Amniotic membrane (AM) or amnion is a thin membrane on the inner side of the fetal placenta; it completely surrounds the embryo and delimits the amniotic cavity, which is filled by amniotic liquid. In recent years, the structure and function of the amnion have been investigated, particularly the pluripotent properties of AM cells, which are an attractive source for tissue transplantation. AM has anti-inflammatory, anti-bacterial, anti-viral and immunological characteristics, as well as anti-angiogenic and pro-apoptotic features. AM is a promoter of epithelialization and is a non-tumorigenic tissue and its use has no ethical problems. Because of its attractive properties, AM has been applied in several surgical procedures related to ocular surface reconstruction and the genito-urinary tract, skin, head and neck, among others. So far, the best known and most auspicious applications of AM are ocular surface reconstruction, skin applications and tissue engineering. However, AM can also be applied in oncology. In this area, AM can prevent the delivery of nutrients and oxygen to cancer cells and consequently interfere with tumour angiogenesis, growth and metastasis.

Human amniotic membrane (hAM) has recently drawn attention as an upcoming anti-cancer therapy. Regarding the strategies which have already investigated, little is known about hAM protein extracts (hAMPE) effect on cancer. So, this work aims to study the effect of hAMPE in metabolic activity of several human cancer cell lines. hAMPE were mechanically obtained, thus avoiding the effect of detergents and other reagents commonly used in protein extraction under the cell lines studied. After quantification of proteins in hAMPE, their effect on the metabolic activity of 21 human cancer cell lines was assessed by 3-(4,5-dimethylthia-zolyl-2)2,5-diphenyltetrazolium bromide (MTT) assay. Our results indicate that there is an inhibition of metabolic activity until 25 and 50% in two and seven cell lines, respectively. Five cell lines proved to be very sensitive to hAMPE, being its metabolic activity more than 50% inhibited. Our results show that hAMPE can inhibit the metabolic activity of some human cancer cell lines. However, research about this cell line-dependent response to hAMPE becomes indispensable.

Recently, three-dimensional (3D) cell culture platforms have emerged as valuable tools with potential to increase the accuracy of in vitro studies. Inspired by the extracellular matrix (ECM) that compose living tissues, these platforms have been designed to support the attachment and growth of cells, therefore providing reliable data on how they behave and respond to stimulus when within their natural environments. Despite the great advances made on the design of such materials, including the development of ECM-based hydrogels such as Matrigel®, recapitulate the complexity and function of native ECMs, while producing cost-effective and safe materials, remains a challenge. METATISSUE is a start-up company founded in 2018 that intends to address this challenge by developing human-derived 3D cell culture platforms. Amniotic membrane (AM) is a placental tissue usually discarded after delivery, thus representing a readily available source of human ECM. Although this tissue has been widely explored in the formulation of scaffolds for tissue engineering, its formulation as a hydrogel has been highly limited by the poor mechanical properties of resulting materials. Therefore, the aim of this work was to develop a photopolymerizable hydrogel derived from human AM with tunable mechanical properties for application in 3D cell culture. Amniotic membrane was isolated from placenta, decellularized and solubilized to produce a solution rich in ECM structural proteins and growth factors, and free of cells. Amniotic membrane methacrylated (AMMA) was produced by reacting the previous solution with methacrylic anhydride in order to obtain two different degrees of modification: AMMA100 (low-modification degree) and AMMA300 (high-modification degree). Finally, AMMA hydrogels were successfully obtained upon irradiation with UV or visible light. AMMA hydrogels were characterized in terms of their biochemical, mechanical and biological properties. Although some ECM components were significantly reduced after processing of AM, the hydrogels obtained were robust and stable. Indeed, characterization studies revealed that were obtained hydrogels with tunable mechanical properties. In vitro assays using human adipose-derived stem cell (hASCs) and human umbilical cord endothelial cells (HUVECs) indicated that these materials were able to support growth of top seeding cells. The encapsulation of stem cells confirmed that AM hydrogels support adhesion, proliferation and invasion of this kind of cells. In sum, this work demonstrated that human AM-derived ECM can be efficiently functionalized with photorespondive groups to produce a versatile and robust hydrogel with potential to be used in 3D cell culture.

In the past few years researchers have witnessed a paradigm shift in the development of biomaterials for drug discovery, tissue engineering, and regenerative medicine. After the great advances resulting from the transition of the 2D to the 3D, the new focus has been to increase the clinical relevance of such systems, as well as avoid the use of animals, by developing platforms that better replicate the human physiology in vitro. In this sense, we envisage the use of human matrices extracted from ethically sourced and readily available tissues as an optimal and promising alternative to currently used approaches. Hereupon, we report for the first time the chemical modification of human ECM proteins from the amniotic membrane (AM) with photoresponsive groups to produce bioinks and hydrogel precursors to engineer customizable platforms that are representative of native tissues and capable of supporting long-term cell culture. Our results demonstrated an efficient decellularization, liquefaction and functionalization of AM-derived ECM with methacryloyl domains (AMMA), with production of stable and versatile hydrogels. Mechanical characterization evidenced an increased compression strength as a function of methacrylation degree and decellularized ECM concentration. Three-dimensional (3D) stem cell culture in the AMMA hydrogels resulted in viable and proliferative cells up to 7 days; moreover, the mouldable character of the hydrogel precursors permits the processing of patterned hydrogel constructs allowing the control over cellular alignment and elongation, or microgels with highly tunable shape.

Perinatal tissues are an abundant source of human extracellular matrix proteins, growth factors and stem cells with proved potential use in a wide range of therapeutic applications. Due to their placental origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Additionally, as a temporary organ, placenta is usually discarded as a medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. Although some of these tissues, such as the amniotic membrane and umbilical cord, have been used in clinical practices, most of them continue to be highly under explored. This review aims to outline the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM), as well as highlight how these solutions can be used to overcome the shortage of adequate scaffolds and cell sources that currently hampers the translation of TERM strategies towards clinical settings. STATEMENT OF SIGNIFICANCE: Stem cells and extracellular matrix derived from perinatal tissues such as placenta and umbilical cord, have drawn great attention for use in a wide variety of applications in the biomedical field. Due to their origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Also they are typically considered medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. This work aims to present and discuss the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM).

Realizou-se a ceratectomia superficial em 28 coelhos, distribuídos em dois grupos. No grupo tratado, composto por 14 animais, utilizou-se a membrana amniótica canina como bandagem, suturada com sua face epitelial voltada contra a superfície corneana. O grupo controle não recebeu tratamento. A avaliação clínica foi realizada 24 horas após a cirurgia, a cada dois dias durante uma semana e a cada quatro dias até 180 dias. Avaliaram-se os efeitos da membrana amniótica como bandagem, comparando-se o tempo de epitelização da córnea entre os grupos e verificando o período necessário para que a córnea apresentasse transparência completa. A opacidade corneana esteve presente em todos os animais durante o período observado. O tratamento instituído resultou clinicamente em neovascularização corneana, opacidade de córnea mais intensa no período inicial de reparação, retardou o processo de epitelização e causou mais dor e desconforto que nos animais do grupo-controle.

ABSTRACT PURPOSE: Sphincter repair is the primary management for fecal incontinence especially in traumatic causes. Regardless of progression in the method and material of sphincter repair, the results are still disappointing. This study evaluates the efficacy of using amniotic membrane during sphincteroplasty regarding its effects in healing of various tissues. METHODS: Rabbits undergone sphincterotomy and after three weeks end to end sphincteroplasty was done. Animals divided to three groups: classic sphincteroplasty, sphincteroplasty with fresh amniotic membrane and sphincteroplasty with decellularized amniotic membrane. Three weeks after sphincteroplasty animals were sacrificed and sphincter complex was sent for histopathologic evaluation. Sphincter muscle diameter and composition of sphincter was evaluated. Before sphincterotomy, before and after sphincteroplasty electromyography of sphincter at the site of repair were recorded. RESULTS: No statistical significant difference was seen between groups even in histopathology or electromyography. CONCLUSION: Although amniotic showed promising effects in the healing of different tissue in animal and human studies it was not effective in healing of injured sphincter.

Introduction: currently, the most relevant applications of perinatal derivatives are in wound healing, for example, the use of amniotic membrane (AM) as a biomaterials source for use in different healing processes. The anastomotic healing compromised by antimetabolic drugs, such as 5-fluorouracil (5-FU), is a potential target of AM. Objective: To evaluate the healing effects of the AM use in rats treated with 5-FU at a dose of 20 mg/kg on the 7th postoperative day, regarding the parameters percentage of type I collagen (mature), cell viability, microvascular density and granulation tissue formation. Method: Thirty-two Wistar rats were submitted to colotomy and colorraphy, separated in 4 groups with 8t rats each and received, by 7 days, different treatments intraperitoneally, once daily from the operation day until sacrifice: 1- saline solution (C), 2- 20 mg/kg 5-FU, 3- 20 mg/kg 5-FU and 4- MA, and MA. Results: Tthe treatment with 5-FU (20 mg/kg – 7 days) induced adverse effects in the anastomotic healing process evidenced by the decrease of type I collagen (mature), cell viability, percentage microvascular density, granulation tissue formation (leukocyte-fibrin crust and angio-fibroblastic proliferation). The use of AM, under these conditions, induced an improvement in type I collagen (mature) percentage. Conclusion: The treatment with 5-FU induced adverse effects on the healing anastomotic process, and the use of AM, under these conditions, induced improvement in type I (mature) collagen percentage.

A membrana amniótica (MA) consolidou-se no tratamento de afecções na superfície ocular. O objetivo deste estudo foi avaliar a viabilidade do implante de MA equina em ceratoplastia lamelar de cães. As membranas amnióticas foram preservadas em glicerina (98%). A eficácia do implante foi acompanhada por avaliação clínica, tempo de cicatrização, resposta inflamatória e reconstrução da arquitetura da córnea. Foram selecionados 12 cães, que foram divididos em quatro grupos de três animais. Em cada animal, foi realizada ceratotomia lamelar com 5 mm de diâmetro, seguida de aplicação do implante de MA. Após cirurgia, os animais foram avaliados em diferentes tempos: 2, 7, 21 e 40 dias. Durante o período de observação, os exames oftalmológicos foram realizados com intervalo de 48 h e, após a última avaliação, os animais foram submetidos à eutanásia. Os olhos foram enucleados, fixados e corados com hematoxilina-eosina (HE), ácido periódico de Schiff (PAS) e picrossirius. Os implantes foram completamente epitelizados em cerca de 10 dias após a cirurgia. Os neovasos apresentaram involução progressiva a partir de 21 dias e não foram detectados ao final de 40 dias pós-cirurgia, restando apenas uma nébula no local da lesão. À microscopia óptica, observou-se resposta inflamatória moderada, presença de epitélio pavimentoso estratificado aos sete dias e epitelização completa aos 21 dias. Aos 40 dias, a membrana basal do epitélio apresentou-se reconstituída. Assim, concluímos que a membrana amniótica equina é viável como implante em córnea de cão, sendo incorporada ao estroma e resultando em restabelecimento parcial da transparência.