Preserved Amniotic Membrane Transplantation for Conjunctival Surface Reconstruction

Objective. To study the efficacy of preserved human amniotic membrane in the reconstruction of conjunctival defect created during surgical removal of conjunctival lesions or symblepharon lysis. Methods. Preserved human amniotic membrane transplantation was performed in 93 eyes of 85 patients for reconstruction of various conjunctival surface problems. The indications for surgery were (1) pterygium excision (54 eyes), (2) conjunctival tumors excision (23 eyes), lysis of symblepharon (13 eyes), and covering a scleral graft (three eyes). Results. Success was noted in 69.9% (65/93) eyes, partially success in 22.6% (21/93) eyes, and failure in 7.5% (7/93) eyes with a mean follow-up of 8.9 months (1–28 months). In pterygium, conjunctival tumor, symblepharon, and scleral graft group, the success rate in each group was 70.3%, 78.3%, 53.8%, and 66.7% respectively. No serious immediate post-operative complications or graft rejection occurred. Conclusion. Amniotic membrane transplantation can be considered an alternative treatment for difficult ocular surface problems, and is effective in promoting epithelial healing, and reducing inflammation and scarring.     

Ophthalmic Applications of Preserved Human Amniotic Membrane: A Review of Current Indications

Preserved human amniotic membrane (AM) is currently being used for a wide spectrum of ocular surface disorders. The AM has a basement membrane, which promotes epithelial cell migration and adhesion. The presence of a unique avascular stromal matrix reduces inflammation, neovascularization and fibrosis. The basic tenets of amniotic membrane transplantation (AMT) are to promote re-epithelialization, to reconstruct the ocular surface and to provide symptomatic relief from surface aberrations. AMT is a useful technique for reconstruction of surface defects resulting from removal of surface tumors and symblephara. AMT has effectively restored a stable corneal epithelium in eyes with, persistent epithelial defects and corneal ulcers. In the setting of acute ocular burns and SJS, AMT has satisfactorily reduced scarring and inflammation. AMT alone may be an effective alternative for partial limbal stem cell deficiency. However remarkable improvements in surface stability have resulted from concurrent AMT and limbal stem cell transplantation, wherein the limbal grafts are obtained from the normal fellow eye, living relative or cadaveric eye. In severe or bilateral cases, well being of the donor eye is a major concern. Currently, the most unique application of preserved human AM in ophthalmology is its use as a substrate for ex-vivo expansion of corneal and conjunctival epithelium. In this novel technique of tissue engineering, epithelial stem cells can be safely harvested and expanded on denuded AM. The resultant composite cultured tissue has been successfully transplanted to restore vision, as well as the structure and function of damaged ocular surfaces.     

Further evaluation of amniotic membrane banking for transplantation in ocular surface diseases

Objective: To define the best conditions foramniotic membrane preparation, storage and banking in its use for cornealreconstruction.Methods: Amniotic membrane pieces were prepared understerile conditions from placentas selected on the basis of donor medical andsocial history, serology, microbiological tests and histology. The pieces werekept at –140 °C but before grafting they werethawed and stored at 4 °C in RPMI medium, to have apreparation usable within 72 h. This procedure was validatedby testing its therapeutic effectiveness in 25 patients 13 of which had cornealulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 bandkeratopathy and 6 corneal stem cell deficiency due to chemical or thermalburns.Results: The preparation showed appreciableanti-inflammatory and analgesic effects. In the absence of corneal stem celldeficiency a stable re-epithelialisation was achieved in 15 out of 19 patients.When the limbus was lesioned, the amniotic membrane decreased vascularizationand increased the number of corneal epithelial cells only in 1 of the 6patients. No adverse reactions attributable to the tissue were recorded.Conclusions: A ready-to-use amniotic membrane preparationstored at 4 °C after cryopreservation has been tested incorneal reconstruction. Like the amniotic membrane thawed immediately beforegrafting, this preparation displayed full therapeutic effect in epithelialdefects with stromal ulceration but without severe limbal stem cell deficiency.In two years banking activity 463 pieces of the preparation were successfullydistributed to 90 Italian hospitals.     

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.     

Reconstruction of a rabbit corneal epithelium on a lyophilized amniotic membrane using tilting air-liquid interface culture followed by tilting submerged culture

Herein, we reconstructed a rabbit corneal epithelium on a lyophilized amniotic membrane (LAM) using a modified version of two Teflon rings (the Ahn’s supporter). We compared the corneal epithelial cells we had differentiated in vitro using air-liquid interface (6 days, 12 days) and submerged (6 days, 12 days) cultures and followed a six-day tilting dynamic air-liquid interface culture with a six-day tilting submerged culture. We characterized the reconstructed corneal epithelium using digital photography, histological imaging, and transmission electron microscopy. The reconstructed corneal epithelium created under air-liquid interface culture exhibited a healthier basal corneal epithelial layer than that created under submerged culture. The reconstructed corneal epithelium on the LAM that was produced using the tilting dymanic culture exhibited a healthy basal layer. We therefore proposed that tilting submerged culture not only supplied nutrients from the medium to the corneal epithelial cells on the LAM, but it also removed the horny layer in the upper part of the reconstructed corneal epithelium, presumably by mimicking the effects of blinking. This study demonstrated that corneal epithelium reconstruction on a LAM using a tilting submerged culture after a tilting air-liquid interface culture may be useful not only for allogeneic or autologous transplantation, but also for in vitro toxicological test kits.     

Amniotic membrane graft: histopathological findings in five cases

Amniotic membrane transplantation (AMT) is an effective treatment for ocular surface reconstruction; however, the mechanisms through which amniotic membrane (AM) exerts its effects as well as its fate after transplantation have not been entirely elucidated and have been investigated only in part. We evaluate the integration of AM in the host cornea in five patients who underwent AMT as the result of Bowen's disease, band keratopathy, radio- or cryotherapy-induced keratopathy, chemical burn or post-herpetic deep corneal ulcer with descemetocele. Due to persistent opacification in four cases and a progressing tumor in one case, penetrating keratoplasty (PK) and enucleation were performed as early as 2 months and up to 20 months after AMT. The corneas were analyzed histopathologically. To evaluate AM remnants, corneas were stained with periodic acid Schiff's reaction (PAS), Alcian blue, and Gomory and Masson trichrome; immunostaining including collagens III and IV antibodies was also performed. None of the corneas showed remnants of AM. In all cases, we observed discontinuity of Bowman's membrane. In three cases, the corneal epithelium was completely restored, ranging from three to six cell layers. In the other two cases, we detected an intense inflammatory reaction with rich neovascularization; the epithelial surface of the central cornea was completely restored, while at the periphery of the cornea goblet mucus-producing cells were present. Although clinically useful in all cases, restoration of a stable corneal epithelium through AMT is limited by the extent and severity of limbal stem cell deficiency (LSCD). The lack of histologically documented AM remnants in our cases seems to explain the efficacy of AMT more through its biological properties than through its mechanical properties.     

Long-term result of autologous cultivated oral mucosal epithelial transplantation for severe ocular surface disease

The present study aimed to investigate the clinical outcomes of autologous cultivated oral mucosal epithelial transplantation (COMET) on human amniotic membrane (AM) for corneal limbal stem cell deficiency (LSCD). In this prospective, noncomparative case series, 20 eyes (18 patients) with bilateral severe ocular surface disease were chosen to undergo COMET on human AM. The primary outcome was clinical success, and the secondary outcomes were the best-corrected visual acuity difference, corneal opacification, symblepharon formation, and complications. The mean patient age was 48.2 ± 15.5 years. The mean follow-up time was 31.9 ± 12.1 months (range 8–50 months). All except one eye exhibited complete epithelialization within the first postoperative week. A successful clinical outcome, defined as a stable ocular surface without epithelial defects, a clear cornea without fibrovascular tissue invasion at the pupillary area, and no or mild ocular surface inflammation, was obtained in 15 of 20 eyes (75 %). The clinical success rate at 1 year was 79.3 %, and that at 4 years (end of follow-up) was 70.5 %. Fourteen of 20 (70 %) eyes exhibited improvement in visual acuity after COMET, and some required subsequent cataract surgery (2 eyes), penetrating keratoplasty (3 eyes), or keratoprosthesis implantation (1 eye). Preoperative symblepharon was eliminated in most eyes (8 of 13, 61.5 %) after COMET combined with eyelid reconstruction when needed. The only complication was corneal perforation (1 eye) induced by a severe eyelid abnormality; treatment with a tectonic corneal graft was successful. COMET can successfully restore ocular surface damage in most eyes with corneal LSCD.     

Amniotic Membrane Transplantation for Ocular Surface Reconstruction

The use of amniotic membrane (or amnion) for transplantation as graft in ocular surface reconstruction is reviewed. This technique has become widespread because of the availability of the amnion, convenience and ease of use, and high and reproducible success rates. The mechanisms of action of the transplantation are varied and include the prolongation and clonogenic maintenance of epithelial progenitor cells, promotion of goblet and non-goblet cell differentiation, exclusion of inflammatory cells with anti-protease activities, suppression of Transforming Growth Factor signaling and myoblast differentiation of normal fibroblasts. The observed clinical effects include facilitation of epithelialization, maintenance of normal phenotypes, and reduction of inflammation, vascularization and scarring. Amniotic membrane transplantation is being increasingly used as graft for various conjunctival and corneal diseases and as a patch in cases of chemical and thermal burns, refractory and recalcitrant keratitis, and most recently as an excellent substrate for expanding epithelial stem cells ex vivo.     

Amniotic membrane transplantation for ocular surface reconstruction

The purpose of this study is to analyze the clinical experience and the effect of human amniotic membrane transplantation on pterygium excision and bullous keratopathy. From January 1999 to January 2001 at University Hospital "Sestre milosrdnice" amniotic membrane transplantation was performed consecutively in 21 eyes: 11 eyes with bullous keratopathy and 10 with recurrent pterygia. In the group with bullous keratopathy epithelization took place in 19.6 days in 72.7% and the reduction of pain was satisfactory. Recurrence rate in group with recurrent pterygia was 20%. Based on the presented results it could be concluded that amniotic membrane transplantation can be considered as an effective alternative for treating severe ocular surface diseases and as an alternative for penetrating keratoplasty if there is a lack of grafts.     

Amniotic membrane transplantation in the treatment of persistent epithelial defect on the corneal graft

It has been shown that amniotic membrane transplantation (AMT) improves healing of the epithelium defects as it serves as a basement membrane for endothelial cells growth, prevents inflammatory cell infiltration and reduces apoptosis in keratocytes. Having in mind the healing properties of AM we investigated the efficacy of AMT in persistent epithelial defect (PED) on the corneal graft. 80 corneal grafts were prospectively followed up for presence of PED 10 months after surgery. PED was detected in 12 cases (15%) having surgery for: rejected graft (n = 4), keratoconus (n = 3), keratoconus following PK on a second eye (n = 3), corneal perforation (n = 1) and Stevens-Johnson keratopathy (n = 1). Epithelial defect (ED) developed 14 +/- 7 days after surgery in 10 cases and 1.5 month in other two. All patients were primarily conservatively treated with subconjuctival steroids and artificial tears for 10 days and systemic steroid therapy if needed after, until the period of 2 weeks. 4 patients were healed. Since ED was unresponsive to all previous treatments for more than 2 weeks, one layer of AM was placed on the corneal lesion in 5 patients, and in 3 cases of deep PED several layers of AM were placed. Healing of the defect was obtained in 7/8 (87.5%) eyes. In 1 patient second AM transplantation was necessary. Mean epithelization time was 2 weeks (range 1-3 weeks) in monolayer and 3 weeks (range 2-4 weeks) for multilayer cases. 5 out of 8 patients retained the same best corrected visual acuity (BCVA) while 3/8 patients improved their vision more than 2 lines. Preoperative corneal thickness of 255 +/- 40 mm increased to 455 +/- 90 mm. AM transplantation facilitates healing of corneal epithelium. PED on the corneal graft unresponsive to conventional treatment can be effectively cured when covered with one or more amniotic membrane layers.     

Amnion and ocular surface problems

The amniotic membrane, the most internal placental membrane, has various properties useful in ophthalmology. Collected on delivery by elective Caesarean section, the amnion is prepared under sterile conditions, and, usually, cryopreserved until its use as a biological bandage or as a substrate for epithelial growth in the management of various ocular surface conditions. Specifically, the amnion is used to : (1) limit formation of adhesive bands between eyelids and eyeball (symblepharon) or the progression of a fibrovascular outgrowth towards the cornea (pterygium) or to (2) facilitate the healing of corneal ulcers, bullous keratopathy, and corneal stem cell deficiency. In this last condition, either hereditary or acquired after a thermal or a chemical burn, corneal stem cells, located at a transitional zone between the cornea and conjunctiva, are lost. These cells are essential for renewal of corneal epithelium in normal and in diseased states. The loss of these cells leaves the corneal surface free for invasion by conjunctival epithelium. Not only, does conjunctival epithelium support the development of vascularisation on the normally avascular cornea, but some conjunctival cells differentiate into mucus secreting goblet cells. Such a change in phenotype leads to loss of corneal transparency and visual disability. The removal of this fibro-vascular outgrowth in combination with transplantation of both amniotic membrane and corneal stem cells are used to treat this condition. The amnion stimulates the proliferation of less differentiated cells which have the potential to reconstruct the cornea. This potential is at the origin of the hypothesis that the amnion may provide an alternative niche for limbal stem cells of the corneal epithelium. It abounds in cytokines and has antalgic, anti-bacterial, anti-inflammatory and anti-immunogenic properties, in addition to allowing, like fetal skin does, wound healing with minimal scar formation. These desirable properties are responsible for the increasing use of amniotic membrane in ophthalmology. The complete understanding of the mechanisms of action of amniotic membrane for ocular surface diseases has yet to be understood. Once revealed by research, they may provide new pharmacological avenues to treat ocular surface diseases.     

Amniotic membrane transplantation for ocular surface reconstruction in neurotrophic corneal ulcera

The purpose of this study is to analyze clinical experience about the effects of human amniotic membrane transplantation in eyes with neurotrophic ulcers. In 11 eyes the application of amniotic membrane was performed since January 1999 because of neurotrophic ulcers. The follow up period was longer than 12 months: 19.7+/-6.0 months. The average healing period after the surgery was 1.6+/-0.6 weeks. All corneas were fluorescein negative even 12 months after operation. Visual acuity after the transplantation was similar to the one before the surgery in 8 eyes. In 3 eyes the visual acuity after the surgery was better than before. Amniotic membrane transplantation can be considered an effective alternative for treating persistent epithelial defects such as neurotrophic ulcers. It has some advantages over corneal transplantation: a relatively simple procedure, no allograft rejection and it could be particularly beneficial in countries where cornea shortage is apparent.     

Silica gel dissication of amniotic membrane with related epithelium cells for ocular surface reconstruction

Cornea reparative regeneration when in various pathological states needs creating certain conditions to intensify the potential of regional stem cells mitotic activity. Aims of the Research. To find out the degree of the epithelium AM preservation after preliminary processing and conservation by means of dehydration over silica gel with further sterilization; to study the effectiveness of clinical treatment of AM conserved in the surroundings with vital ability epithelium and AM dried over silica gel. Materials and methods. There was carried out an investigation of 18 samples of native amnion treated with antibiotics and 18 total surface amnion samples conserved by drying over silica gel and then sterilized by gamma rays. Clinical experiments were carried out on patients with severe chemical and thermal burns – 18 people (21 eyes). After the burn trauma all the patients underwent the standard procedure of necrectomy, the covering of the eyeball with amniotic membrane dried over silica gel. Conclusion. The drying out of the amniotic membrane over silica gel on frames without being fixed on nitrocellulose paper makes the process of the amniotic membrane conservation simpler and makes it possible to preserve its unique biological qualities. The effectiveness of the regeneration of epithelium tissue of the eyeball surface with amniotic membrane dried over silica gel without the vital capacity cells of the epithelium layer is analogous to the regeneration of epithelium cells with amniotic membrane with vital capacity cells. With eye burns AM coverage hinders the formation of rough conjunctiva cicatrix, provides a favorable out-of-cell matrix substrate for epithelium migration and leads to quicker regeneration of one's own epithelium, makes further visual rehabilitation simpler. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regulation of Connexin43 Gap Junction Protein Triggers Vascular Recovery and Healing in Human Ocular Persistent Epithelial Defect Wounds

Transiently blocking the expression of the gap junction protein connexin43 using antisense oligodeoxynucleotides or blocking hemichannels with connexin mimetic peptides has been shown to significantly improve outcomes in a range of acute wound models. Less is known about their likely effects in nonhealing wounds. In the eye, prolonged inflammation and lack of epithelial recovery in nonhealing corneal epithelial wounds may lead to corneal opacity, blindness or enucleation. We report here the first human applications of antisense oligodeoxynucleotides that transiently block translation of connexin43 in a prospective study of five eyes with severe ocular surface burns (persistent epithelial defects), which were unresponsive to established therapy for 7 days to 8 weeks prior to treatment. Connexin43-specific antisense oligodeoxynucleotide was delivered in cold, thermoreversible Poloxamer407 gel under either an amniotic membrane graft or a bandage contact lens. The connexin43-specific antisense application reduced inflammation within 1–2 days, and in all five eyes complete and stable corneal reepithelialization was obtained. Recovery of the vascular bed and limbal reperfusion appeared to precede corneal epithelial recovery. We conclude that connexin modulation provides a number of benefits for nonhealing ocular burn wounds, one of which is to promote vascular recovery.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6 +/- mouse model of epithelial wound-healing delay

Background  

Congenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells.     

Amniotic membrane in ophthalmology: properties,preparation, storage and indications for grafting—a review

The use of amniotic membrane in ophthalmic surgery and other surgical procedures in the fields of dermatology, plastic surgery, genitourinary medicine and otolaryngology is on the increase. Furthermore, amniotic membrane and its epithelial and mesenchymal cells have broad use in regenerative medicine and hold great promise in anticancer treatment. Amniotic membrane is a rich source of biologically active factors and as such, promotes healing and acts as an effective material for wound dressing. Amniotic membrane supports epithelialization and exhibits anti-fibrotic, anti-inflammatory, anti-angiogenic and anti-microbial features. Placentas utilised in the preparation of amniotic membrane are retrieved from donors undergoing elective caesarean section. Maternal blood must undergo serological screening at the time of donation and, in the absence of advanced diagnostic testing techniques, 6 months postpartum in order to cover the time window for the potential transmission of communicable diseases. Amniotic membrane is prepared by blunt dissection under strict aseptic conditions, then is typically transferred onto a nitrocellulose paper carrier, usually with the epithelial side up, and cut into multiple pieces of different dimensions. Amniotic membrane can be stored under various conditions, most often cryopreserved in glycerol or dimethyl sulfoxide or their mixture with culture medium or buffers. Other preservation methods include lyophilisation and air-drying. In ophthalmology, amniotic membrane is increasingly used for ocular surface reconstruction, including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries.     

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.