Amniotic Tissue Research Articles

Zika virus infection and the nervous system

Zika virus is an arbovirus from the family of flaviviruses, which is transmitted by the mosquito Aedes aegyptii and also by the Asian mosquito Aedes albopticus. The largest observed Zika virus epidemic is currently taking place in North and South America, in the Caribbean, southern USA and Southeast Asia. In most cases the infection is an unspecific, acute, febrile disease. Neurological manifestations consist mainly of microcephaly in newborns and Guillain-Barré syndrome but other rare manifestations have also become known in the meantime, such as meningoencephalitis and myelitis. Therefore, the Zika virus, similar to other flaviviruses, has neuropathogenic properties. In particular, the drastic increase in microcephaly cases in Brazil has induced great research activities. The virus is transmitted perinatally and can be detected in the amniotic fluid, placenta and brain tissue of the newborn. Vaccination or acausal therapy does not yet exist. The significant increase in Guillain-Barré syndrome induced by the Zika virus was observed during earlier outbreaks. In the meantime, scientifically clear connections between aZika virus infection and these neurological manifestations have been shown. Long-term studies and animal models should be used for a better understanding of the pathomechanisms of this disease.

Human Somatic Stem Cell Neural Differentiation Potential.

Human somatic stem cells can be identified and isolated from different types of tissues and are grouped here based on their developmental maturation and ability to undergo neural differentiation. The first group will represent afterbirth somatic tissues, which are perinatal stem cells including placental blood and tissue, amniotic fluid and tissue, and umbilical cord blood- and umbilical cord tissue-derived cells. The second group of cells discussed in this chapter is the adult stem cells, generally those in a transient period of development, thus placing them in the special position of transitioning from the perinatal to young somatic tissue, and they include the menstrual blood-, the peripheral blood-, and the bone marrow-derived stem cells.

Cryopreserved Amniotic Membrane Improves Clinical Outcomes Following Microdiscectomy.

Prospective, randomized controlled trial. To compare pain, physical/mental functional recovery and recurrent herniation for patients following lumbar microdiscectomy with and without the use of a cryopreserved amniotic tissue graft. Although microdiscectomy procedures are routinely successful for patients with lumbar radiculopathy due to herniated disc disease, residual low back pain, and recurrent herniation remain unsolved clinical problems. Following Investigated Review Board approval, 80 subjects were randomized in a 1:1 ratio to either receive cryopreserved amniotic (cAM) tissue or no tissue following elective lumbar microdiscectomy surgery. cAM grafts were applied to the annular defect at the conclusion of the procedure. Patients provided preoperative and postoperative clinical assessment data out to 24 months using the Oswestry Disability Index (ODI), Short Form-12 (SF-12) Health Survey, and Visual Analog Pain Scale for back and leg pain. Patients with symptomatic recurrent disc herniation were recorded. In total, 48 males and 32 females with an average age of 47.2 years were included. Mean ODI scores for subjects treated with cAM graft demonstrated statistically greater improvement at 6 weeks (14.49 vs. 21.82; P=0.05) and 24 months (6.62 vs. 14.40; P=0.02) compared with controls. Similarly, SF-12 Physical Component Scores demonstrated statistically greater gains in the cAM group at both the 6 weeks and 24 months. None of the subjects in the cAM graft group sustained a recurrent herniation at the same surgical level, whereas 3 patients in the control group sustained a recurrent herniation at the same surgical level, with 2 requiring fusion to manage persistent pain. The data demonstrate statistically superior clinical outcomes following lumbar microdiscectomy as measured by ODI and SF-12 (physical composite scale) and a lower rate of recurrent herniation with the use of a cAM tissue graft compared with traditional microdiscectomy.

Amniotic therapeutic biomaterials in urology: current and future applications.

To examine the rationale and applications of amniotic tissue augmentation in urological surgery. Published literature in English-language was reviewed for basic science and clinical use of amniotic or amnion-chorionic tissue in genitourinary tissues. Basic science and animal studies support the likely benefit of clinical applications of amnion-derived tissues in a variety of urologic interventions. The broad number of properties found in amniotic membrane, coupled with its immunologically privileged status presents a number of future applications in the urological surgical realm. These applications are in their clinical infancy and suggest that further studies are warranted to investigate the use of these products in a systematic fashion.

Rehabilitation Following Limbal Stem Cell Deficiency: Current Treatment Options and Future Developments

Limbal stem cell deficiency (LSCD) is a condition caused by the loss of corneal epithelial regenerative potential. The treatment of this condition is still a challenge. It results from various conditions both intrinsic as well as extrinsic. LSCD can be either uni- or bilateral and either partial or total. Today treatment options include a variety of techniques including transplantation of amniotic membrane and limbal tissue or tissue engineered cell sheets. This article summarizes the current techniques to treat LSCD and upcoming developments.

Application of Amniotic Membrane Improves Repair in a Diabetic Animal Model for Delayed Tendon Healing

Category: Basic Sciences/Biologics, Diabetes Introduction/Purpose: Tendon injuries often heal with significant scar formation and compromised biomechanical function. For diabetics, these injuries are further complicated by changes in the extracellular matrix of the tendon, leading to higher incidences of injury and slower healing. Defective or delayed healing is a result of several factors including an impaired ability to form a collagen matrix, compromised angiogenesis, and inadequate production of growth factors. Consequently, complications and re- rupture rates are higher in diabetic patients. Amnion-derived cells resulted in improved tendon healing in an otherwise healthy animal model, in part due to their ability to provide numerous regenerative cytokines to the repair site. The purpose of this study was to evaluate the effect of amniotic tissues on tendon healing in a diabetic model with impaired healing. Methods: For this study BBZDR/WOR animals, an insulin dependent Type II diabetic rodent model, were used. After appropriate anesthesia, a full thickness injury was made through the Achilles tendon; immediately following injury, the tendon was repaired using the modified Kessler method. Repaired tendons were wrapped with a 0.5 × 0.5 cm section of either a fresh hypothermically stored human amniotic membrane (HSAM), a dehydrated human amnion chorion membrane (dHACM), or left unwrapped as a control. Contralateral tendons were used as sham controls, which were exposed then immediately closed. Tendons were retrieved at 14 or 28 days and evaluated using histology, immunohistochemistry, and qPCR. At 28 days, both experimental tendons and contralateral controls from five rats per group were harvested and tested to failure and peak force, stiffness, energy uptake, and displacement at rupture were then determined. Results: At day 14, histological evaluation found significant increases in cellular recruitment at the site of injury in both dHACM and HSAM treated animals (p=0.0001). qPCR and immunofluorescence results confirmed several biomarkers implicated in tendon repair were highly expressed in the treatment groups, but not in untreated controls. In dHACM treated animals, TNF-a, TGFß-1, IL6, FLAP, Tenascin-C, and Scleraxis expression was elevated at 14 days. Control animals with only primary repair resulted in a tendon repair failure rate of 20%; whereas animals treated with HSAM or dHACM had a 6% and 0% failure rate, respectively. dHACM treated tendons also had significant improvements in biomechanical properties compared to controls including increases in the max force, stiffness and strain (47±21%, 146±90%, and 59±35% respectively). Conclusion: Augmentation of tendon repair with placental-derived membranes may improve diabetic tendon repair and reduce re-rupture rates. This study presents compelling data suggesting chorion-containing dHACM is more efficacious than the amnion- only HSAM for tendon repair. The cause for this difference is unclear, we hypothesize that cytokine content, important for tendon healing, within the chorion may be responsible. Components of placental tissue contribute different cytokines, ECM and cell populations—this study highlights that differences in components-delivered and processing techniques may impact outcomes and results may vary for different clinical applications. This study highlights a promising treatment option for a clinically challenging population.

Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

Construction of tissue-engineered skin by culture of human amniotic epithelial cells on human de-epidermized dermis for the repair of skin defects in nude mice: an experimental study

Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED) . Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs, and a two-step collagenase digestion was used to treat healthy children′s prepuce tissues to prepare fibroblast suspensions. When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2, they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissue-engineered skin. A total of 20 healthy male nude mice aged 3-4 weeks were enrolled into this experiment, and full-thickness skin defects were made on the middle of the back of mice. Then, these mice were randomly divided into 2 groups by using a lottery method, and reconstructed full-thickness tissue-engineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively. The whole body and transplantation sites of the nude mice were observed on day 7, 14, 21 and 28 after transplantation, the wound healing time and rate were compared between the above two groups, and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination. Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen-4 (SSEA-4) . After 2-week organ culture, the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium, and the histological structure of the epidermis was similar to that of the normal human skin. Compared with the control group, the tissue-engineered skin group showed significantly higher wound healing rates on day 7, 14 and 21 after transplantation (57.49% ± 6.11% vs. 22.93% ± 4.26%, 92.80% ± 3.10% vs. 54.57% ± 7.94%, 98.83% ± 0.25% vs. 91.16% ± 4.79%, respectively; n= 10, t= 27.36, 32.23, 11.80, respectively, all P < 0.001) , shorter wound healing time[ (21.51 ± 1.51) d vs. (28.80 ± 1.14) d, n= 10, t= 42.23, P < 0.001], with the color of skin grafts closer to that of autologous skin on day 28 after transplantation. Histological examination revealed distinct stratification of the epithelium, obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group, but thin epithelium with some defects, indistinct stratification of the dermis, and inflammatory cell infiltration in the control group. Conclusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation, resulting in a more favorable healing of wounds, and is expected to serve as a kind of ideal tissue-engineered skin. Key words: Tissue engineering; Epithelial cells; Dermis; Skin transplantation; Wound healing; De-epidermized dermis; Human amniotic epithelial cells

Amnion Membrane in Diabetic Foot Wounds: A Meta-analysis.

Background:Amniotic membrane is tissue obtained from human placenta rich in cytokines, growth factors, and stem cells that possess the ability to inhibit infection, improve healing, and stimulate regeneration.Methods:A meta-analysis was performed examining randomized controlled trials comparing amniotic tissue products with standard of care in nonhealing diabetic foot ulcers including PubMed, Cochrane Central Register of Controlled Trials, and the Cochrane Database of Systematic Reviews.Results:A search of 3 databases identified 596 potentially relevant articles. Application of selection criteria led to the selection of 5 randomized controlled trials. The 5 selected randomized controlled trials represented a total of 311 patients. The pooled relative risk of healing with amniotic products compared with control was 2.7496 (2.05725–3.66524, P < 0.001).Conclusions:The current meta-analysis indicates that the treatment of diabetic foot ulcers with amniotic membrane improves healing rates in diabetic foot ulcers. Further studies are needed to determine whether these products also decrease the incidence of subsequent complications, such as amputation or death, in diabetic patients.

Contemporary Methods Allowing for Safe and Convenient Use of Amniotic Membrane as a Biologic Wound Dressing for Burns.

Partial-thickness burns involve damage to the upper layer of skin, which leaves nerve endings exposed, and therefore represent the most painful of several categories of thermal injuries. Historically, partial-thickness burns were treated conservatively by debriding the blisters, by daily tubbing and washing, and the application of new bandages with topical medications. Human amniotic membrane has been used for centuries as a biological wound dressing. In the past 20 years, there has been an increasing body of literature addressing the use of amniotic tissue in chronic wounds and burns. In this review, we present an overview of the use of amniotic membrane in the treatment of burns including processing methods and early clinical use. We believe that amniotic membranes have great potential in improving burn wound care in the future. Standardized processing methods and terminal sterilization ensure safety and allow the material to be available for use by health care providers around the world in clinical trials and for patient care.

Does a prenatal bacterial microbiota exist?

Does a prenatal bacterial microbiota exist?

Amniotic Derived Stem Cells: Role and Function in Regenerative Medicine

Stem cells are undifferentiated biological cells that have the ability to differentiate into any specialized cells capable of cell division through mitosis. Amniotic stem cells (ASCs) are collectively a mixture of stem cells that can be obtained from amniotic fluid and tissue. Amniotic fluid is commonly called a pregnant woman’s water. It is the protective liquid contained within the amniotic sac that lubricates the embryo or fetus that is generated by the maternal blood plasma, which is the liquid component that contains all of the blood derived cells in suspension. The amniotic fluid is absorbed through the fetal tissue and skin until the 20-25th week of pregnancy when the fetal skin begins to keratinize. This coincides with the fetal gut and its capacity to perform amniotic absorption.

Endoscopic Discectomy with Amniotic Allograft Tissue Implantation for Treatment of Lumbar Disc Herniation

Endoscopic Discectomy with Amniotic Allograft Tissue Implantation for Treatment of Lumbar Disc Herniation

Isolation, Culture, Identification and Differentiation of Canine Amniotic Mesenchymal Stem Cells in Vitro

Recent years, people are paying more and more attention to amniotic membrane tissue which has a rich resource and no ethical restrictions. Canine amniotic mesenchymal stem cells (cAMSCs) were collected by enzyme digestion method from canine amniotic membrane tissues. The cell morphology was continuously observed at different passages and cell growth curves of passage 3 and passage 9 were drawed. Stem cell related proteins and genes were identified by immunofluorescence and RT-PCR. The multiple differentiation potential of cAMSCs was identified by osteogenesis and adipogenesis. This study showed that the cAMSCs was long fusiform and could be continuously subcultured in vitro. The immunofluorescence results showed that Vimentin and SSEA-4 were positively expressed. The RT-PCR results showed that stem cell related genes OCT4, SOX2 and NANOG were positively expressed too. The cAMSCs could be induced to osteoblasts and adipocytes in vitro. The markers associated with osteogenesis and adipogenesis, such as COL1A1 and LPL, were positively expressed after differentiation. The results showed that the cAMSCs could be successfully isolated and cultured in vitro. Stem cell related proteins and genes were positively expressed. And the cAMSCs has the multiple differentiation potential. This study showed that cAMSCs could be a rich source of stem cells in veterinary. Furthermore, cAMSCs may be useful as a cell therapy application for veterinary regenerative medicine.

Amniotic Fluid, Cells, and Membrane Application

Orthobiologics is a rapidly growing field, as the potential of stem cells becomes better understood. Amniotic tissue has a long history of clinical use and its anti-inflammatory and paracrine function makes it an attractive source for cells. The use of amniotic membrane for cartilage damage has been evaluated primarily in preclinical settings. Multiple in vitro studies have shown that amniotic membrane and amniotic mesenchymal stem cells can produce a chondrocyte phenotype with accumulation of glycosaminoglycans, collagen, and chondrogenic markers. Both autologous and allogeneic sources are available, with the latter having the benefit of decreased morbidity to the patient. A wide array of placental-derived allograft tissue forms are currently available in both tissue and injectable formats. Sheets generally consist of one or more intact layers of placental membrane, namely amnion, amnion and chorion, double layer amnion, or umbilical cord. Liquid products consist of morselized tissues such as amnion or chorion or both, suspensions containing cells such as amniotic fluid stem cells, purified variants of amniotic fluid, or some combination. Clinical and basic science investigations are underway to define the cellular mechanisms of action and appropriate clinical indications for the use of these placental-derived products.

158 - Improved Outcome Function following Lumbar Microdiscectomy is Achieved with the Use of an Amniotic Tissue Graft

158 - Improved Outcome Function following Lumbar Microdiscectomy is Achieved with the Use of an Amniotic Tissue Graft

Microcefalia e Zika Vírus: características e associações

Objetivo: Discutir a associação entre microcefalia e a infecção materna por Zika Vírus. A microcefalia é o tamanho da cabeça menor do que o esperado em comparação com bebês do mesmo sexo e idade. Entre as causas conhecidas, estão as infecções congênitas. O aumento de casos entre outubro e novembro de 2015 no nordeste brasileiro, que coincidiu com a presença da circulação de novo vírus no país, em maio do mesmo ano, criou a hipótese de associação entre a microcefalia e a infecção materna durante a gravidez. O Zika Vírus é um arbovírus similar ao da Febre Amarela e da Dengue, transmitido principalmente através da picada do Aedes aegypti. A provável transmissão por relação sexual e transfusão de sangue, além de outros vetores como o Aedes albopictus e possivelmente até o pernilongo (Culex sp) aumentam a necessidade de cuidados preventivos em relação à infecção. O exame para detecção viral idealmente é realizado até o quinto dia após o início dos sintomas. Sorologias ainda não são amplamente disponíveis no Brasil. Métodos: Revisão narrativa da literatura. Conclusão: A associação entre casos de microcefalia e o Zika Vírus é embasada nos relatos de relação têmporo-espacial, padrão de alterações neurológicas associado a malformações congênitas, presença do RNA viral no líquido amniótico e nos tecidos de fetos. As respostas definitivas de causalidade serão possíveis após pesquisas e disponibilidade de exames laboratoriais. As evidências até agora apoiam fortemente esta hipótese e todas as medidas preventivas devem ser estimuladas.

The Use of Cryo-Preserved Umbilical Cord Plus Amniotic Membrane Tissues in the Resection of Tarsal Coalition

Category: Hindfoot Introduction/Purpose: Tarsal coalition can be a common underlying cause of painful flatfoot deformity in adolescent and adult patient populations. When conservative options fail, surgical resection becomes the mainstay of treatment. Adequate resection leaves a void, often filled with bone wax, fat graft or adjacent muscle or tendon. Interposition grafting is designed to provide a physical barrier between the ends of the excised segment and deter recurrence or bony overgrowth. No definitive graft choice has been shown to be superior to another. The goal of this study was to evaluate the use commercially available umbilical cord plus amniotic membrane tissues as an interposition graft with functional and radiological outcomes at one year follow-up. Methods: Retrospective review was performed of patients who had resection of tarsal coalition at our institution. Patients who had interposition of umbilical cord plus amniotic membrane tissues (Amniox Medical, Atlanta GA) at the site of the resection were included in this review. Postoperative data collection included VAS scores, Foot Function index scores, and radiographic review. Radiographic review included postoperative CT scans in all patients. Assessment of hindfoot ROM was noted on physical examination at last follow up visit. Results: Fourteen patients (8 male and 6 female; median age 28.5 years at time of evaluation) were enrolled in the study. Of these patients, 7 were calcaneonavicular coalitions and 7 were talonavicular coalitions. Average follow up occurred at 18 months (range 9-32 months). Interposition of umbilical cord plus amniotic membrane tissues was performed at the site of the resection. Median improvement in pain visual analog score (VAS) was 89.75%, with no post-operative infection or wound complications. Post-operative CT scan review demonstrated evidence of osteoarthritis in two patients (one at talonavicular and one subtalar joint). Four of 14 patients (29%) were found to have recurrence at follow-up. The median post-operative Foot Function Index score was 46 (range 30-110) and 64% of patients had improvement in range of motion. Conclusion: Based on radiological and functional outcomes at an average 19 month follow up, the interposition of umbilical cord plus amniotic membrane tissues after tarsal coalition resection appears to be a viable option in treating these patients. No adverse reactions were found and recurrence rate was noted to be 29%.

Optimizing amniotic membrane tissue banking protocols for ophthalmic use.

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco's modified eagle's medium (DMEM) and glycerol at -80°C and at -196°C for a period of 6weeks and at 4°C in 70% alcohol for 6weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5×5cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at -196 and -80°C for 6 weeks. Similar sections were also stored at 4°C in 70% alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10% FBS in humidified atmosphere of 5% carbon dioxide at 37°C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8×10(6); 1.59, after 6weeks in DMSO:FBS at -196°C 3.0×10(6); 2.38 and at -80°C 2.1×10(6); 1.60, in DMEM:Glycerol at -196°C 3.6×10(6); 2.33 at -80°C 23×10(6); 1.66 and at 4°C 3.3×10(6); 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at -196°C showed morphology similar to fresh AM in both preservation media and AM stored at -80°C showed disruption of the stromal matrix. At 4°C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at -196°C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.

Pterígio e sua abordagem cirúrgica com membrana amniótica

A patogênese do Pterígio está fortemente relacionada à exposição à luz ultravioleta, ressecamento, inflamação crônica e exposição a vento e poeira; substâncias irritantes também são consideradas fatores predisponentes na histopatologia na degeneração elástica do colágeno estromal e tecido fibrovascular subepitelial.A prevalência aumenta progressivamente com a proximidade à linha do equador.O método de correção cirúrgica considerado pela comunidade médica e pela literatura é o transplante de conjuntiva; entretanto exponho aqui minha experiência e de nossa equipe em aproximadamente 6.000 casos desde 1996, realizados com membrana amniótica. As vantagens na utilização desse método são em primeiro lugar a economia do tecido conjuntival do paciente, o mesmo índice de recidiva do transplante livre de conjuntiva ± 4%, aspecto estético igual ou melhor e a obtenção infinda desse tecido. A técnica utilizada nos últimos quatro anos inclui anestesia subtenoniana com Levobupivacaina 0,50% 5mg/mL, a ampla excisão do corpo do pterígio, a retirada da cabeça por dissecção ou arrancamento, uso de mitomicina 0,02 por 2 minutos e lavado intensamente com soro e uso da cola de fibrina para posicionamento do retalho de membrana amniótica.Casos de Pterígio primário, recidivados, ou extensos assim como os pterígios duplos nasal e temporal que requerem muito tecido, são facilmente reparados com retalhos de membrana amniótica e não poderiam ser feitos com retalhos conjuntivais pela extensão de tecido requerida.Os retalhos de membrana amniótica são preparados a partir de placentas obtidas por parto cesáreo, em pacientes com pré-natal completo, com exames para hepatites, sífilis e HIV e colocados em retalhos de papel de filtro e estocados em geladeira embebidos em solução de antibiótico e glicerina (100%) e hidratados com soro ou ringer no momento da cirurgia. O posicionamento correto da membrana amniótica é com a parte estromal voltada para a superfície ocular (enxerto) e essa servirá como membrana basal ao crescimento de conjuntiva que apresenta estereótipo indistinguível da conjuntiva normal.O pós-operatório inclui curativo oclusivo por 3 dias com 2 trocas diárias com colírio e pomada de antibiótico e corticoide. Após os 3 dias é receitado colírio de Hialuronato de sódio 2mg/mL de 2 em 2 horas, mais, colírio de associação antibiótico, mais, corticosteroide 4 vezes ao dia por 1 semana.É sabido que há polêmicas em vários itens sobre cirurgia de pterígeo – entre eles:Anestesia: alguns sugerem anestesia retrobulbar, outros peribulbar, outros subtenoniana (nossa preferência), subconjuntival, somente tópica e há até os que realizam cirurgia sob anestesia geral. A experiência, após anos de testes nas várias modalidades, mostrou que a anestesia subtenoniana com Levobupivacaína sem conservante 3 ml proporciona um pouco mais de conforto nesse pós operatório de sintomatologia tão intensa.Pós operatório: no pós operatório utilizo para analgesia anti-inflamatório não hormonal), associado à Tylex®, Dipirona gotas (até de 4/4 horas).Cola ou Sutura: Polêmica também é a escolha de cola ou sutura na fixação do retalho, seja conjuntival, seja de membrana amniótica. Minha experiência com cola data de 3 anos e utilizamos apenas cola, com preferência a Tissucol® (Baxter®) – que tem funcionado melhor. Anteriormente usávamos sutura com nylon 10.0. Alguns colegas associam alguns pontos de sutura à cola (4 pontos), mas a experiência com cola e membrana mostrou que não é necessário suturar.Membrana amniótica: Amnion ou porção interna das membranas placentarias consiste de uma membrana basal e matriz estromal avascular. A composição da membrana basal é semelhante à da conjuntiva, e é considerada como substrato ideal para garantir o crescimento de tecido conjuntival e suporte de um retalho conjuntival. Nos casos nos quais as diversas recidivas impõem um tratamento cirúrgico com todos os recursos a membrana amniótica mostrou ser de grande valia.A escolha desse tecido na reconstrução da conjuntiva em Exérese de Pterígeo e em outras lesões conjuntivais deve-se às propriedades da membrana facilitando a epitelização, mantendo fenótipo epitelial idêntico ao da conjuntiva e ao mesmo tempo reduzindo inflamação, vascularização e cicatrização.Também é importante ressaltar a facilidade de obtenção desse tecido e a preservação da conjuntiva para outros procedimentos futuros como recobrimento conjuntival, cirurgias de glaucoma, entre outros, especialmente o excelente resultado estético e funcional assim como o baixo índice de recidiva.Nas figuras 1 a 8 ilustra-se, caso de Pterígeo extenso que cobria a córnea em olho direito e bilateral, porém menos extenso no olho esquerdo e seu resultado final.