use amniotic membrane graft and Group 2 BPF. Both groups were evaluated preoperatively and postoperatively. Incisal opening, epithelialization and fibrosis were evaluated after one month of surgery. Chi square and Student t tests were used. Results: According to the presence of smoking habits, the highest frequencies were for smoking (30%) and betel leaf areca nut with tobacco (30%). Regarding the diameter of oral leukoplakia, in 40% of the participants it was 2x3 cm2. In Group1, after one month of surgery preoperative and postoperative inter-incisal opening values were 44.20 ± 3.37 and 42.05 ± 3.47 (p<0.001). In Group 2, preoperative and postoperative inter-incisal opening values were 44.09 ± 3.32 and 43.01±3.38 (p>0.05). When FFAM was used complete epithelialization in 70% and incomplete epithelialization in 30% patients. When BPF was used the results were almost similar. Fibrosis occurred in 30% in Group 1. There were no complications like flap necrosis, infectiona and hematoma formation. Conclusion: Incisal opening was significantly better in Fresh Frozen Amniotic Membrane Group, epithelialization and fibrosis were almost same in both groups after surgical excision of oral leukoplakia.

Amniotic membrane as a biological scaffold for dental pulp stem cell transplantation in ocular surface reconstruction / Membrana amniótica como substrato biológico para o transplante de células-tronco da polpa do dente de leite na reconstrução da superfície ocular

ABSTRACT Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.

RESUMO Objetivos: Avaliar a capacidade das células-tronco imaturas da polpa do dente de leite que são células-tronco mesenquimais de origem da crista neural, de se diferenciarem no epitélio corneano para fins de transplante de córnea e engenharia de tecidos quando cultivadas em membrana amnióticas desepitelizadas. Métodos: Foram comparamos so imunofenótipo (ABCG2, CK3/12 e vimentina) de células cultivadas em membranas amnióticas ou em superfícies plásticas sob condições livres de soro ou em meios de cultura contendo soro ou componentes de substituição de soro. Resultados: Células-tronco imaturas da polpa do dente de leite cultivadas sobre membrana amniótica em condições basais são capazes de manter seu estado indiferenciado. Nossos dados também sugerem que o meio de cultura utilizado no presente trabalho pode modular a expressão de marcadores de células-tronco imaturas da polpa do dente de leite, induzindo a diferenciação epitelial destas células in vitro. Conclusão: Nossos resultados sugerem que a membrana amniótica é uma boa escolha para o crescimento e transplante de células-tronco mesenquimais, particularmente as células-tronco imaturas da polpa do dente de leite, na reconstrução da superfície ocular.

Adequação da assistência pré-natal em casa de parto e causas associadas com as transferências hospitalares / Adecuación de la asistencia pre-natal em casa de parto y causas asociadas com las transferências hospitales / Adequacy of prenatal assistance in birth houses and causes associated with hospital transfers

Resumo OBJETIVO Analisar a adequação da assistência pré-natal em Casa de Parto e as causas associadas com as transferências maternas e dos recém-nascidos para o hospital. MÉTODOS Estudo transversal dos atendimentos da única Casa de Parto, de 2009 a 2014, no Rio de Janeiro. As análises estatísticas foram pelo Teste χ2 e Razão de Prevalência (RP). RESULTADO A assistência pré-natal adequada predominou (42,8%) e não houve associação (p=0,55) com as transferências. A transferência materna é causada pela bolsa amniótica rota (RP= 2,09; IC 95% 1,62-2,70) e batimentos cardíacos fetais alterados (RP= 3,06; IC 95% 2,13-4,39). As transferências do recém-nascido estão associadas com a presença de mecônio no líquido amniótico (RP= 2,40; IC 95% 1,30 - 4,43); Apgar abaixo de 7 (RP= 5,33; IC 95% 2,65-10,73) e assistência ventilatória ao nascer (RP= 9,41; IC 95% 5,52-16,04). CONCLUSÃO As intercorrências na assistência intraparto são as causas associadas com as transferências.

Resumen OBJETIVO Analizar la adecuación de la asistencia prenatal en Casa de Parto y las causas asociadas con las transferencias maternas y de los recién nacidos para al hospital. MÉTODOS Investigación transversal de los atendimientos de la única Casa de Parto, de 2009 a 2014, en Río de Janeiro. Los análisis estadísticos fueron por el Test χ2 y Razón de Prevalencia (RP). RESULTADO La asistencia prenatal adecuada predominó (42,8%) y no hubo asociación (p = 0,55) con las transferencias. La transferencia materna es causada por la bolsa amniótica ruta (RP = 2,09, IC 95% 1,62-2,70) y batimientos cardíacos fetales alterados (RP = 3,06, IC 95% 2,13-4,39). Las transferencias del recién nacido se asocian con la presencia de meconio en el líquido amniótico (RP = 2,40; IC 95% 1,30 - 4,43); Apgar abajo de 7 (RP= 5,33; IC 95% 2,65-10,73) y asistencia ventilatoria al nacer (RP=9,41; IC 95% 5,52-16,04). CONCLUSIONES Las intercurrencias en la asistencia intraparto son las causas asociadas con las transferencias.

Abstract OBJECTIVE To analyze the adequacy of prenatal care in a Birth Center and the causes associated with maternal and newborn transfers to the hospital. METHODS Cross-sectional study of the care provided at the only Birth Center in Rio de Janeiro, from 2009 to 2014. Statistical analyzes were based on the χ2 test and Prevalence Ratio (PR). RESULTS Suitable prenatal care was predominant (42.8%) and there was no association (p = 0.55) with the transfers. Maternal transfer is caused by the ruptured amniotic sac (PR = 2.09, 95% CI 1.62-2.70) and altered fetal heart rates (PR = 3.06, 95% CI, 2.13-4.39). Newborn transfers are associated with the presence of meconium in the amniotic fluid (PR = 2.40, 95% CI 1.30-4.43); Apgar below 7 (PR = 5.33, 95% CI 2.65-10.73); and ventilatory assistance at birth (PR = 9.41, 95% CI 5.52-16.04). CONCLUSION Complications during intrapartum care are the causes associated with transfers.

A New Amniotic Membrane for Placement during Pterygium Surgery

PURPOSE: We introduce a new amniotic membrane (AM) for placement during pterygium surgery. CASE SUMMARY: After excision of the pterygium, we measured the size of the defect with reference to the side opposite the defective area and prepared an AM with margins 1.5–2.0 mm greater than the defect size. The AM was first sutured vertically, with reference to the opposite side of the defect. Then we sutured the upper and lower horizontal axes, and positioned the eye, from the front, slightly away from the direction of the opposite side of the defect. The AM was cut by reference to its boundary at the limbus, and three fixation sutures were placed. CONCLUSIONS: Appropriate AM sizing is important in terms of AM transplantation; the AM is non-elastic and easily torn. Our technique transplants a correctly sized AM and anchors it firmly.

Propofol Suppresses LPS-Induced Inflammation in Amnion Cells via Inhibition of NF-κB Activation

BACKGROUND: Preterm labor is a leading risk factor for neonatal death and long-term impairment and linked closely with inflammation. Non-obstetric surgery is occasionally needed during pregnancy and the anesthetic drugs or surgery itself can give rise to inflammation. Here, we examined the influence of propofol pretreatment on the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) after lipopolysaccharide (LPS) stimulation. In addition, we evaluated the expression of pro-inflammatory cytokines and nuclear factor kappa B (NF-κB). METHODS: Human amnion-derived WISH cells were used to investigate the effect of propofol on the LPS-induced expression of inflammatory substances involved in preterm labor. For the experiment, WISH cells were pretreated with various concentrations propofol (0.01–10 µg/ml) for 1 h and then treated with LPS (1 µg/ml) for 24 h. Cytotoxicity was evaluated using MTT assay. PGE2 concentration was assessed by ELISA. Protein expressions of COX-2, PGE2 and NF-κB were analyzed by western blotting analysis. RT-PCR was used for analysis of mRNA expression of COX-2, PGE2, interlukin (IL)-1β and tumor necrosis factor (TNF)-α. RESULTS: Propofol showed no cytotoxicity on the WISH cells. LPS-induced PGE2 production and COX-2 and PGE2 expression were decreased after propofol pretreatment. Propofol also attenuated the LPS-induced mRNA expression of IL-1β and TNF-α. Moreover, the activation of NF-jB was inhibited by propofol pretreatment on LPS-stimulated WISH cells. CONCLUSION: We demonstrated that propofol suppresses the expression of inflammatory substances enhanced by LPS stimulation. Furthermore, this inhibitory effect of propofol on the inflammatory substance expression is mediated by suppression of NF-κB activation.

The Isolated Oligohydramnios

Amniotic fluid is an indicator of normal placental function and is essential for normal fetal lung maturation. Amniotic fluid index (AFI) is the most preferred method of amniotic fluid measurement in pregnancy although single deepest pocket (SDP) is also used. To measure AFI, the examiner divides the uterus into four equal quadrants. AFI is the sum of deepest pocket from each quadrant. The normal AFI ranges between 5~24 cm while any value above 24 cm is considered as hydramnios and that below 5 cm is indicated as oligohydramnios. An adequate volume of amniotic fluid is critical to allow normal fetal movement and growth, while also cushioning the fetus and umbilical cord. Regardless of the etiology, oligohydramnios may inhibit these processes and may lead to fetal deformation, umbilical cord compression, and death in some instances. Oligohydramnios can be due to underproduction, loss, or sometimes, isolated. Isolated oligohydramnios has been found to be responsive maternal hydration and is neither a malformation of the urinary system in the fetus, nor a rupture of amnion and due to genetic cause. The author would like to introduce a way to increase amniotic fluid volume in isolated oligohydramnios which is expect to improve the perinatal outcomes.