Application of Preserved Human Amniotic Membrane for Corneal Surface Reconstruction

Objective: To evaluate the efficacy of preserved human amniotic membrane transplantation for reconstruction of the corneal surface diseases. Methods: Preserved human amniotic membrane transplantations were performed in 84 eyes of 78 patients for corneal surface reconstruction. The indications were limbal stem cell deficiency from Steven–Johnson syndrome, chemical burn and herpes keratitis (27 eyes), bullous keratopathy (26 eyes), persistent epithelial defect and dellen (17 eyes), band keratopathy (11 eyes), preparing for prosthesis (1 eye), corneal ulcer (1 eye) and acute chemical burn (1 eye). Results: Success was noted in 83.3% (70/84) eyes, partial success in 13.1% (11/84) eyes, and failure in 3.6% (3/84) eyes for an average follow-up of 10.5 months (3 – 29 months). No patient developed major immediate post-operative complications. Conclusion: Amniotic membrane transplantation can reduce inflammation, promote corneal epithelial healing, and decrease irritation in corneal surface problems.     

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.     

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.     

Comparison of characteristics of cultured limbal cells on denuded amniotic membrane and fresh conjunctival, limbal and corneal tissues

This study aimed to evaluate proposed molecular markers related to eye limbal stem cells (SC) and to identify novel associated genes. The expression of a set of genes potentially involved in stemness was assessed in freshly prepared limbal, corneal and conjunctival tissues. PAX6, AC133, K12 and OCT4 were detected in all the tissues and p63(+)/K3(-)/K12(+)/Nodal(+)/Cx43(+) were expressed in conjunctival, p63(-)/K3(+)/K12(+)/Nodal(-)/Cx43(+) in corneal, and p63(+)/K3(-)/K12(-)/Nodal(-)/Cx43(-) in limbal tissues. Limbal explants were cultured on human amniotic membrane for 21 days. The cells expressed p63 but not K3, K12, Nodal and Cx43, however, the expression of K3, K12 and Cx43 was detected, and p63 and the high BrdU-labeling index decreased with more culture. Ultrastructure analysis of the cultured cells showed typically immature organization of intracellular organelles and architecture. Our data suggest that limbal, corneal and conjunctival tissues are heterogeneous with some progenitors. Also, the expression of traditional SC markers may not be a reliable indicator of limbal SC and there is an increasing need to determine factor(s) involved in their stemness.     

Safety and efficacy of human amniotic membrane in primary pterygium surgery

Grafts made from human amniotic membrane are used to prevent recurrence of pterygium after excision. The success of the procedure can be affected by the quality of preparation and preservation of the grafts. We prospectively evaluated the safety and efficacy of cryopreserved amniotic membrane prepared at the research tissue bank of the Biotechnology Research Center in Tripoli, Libya, and used as adjunct therapy in primary pterygium excision. Twenty-six patients (15 males and 11 females) aged 21–78 years and indicated for primary pterygium excision were transplanted at the Tripoli Eye Hospital with the amniotic membrane grafts. Sixteen patients (62 %) were available for all three follow-up visits scheduled at 1, 3 and 6 months post-surgery. By the third visit, two patients (12.5 %) developed granuloma and three (18.8 %) had pterygium recurrence. The grafts were used after cryopreservation for ≤180 days or >180 days, but statistical analysis showed that the complications were not associated with the length of storage. Moreover, the high rate of complications in this study was not caused by use of cryopreserved AM. In conclusion, locally produced cryopreserved AM is safe as an adjunct therapy for treatment of primary pterygium excision.     

The change of cytokines in tear and blood after different pterygium operation

Pterygium is an invasion of altered ocular tissue into the cornea. Bone marrow-derived stem cells have been reported to be involved in wound healing under chemotactic factors after pterygium removal and pain may act as a trigger signal. We evaluated the change of systemic and local chemotactic factors that could affect the mobilization and migration of BMSCs to the wound bed after conventional bare sclera pterygium excision. We also applied temporary amniotic membrane patch after pterygium removal, and compared the changes of cytokines with those of conventional bare sclera excision group. Substance-P (SP), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) were measured in plasma and tear using ELISA and migrating CD34⁺ cells by flow cytometry. The results showed that post-operative pain was much reduced (p < 0.05), and SP, VEGF and SCF kept consistently lower levels in plasma after temporary amniotic membrane application. Circulating CD34⁺ cells increased slightly in the temporary amniotic membrane patch group compared with marked increase in the bare sclera group. Thus, the application of a temporary amniotic membrane after pterygium removal might be an effective therapeutic means by controlling pain and excessive infiltration of bone marrow-derived stem cells.     

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.     

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.     

Signaling roadmap to epithelial–mesenchymal transition in pterygium,TWIST1 centralized

Pterygium as a complex disease shares common features with other malignant cells in its onset recurrence and especially epithelial–mesenchymal transition (EMT) transition. Although using different approaches including conjunctival autografts, amniotic membrane, radiotherapy, mitomycin C (MMC) has shown promising insights in the inhibition of pterygium recurrence, it needs to be investigated in more details in molecular pathways to present adjuvant target therapy. In this study, we aimed to evaluate the expression of and then illustrate the role of signaling pathways on EMT in pterygium. Using real-time polymerase chain reaction, the twist-related protein 1 (TWIST1) expression was compared in primary pterygium and normal conjunctiva. This study assessed the mRNA expression, as well as the association between the clinicopathological indices and the gene expression level. The expression level of TWIST1 was overexpressed in 36% of our cohort ( n = 76). There was a significant positive correlation between recurrence with grade T, grade V and a significant negative correlation with growth activity. Our vast literature review on different signaling pathways in pterygium showed that EMT has centralization role in recurrence of this disease. Our data confirmed that EMT is important in the recurrence of pterygium samples and different signaling pathways end up activating the EMT markers. It is suggested to evaluate the environmental factors and their correlation with molecular markers to select favorable treatment for this kind of diseases.     

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.     

USE OF FREE GRAFTS IN CORRECTION OF RECURRENT PTERYGIA,PSEUDOPTERYGIA AND SYMBLEPHARON

Use of conjunctival material for grafting over areas of the eye denuded by removal of a pterygium, pseudopterygium or symblepharon gives better results cosmetically, and in some instances functionally, than either thin split skin or mucous membrane from the mouth. A technique for grafting with conjunctiva is described.     

Use of free grafts in correction of recurrent pterygia,pseudopterygia and symblepharon

Use of conjunctival material for grafting over areas of the eye denuded by removal of a pterygium, pseudopterygium or symblepharon gives better results cosmetically, and in some instances functionally, than either thin split skin or mucous membrane from the mouth. A technique for grafting with conjunctiva is described.     

老年翼状胬肉患者翼状胬肉切除与角膜缘干细胞移植联合治疗的疗效观察

摘要 目的：分析评估老年翼状胬肉患者实施翼状胬肉切除与角膜缘干细胞移植联合治疗的临床效果。方法：回顾性纳入的本院2018年08月至2020年07月期间295例翼状胬肉患者（共442只眼）的临床资料,根据术式差异将患者分为联合组（150例,共224只眼）和参照组（145例,共218只眼）,参照组病患仅择取翼状胬肉切除手术,联合组病患于以上基础实施角膜缘干细胞移植术,比较两组病患术前及术后各时间点（1周、1个月、3个月）的BUT、SIt、UCVA、CAD、CAA,统计病患术后3个月的复发情况。结果：两组BUT在术后1周较术前显著缩短（P<0.05）,而在术后1月和3月较术前显著延长（P<0.05）,两组BUT在术后1周对比差别明显（P<0.05）,而其他时间点对比没有明显差别（P>0.05）。两组术前术后SIt对比没有明显差别（P>0.05）,术后1月和3月的两组UCVA均高于术前（P<0.05）,而两组在各时间点UCVA对比没有明显差别（P>0.05）,两组术后各时间点的角膜CAD较术前显著性降低（P<0.05）,而两组角膜CAD在各时间点无明显差别（P>0.05）。两组术后CAA术后3月与术前比较,WR比例显著降低,AR、OA比例显著增加（P<0.05）,而组间对比没有明显差别（P>0.05）。术后复发率联合组（6.67%）与参照组（32.26%）对比有明显差别（P<0.05）。结论：与翼状胬肉切除术比较 ,老年翼状胬肉病患实施翼状胬肉切除与角膜缘干细胞移植术联合治疗对患者早期眼表无较大影响,两种术式均不影响泪液分泌,且能够改善裸眼视力、散光度及轴向,但联合治疗在降低术后复发率方面优势明显。     

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.     

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.     

翼状胬肉切除联合自体角膜缘干细胞移植术对翼状胬肉患者视觉质量和泪膜功能的影响

目的:探讨翼状胬肉切除联合自体角膜缘干细胞移植术治疗翼状胬肉的疗效及对视觉质量和泪膜功能的影响。方法:选取2017年2月～2018年12月期间我院收治的翼状胬肉患者201例,根据乱数表法将患者随机分为对照组(n=100)和研究组(n=101),对照组给予翼状胬肉切除术治疗,研究组在此基础上联合自体角膜缘干细胞移植术治疗。比较两组临床疗效、视觉质量、泪膜功能、并发症以及角膜上皮修复时间。结果:研究组术后6个月的临床总有效率为90.10%(91/101),显著高于对照组的73.00%(73/100)(P0.05)。研究组角膜上皮修复时间均短于对照组(P0.05)。两组术后6个月角膜散光度降低,且研究组低于对照组(P0.05);角膜水平曲度、角膜垂直曲度、视力、泪膜破裂时间(BUT)、基础泪液分泌试验(SIT)均升高,且研究组高于对照组(P0.05)。研究组随访期间并发症发生率低于对照组(P0.05)。结论:翼状胬肉患者在翼状胬肉切除术的基础上联合自体角膜缘干细胞移植术治疗,疗效确切,可有效改善患者的视觉质量和泪膜功能,并可减少术后并发症的发生率,临床应用价值较高。     

Histopathology of the pterygium in population on Croatian Island Rab

In the 1975-2004 period, 130 cases of pterygium were estimated, 83 males and 47 females, on a small island Rab. Island Rab is located in the north part of the Adriatic sea, which has very high index of sun radiation. Pterygium is usually histopathologically defined as a hyperplasia of conjunctival tissue, elastoid degeneration of subepithelieum and fragmentation of Bowman's membrane. Our histopathological findings in 73 eyes are following: conjunctiva with neovascularisation, leukocyte margination and subepithelial basophilic degeneration, proliferation of conjunctival tissue, acanthosis and squamous metaplasia, and mucinous elements, focaly present plasma cells, focuses of increased pigmentisation in basal epithelial layers, tenon capsule with edema, diffuse neovascularisation and fragmentation of fibrils. These findings suggest that in severe cases of pterygium histopathologically exists precancerosis. In conclusion, on the basis of these histopathological findings, especially in the Tenon capsule, we suggest that for surgical procedure of pterygium the excision of the Tenon capsule, extensively under pterygium, is necessary.     

Successful Reconstruction of Damaged Ocular Outer Surface in Humans Using Limbal and Conjuctival Stem Cell Culture Methods

When the ocular outer surface is badly damaged, subsequent corneal transplantation fails due to the absence of basal cells that are needed to support the graft. With the realization that the limbus and the conjunctiva have adult stem cells that can be cultured, it has been possible for us to explant culture these on de-epithelized human amniotic membrane, and to graft the resulting viable and transparent epithelium to 125 needy human patients with success. Ultrastructural, histological, biochemical and immunological assays establish the identity of the cells and the tissue formed.