The Fate of Limbal Epithelial Progenitor Cells during Explant Culture on Intact Amniotic Membrane.

• Li W,
• Hayashida Y,
• He H,
• Kuo CL,
• Tseng SC.

Ocular Surface Center, and TissueTech, Inc., Miami, Florida.

PURPOSE: The clinical success of treating corneas with total limbal stem cell deficiency using limbal biopsy explants cultured on intact amniotic membrane (iAM) relies on ex vivo expansion of limbal epithelial progenitor cells. However, the ultimate fate of limbal epithelial progenitor cells in the explant remains unclear. METHODS: Human limbal explants were cultured on iAM for 2 weeks and then removed and transferred to a new iAM until passage 3. The outgrowth surface area of each passage was measured and compared. For each passage, clonogenicity on 3T3 fibroblasts feeder layers was compared among progenitor cells removed from the outgrowth, the explant surface, and the remaining stroma. Cryosections of the explant and the outgrowth were detected with p63, vimentin, pancytokeratin, and the basement membrane components type VII and IV collagen and laminin 5 antibodies. RESULTS: The outgrowth surface area significantly decreased from passage (P)1 to P3. The total number of epithelial cells that were isolated from the explant surface also decreased from before culture (P0) to P1, became stable from P1 to P2, but was uncountable at P3. Clonogenicity significantly declined from P1 to P3 for the epithelium derived from the explant surface and the outgrowth epithelium; the extent was less in the former than in the latter at P2 and P3. In addition, groups of epithelial cells invaded the limbal stroma of the explants from P1 to P3; p63(+)/pancytokeratin(-) and p63(+)/vimentin(+) cells also presented in the limbal stroma. Increasing fibroblast, but not epithelial, colonies were observed from cells isolated from the remaining limbal stroma when seeded on 3T3 fibroblast feeder layers from P1 to P3. CONCLUSIONS: During ex vivo expansion on iAM, some limbal epithelial progenitor cells indeed migrate onto iAM from the explant surface, whereas some also invade the limbal stroma, very likely undergoing epithelial-mesenchymal transition. This new information should be taken into account in formulating new strategies to improve the expansion protocol.

PMID: 17251456 [PubMed - in process]

Role of amniotic membrane graft for ocular chemical and thermal injuries.

• Tejwani S,
• Kolari RS,
• Sangwan VS,
• Rao GN.

Comprehensive Ophthalmology Services, L. V. Prasad Eye Institute, L. V. Prasad Marg, Banjara Hills, Hyderabad. sushma@lvpei.org

PURPOSE: To evaluate the results of amniotic membrane transplantation (AMT) for ocular surface reconstruction in chemical and thermal injuries. METHODS: Retrospective review of case records of patients who had undergone AMT for chemical injuries (January 1998 to May 2001). RESULTS: Seventy two eyes of 69 patients were studied of which 24 were acute cases (median-2 days, range, 1-20 days) and 48 were chronic cases (median-12.4 months, range, 1.02-95.8 months). Mean age was 22.4 years (SD +/- 13.34 years) and average follow up duration was 7.8 months (SD +/- 7.1). Main clinical findings were symblephara (52.8%), corneal vascularization (51.3%), conjunctivalization (45.8%), Limbal ischemia (45.8%), Limbal stem cell deficiency (55.5%) and epithelial defect (48.6%). 18 cases were due to acid injuries (5 acute, 13 chronic), 52 were due to alkali (18 acute and 34 chronic) and 2 cases were due to thermal burns (1 each acute and chronic). Overall success rate was 87.5% in acute cases and 72.9% in chronic cases. Indication-wise success rates were 94.3% for epithelial defect healing, 88.2% for symptomatic relief, 59.7% for ocular surface reconstruction, and 55% for improving limbal stem cell function. Success was not achieved in any outcome measure in 1/24 (4.2%) in acute group and 6/48 (12.5%) in chronic group. CONCLUSION: AMT helps in ocular surface reconstruction, promotes rapid epithelial healing and partially restores limbal stem cell function. It can be considered as an effective modality for the ocular surface restoration in chemical and thermal injuries in selected cases. Success rates in acute and chronic cases are comparable.

PMID: 17198009 [PubMed - in process]

A novel method for preserving cultured limbal epithelial cells.

• Utheim TP,
• Raeder S,
• Utheim OA,
• Cai Y,
• Roald B,
• Drolsum L,
• Lyberg T,
• Nicolaissen B.

Center for Eye Research, University of Oslo, Department of Ophthalmology, Norway.

BACKGROUND/AIMS: To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue engineered epithelia used to treat patients with limbal stem cell deficiency. METHODS: Limbal epithelial cells were cultured for three weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for one week at 23 degrees C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry. RESULTS: The viability of the preserved epithelia was 84+/-20%, and no statistically significant difference was found compared to non-preserved epithelia. In general, the cell borders were maintained, the nuclei demonstrated no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained. CONCLUSIONS: Cultured limbal epithelial cells can be preserved in organ culture medium for one week at room temperature while maintaining the original layered structure and undifferentiated phenotype.

PMID: 17124242 [PubMed - as supplied by publisher]

[Advantages of amniotic membrane transplantation in eye surface diseases]

• Muraine M,
• Gueudry J,
• Toubeau D,
• Gardea E,
• Verspyck E,
• Menguy E,
• Brasseur G.

Service d'Ophtalmologie, Hopital Charles Nicolle, Rouen.

Amniotic membrane transplantation is now a widely adopted technique in the field of eye surface diseases. Depending on the indication, the amniotic membrane can be used as either a graft or a patch. When used as a graft, the amniotic membrane serves as a substrate for regrowth of deficient epithelium; the aim is to integrate this membrane. The basal membrane reinforces the adhesion and differentiation of the corneal epithelial cells, facilitates their migration, and prevents their apoptosis. When used as a patch, the amniotic membrane is sutured epithelium-down so as to maximize the concentration of biological factors delivered by this membrane: the membrane covers the diseased cornea and acts as a biological bandage and analgesic. The best indications for amniotic membrane grafts are acute chemical burns and trophic corneal ulcers refractory to all medical treatment. When these ulcers are perforated or in the early stages of perforation, it is best to use multiple layers of amniotic membrane, restoring the thickness of the cornea. In cases of confirmed limbal deficiency, amniotic membrane grafts may be a useful complement to the necessary limbal stem cell grafts. In the future, amniotic membranes will provide an indispensable support for the expansion of cultured stem cells. Amniotic membrane grafts may also be used to reconstruct the conjunctiva following the exeresis of symblepharons or conjunctival tumors. However, the use of this technique is currently limited to diseases with little inflammation and no extensive fibrosis.

PMID: 17115002 [PubMed - in process]

Analysis of corneal surface evolution after moderate alkaline burns by using impression cytology.

• Lopez-Garcia JS,
• Rivas L,
• Garcia-Lozano I,
• Murube J.

Ophthalmology Services, Hospital Cruz Roja, Madrid, Spain. docsantilopez@hotmail.com

PURPOSE: To compare corneal surface evolution after moderate alkaline burns by impression cytology in patients treated with medical therapy or with amniotic membrane transplantation (AMT). METHODS: A prospective study of 24 eyes from 18 patients (13 men and 5 women) with moderate alkaline burns was performed. All patients were divided according to the clinical ocular severity and the therapy used. Twelve eyes were treated surgically with AMT and the other 12 eyes received only medical therapy. Corneal cytology was obtained immediately after the burns, and 1, 2, 5, and 9 months later. We differentiated between samples obtained from affected areas and areas not affected by the burns. Cellular size, nuclear size, and nuclear-cytoplasmic (N:C) ratio were examined in corneal epithelial cells, as was the presence of goblet cells in corneal epithelium. RESULTS: Nuclear size, cellular size, and N:C ratio in non-burn-affected corneal areas had no significant alterations in comparison with normal eyes. In contrast, in burn-affected corneal areas, these parameters were significantly worse, and the presence of goblet cells in corneal epithelium was frequent 1 month after severe burns. Cellular size, nuclear size, N:C ratio, and corneal conjunctivalization improved during the study in all patients, but corneal reepithelialization occurred earlier in patients treated with AMT than in patients with only medical therapy. CONCLUSION: Morphologic and morphometric analysis of corneal cells by impression cytology after ocular burns permits the establishment of cellular reepithelialization patterns in relation with limbal deficiency level and with clinical ocular severity. AMT improves corneal reepithelialization earlier than medical therapy in moderate alkaline burns.

PMID: 17102665 [PubMed - indexed for MEDLINE]

Technique of cultivating limbal derived corneal epithelium on human amniotic membrane for clinical transplantation.

• Fatima A,
• Sangwan VS,
• Iftekhar G,
• Reddy P,
• Matalia H,
• Balasubramanian D,
• Vemuganti GK.

Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, L.V. Prasad Eye Institute, Banjara Hills, Hyderabad - 500 034, India.

BACKGROUND: The technique of transplantation of cultivated limbal epithelium rather than direct limbal tissue isa novel method of "cell therapy" involved in reconstructing the ocular surface in severe limbal stem cell deficiency [LSCD], caused by chemical burns. AIM: To describe a simple feeder-cell free technique of cultivating limbal epithelium on human amniotic membrane[HAM]. MATERIALS AND METHODS: The limbal tissues (2 mm) were harvested from patients with LSCD. These tissues were proliferated in vitro on HAM supplemented by human corneal epithelial cell medium and autologous serum. Cultures covering more > or = 50% area of 2.5 x 5 cm HAM were considered adequate for clinical use. The cultured epithelium was characterized by histopathology and immunophenotyping. RESULTS: A total of 542 cultures out of 250 limbal tissues were cultivated in the laboratory from January 2001 through July 2005. The culture explants showed that clusters of cells emerging from the edge of the explants in one-three days formed a complete monolayer within 10-14 days. In 86% of cultures (464 of 542), the growth was observed within one-two days. Successful explant cultures were observed in 98.5% (534 of 542 cultures) with 91% explant cultures showing an area of > or = 6.25 cm2 (6.25 - 12.5 cm2 range). The cultivated epithelium was terminated between 10-14 days for clinical transplantation. The problems encountered were inadequate growth (2 of 542) and contamination (2 of 542). CONCLUSIONS: We demonstrate a simple technique of generating a sheet of corneal epithelium from a limbal biopsy. This new technique could pave the way for a novel form of cell therapy.

PMID: 17102542 [PubMed - indexed for MEDLINE]

Regulation of corneal angiogenesis in limbal stem cell deficiency.

• Ma DH,
• Chen JK,
• Zhang F,
• Lin KY,
• Yao JY,
• Yu JS.

Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan, ROC. davidhkma@yahoo.com

Corneal angiogenesis is associated with a variety of corneal diseases, and is sometimes vision threatening. In recent years, with the discovery of major pro- and anti-angiogenic factors in the cornea, details of the angiogenic process are gradually unveiled. Of note, corneal inflammation and neovascularization associated with severe limbal stem cell (LSC) deficiency is a clinically challenging issue in that the condition persists long after the initial insult, and will not improve without transplantation of LSCs. However, to date the molecular mechanism by which LSC transplantation restores corneal avascularity is not fully understood. In addition to discussing major pro-angiogenic factors involved in corneal neovascularization, this review article also focuses on possible molecular mechanisms underlying persistent inflammation and neovascularization following severe LSC deficiency, and anti-angiogenic factors expressed by human limbo-corneal epithelial cells (HLCECs). Most of the recently discovered corneal anti-angiogenic factors belong to extracellular matrix proteins that acquire angio-inhibitory activity only after proper proteolytic processing. Our recent findings showed that the secretion of endostatin (derived from basement membrane collagen XVIII) and restin (from collagen XV) by HLCECs were enhanced when HLCECs were cultivated on amniotic membrane (AM). This adds to the advantage of transplanting ex vivo expanded HLCECs cultivated on AM in that the anti-angiogenic activity of the epithelial cells is augmented in a physiological way. Furthermore, proteomic profiling of HLCECs and human conjunctival epithelial cells (HCECs) identified a 14-3-3 protein (stratifin) preferentially expressed by HLCECs. In addition to functioning as a cell cycle controller, keratinocyte-derived stratifin induces MMPs which are involved in the generation of restin (by MMP-1) and endostatin (by MMP-3). These findings highlight the significance of delicate epithelial-matrix interactions in the maintenance of corneal avascularity.

PMID: 17079182 [PubMed - indexed for MEDLINE]

Plasticity of epidermal adult stem cells derived from adult goat ear skin.

• Yang X,
• Qu L,
• Wang X,
• Zhao M,
• Li W,
• Hua J,
• Shi M,
• Moldovan N,
• Wang H,
• Dou Z.

Shaanxi Branch of National Stem Cell Engineering Center, Northwest A&F University, YangLing, Shaanxi Province, China.

Here we report the isolation and characterization of pluripotent stem cells from adult goat skin. We found that these primary cells have the properties of embryonic stem cells (ESC), including the expression of appropriate immunological markers and the capability of forming embryoid bodies. The subcultured cells also show the characteristics of stem cells, such as the expression of CK19, beta(1-)integrin, P63, and formation of holo-clones in culture. Therefore, we termed these cells epidermal adult stem cells (EpiASC), although their origin was not identified. We have shown that clones of individual EpiASC proliferate and differentiate in culture to produce neurons, cardiomyocytes, osteoblasts, and occytes. Further, we cultivated EpiASC on bioengineered dermis and denuded human amniotic membrane (HAM), to reconstruct artificial skin and corneal epithelium. We successfully transplanted those artificial tissues in goats with acute full-thickness skin defect (AFTSD) and limbal stem cell deficiency (LSCD), respectively. Our results showed that indeed EpiASC reconstructed the skin (hair was observed in restored areas), and repaired the damaged cornea of goats with total LSCD. These data confirm that EpiASC can differentiate into different functional cell types in vivo or in vitro. Due to their high degree of inherent plasticity, and to their easy accessibility for collection from the skin, EpiASC are excellent candidate sources for diverse cell therapies. (c) 2006 Wiley-Liss, Inc.

PMID: 16998851 [PubMed - in process]

Establishment of a cultivated human conjunctival epithelium as an alternative tissue source for autologous corneal epithelial transplantation.

• Tanioka H,
• Kawasaki S,
• Yamasaki K,
• Ang LP,
• Koizumi N,
• Nakamura T,
• Yokoi N,
• Komuro A,
• Inatomi T,
• Kinoshita S.

Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. htanioka@eye.opth.kup-m.ac.jp

PURPOSE: The corneal epithelium is essential for maintaining corneal transparency, and efforts have been made to develop improved techniques for corneal epithelial transplantation in patients with total limbal failure. We evaluated the suitability of transplanted cultivated human conjunctival epithelium (HCjE) as a corneal epithelium replacement in rabbits with total corneal and limbal deficiency. METHODS: HCjE cells, cultivated on human amniotic membrane (AM) to confluence and exposed to an air-liquid interface (air-lifted), were transplanted onto denuded rabbit corneas and monitored for 2 weeks. The cultivated HCjE sheet and the engrafted epithelium were analyzed by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The transplanted HCjE remained transparent, smooth, and without epithelial defects during the follow-up period. Both the cultivated HCjE cells and the engrafted epithelium manifested five to six layers of stratified squamous epithelium similar in morphology to normal corneal epithelium. The basal cells expressed the putative stem cell markers (ABCG2 and P63) and hemidesmosome and desmosome component proteins. The cytokeratins (CK4, CK13, CK3, and CK12) and MUC4 were found in the engrafted epithelium. However, MUC5AC was not expressed. The results indicate that HCjE cultivated on AM has the potential to be used as an alternative corneal epithelium. CONCLUSIONS: The transplantation of cultivated HCjE sheets is a promising technique for the treatment of eyes with limbal failure.

PMID: 16936093 [PubMed - indexed for MEDLINE]

[Congenital aniridia keratopathy treatment]

• Lopez-Garcia JS,
• Garcia-Lozano I,
• Rivas L,
• Martinez-Garchitorena J.

Hospital Central de Cruz Roja, Madrid, Espana. docsantilopez@hotmail.com

OBJECTIVE: To attempt to review the aniridia keratopathy pathogenesis and establish a treatment protocol according to the severity of the symptoms. METHODS: Personal experience in aniridic keratopathy management and a bibliography review. RESULTS: The ocular manifestations of this anomaly include defects of the cornea, glaucoma, lens subluxation, cataracts, hypoplasia of the iris, fovea and optic nerve, amblyopia and nystagmus. The keratopathy occurs in a 20% of patients with aniridia. The correct PAX6 expression is necessary for normal corneal development, limbal stem cell activity and correct corneal epithelial cell migration and adhesion. CONCLUSIONS: The management of ocular surface diseases due to limbal stem cell deficiency in aniridia is complex but has changed in recent years, as an understanding of the limbal stem cells and their microenvironment has modified the therapeutic approach. The use of autologous serum eye drops, amniotic membrane transplantation, limbal transplantation or cultivated limbal cell transplantation have all been reported as a treatment for several ocular surface diseases.

PMID: 16933167 [PubMed - indexed for MEDLINE]

Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease.

• Nakamura T,
• Inatomi T,
• Sotozono C,
• Ang LP,
• Koizumi N,
• Yokoi N,
• Kinoshita S.

Department of Ophthalmology, Kyoto Prefectural University of Medicine, Graduate School of Medicine, Kyoto, Japan.

PURPOSE: To evaluate the use of autologous serum (AS)-derived cultivated corneal epithelial transplantation for the treatment of severe ocular surface disease. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Nine eyes from 9 patients with total limbal stem cell deficiency were studied. These consisted of 2 eyes with Stevens-Johnson syndrome, 1 with chemical injury, 1 with ocular cicatricial pemphigoid, 1 with Salzmann corneal dystrophy, 1 with aniridia, 1 with graft-versus-host disease, and 2 with idiopathic ocular surface disease. METHODS: Autologous serum obtained from patients was used for cultivating corneal epithelial cells on an amniotic membrane substrate. These AS-derived corneal epithelial equivalents were compared with those derived from fetal bovine serum (FBS)-supplemented medium. At the time of surgery, complete removal of the corneal pannus and conjunctiva up to 3 mm from the limbus was performed. Allogeneic (7 cases) and autologous (2 cases) AS-derived cultivated corneal epithelial equivalents were transplanted onto the ocular surface. Postoperative follow-up included serial slit-lamp examinations with fluorescein staining, as well as photographic documentation. MAIN OUTCOME MEASURES: Ocular surface reconstruction with corneal epithelialization, graft integrity, visual acuity, and postoperative complications. RESULTS: The corneal epithelial sheets cultivated in AS- and FBS-supplemented media were morphologically similar, and demonstrated the normal expression of tissue-specific keratins and junctional specialization assembly proteins. After transplantation, complete corneal epithelialization was achieved within 2 to 5 days. All eyes demonstrated an improvement in visual acuity by > or =2 lines. During the follow-up period of 14.6+/-4.36 months, the corneal surface of all patients remained stable and transparent, without significant complications. CONCLUSIONS: Transplantation of AS-derived cultivated corneal epithelial equivalents was shown to be a feasible method of treating patients with severe ocular surface disease. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

PMID: 16905193 [PubMed - indexed for MEDLINE]

[Amnion and ocular surface problems]

• Giasson CJ,
• Bouchard C,
• Boisjoly H,
• Germain L.

Ecole d'Optometrie, Universite de Montreal, CP 6128, Succursale Centre-ville, Montreal, Quebec, J1H 5N4 Canada. giassonc@opto.umontreal.ca

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.

PMID: 16828041 [PubMed - indexed for MEDLINE]

Interleukin-1 receptor antagonist (IL-1RA) prevents apoptosis in ex vivo expansion of human limbal epithelial cells cultivated on human amniotic membrane.

• Sun CC,
• Su Pang JH,
• Cheng CY,
• Cheng HF,
• Lee YS,
• Ku WC,
• Hsiao CH,
• Chen JK,
• Yang CM.

Department of Ophthalmology, Chung Gung Memorial Hospital, Keelung, Taiwan.

Stem cells of the corneal epithelium have been found to be located exclusively at the anatomical junction between the cornea and the conjunctiva, the limbus. Ex vivo expanded limbal epithelial cells on amniotic membrane (AM) are capable of restoring the corneal surface with limbal stem cell deficiency. Recent studies indicate that intact AM preserves the limbal epithelial phenotype and that distinct epithelial morphology is noted among various culture matrix. However, the factors in response to the interaction between limbal epithelial cells and AM were not well understood. Using Annexin V-fluorescein isothiocyanate staining, we found that human limbal epithelial cells expanded on intact human AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. To identify the anti-apoptotic factors, we performed cDNA microarray analysis and showed that interleukin-1 receptor antagonist (IL-1RA) was overexpressed in cultures on intact AM, which was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (Q-PCR) and enzyme-linked immunosorbent assay. In addition, we also noted that the phenomenon of apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL-1RA protein into the media, whereas apoptosis of limbal epithelial cells cultivated on intact AM could be induced by exogenous neutralizing IL-1RA neutralizing antibody. These results demonstrated that intact human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. IL-1RA might be a candidate mediator to exert as an anti-apoptotic molecule during the interaction between human limbal epithelial cells and intact human AM.

PMID: 16741227 [PubMed - indexed for MEDLINE]

Transplantation of reconstructed corneal layer composed of corneal epithelium and fibroblasts on a lyophilized amniotic membrane to severely alkali-burned cornea.

• Jang IK,
• Ahn JI,
• Shin JS,
• Kwon YS,
• Ryu YH,
• Lee JK,
• Park JK,
• Song KY,
• Yang EK,
• Kim JC.

Department of Ophthalmology, Chung-Ang University Hospital, Seoul, Korea.

The purpose of this article was to evaluate the graft efficacy of reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on a lyophilized amniotic membrane (LAM), in a severely alkali-burned corneal model. After biopsy specimens were obtained from the left eyes of 24 rabbits, the corneal epithelial cells and fibroblasts were expanded in vitro and the corneal layer was reconstructed on LAM. Thirty-six eyes of rabbits underwent alkali burn (1 N NaOH, 30 s) to create a limbal deficiency and a deeply damaged corneal stroma. Four weeks later, group 1 underwent a graft of the reconstructed corneal layer composed of autologous corneal epithelium and fibroblasts on LAM. Group 2 was transplanted with a graft of the reconstructed autologous corneal epithelium, and group 3 served as a control without surgery. Wound healing and stabilization of the ocular surfaces occurred much faster in group 1 than in groups 2 and 3. The eyes in group 3 revealed typical limbal deficiencies with conjuctivalization and persistent corneal epithelial defects. However, the corneas in group 1 developed only mild peripheral neovascularization. Immunohistochemical staining in group 1 demonstrated that p63, cytokeratin 3, E-cadherin, transforming growth factor (TGF)-beta1, and collagen IV were expressed strongly in the corneal epithelium and basement membrane. On the basis of these results, transplantation of the reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on LAM, partially accelerated the recovery of the alkali-injured rabbit ocular surface, and might be useful therapeutically for the treatment of patients with severely damaged cornea.

PMID: 16734593 [PubMed - indexed for MEDLINE]

Combination of serum eye drops with hydrogel bandage contact lenses in the treatment of persistent epithelial defects.

• Schrader S,
• Wedel T,
• Moll R,
• Geerling G.

Department of Ophthalmology, UK-SH Campus Lubeck, Klinik fur Augenheilkunde, Ratzeburger Allee 160, 23538, Lubeck, Germany. mail@stefanschrader.de

BACKGROUND: The treatment of persistent epithelial defects (PED) with autologous serum eye drops is often combined with conventional medication such as artificial tears and topical antibiotics, but until now no report exists on the use of a bandage contact lens (BCL) in combination with autologous serum eye drops in the treatment of PEDs. We report six eyes (five patients) which were all treated with autologous serum eye drops in combination with an FDA group IV hydrogel contact lens. METHODS: Five patients aged 36-88 years, were suffering from six PEDs for 73.5+/-46.9 days due to rheumatoid sterile corneal ulcer (n=1), neurotrophic keratopathy (n=3) or partial limbal stem cell deficiency (n=1). All patients had been unsuccessfully treated with conventional therapy before. Three of them had already had an amniotic membrane transplantation and two had undergone a keratoplasty; however, the epithelial defect persisted or recurred. In all cases, an FDA group IV hydrogel contact lens (Biomedics 55, ocufilcon D, 55% water content) was fitted and serum eye drops applied 8 times a day. RESULTS: The PED healed in five of six eyes after a treatment period of 14.2+/-8.9 days. In one eye the PED became smaller, but it took 90 days until the lesion healed completely. In three eyes (two patients) white deposits appeared on the surface of the BCL during the treatment after 12.3+/-5.1 days. Because no signs of inflammation were observed and since the epithelial defect improved, a new identical lens was applied and the medication continued unaltered. The surface of contaminated and non-contaminated BCLs were analyzed by scanning electron microscopy and SDS gel-electrophoresis. The scanning electron microscopic examination presented a coating of amorphous material with a wrinkled appearance and many corpuscular deposits. There was no indication of bacterial colonisation. The SDS gel-electrophoresis showed a small band at 65 kDa, probably albumin. CONCLUSION: These findings suggest that the combination of a therapeutic contact lens and serum eye drops can be successfully used in the treatment of persistent epithelial defects. Deposition of albumin may occur on the surface of the contact lenses, which, in the small group presented here, caused no unwanted effects.

PMID: 16544115 [PubMed - in process]

Clinical outcome of autologous cultivated limbal epithelium transplantation.

• Sangwan VS,
• Matalia HP,
• Vemuganti GK,
• Fatima A,
• Ifthekar G,
• Singh S,
• Nutheti R,
• Rao GN.

Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, L. V. Prasad Eye Institute, Banjara Hills, Hyderabad, India. vsangwan@lvpei.org

PURPOSE: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. METHODS: Eighty-six patients' records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome and possible factors affecting outcome and complications. RESULTS: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty-two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3-82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1-36.7) were considered failures and 10 patients were lost to follow-up. CONCLUSION: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases.

PMID: 16531667 [PubMed - indexed for MEDLINE]

Comment on:

Am J Ophthalmol. 2005 Aug;140(2):223-30.

Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency.

• Mittal V,
• Sangwan VS,
• Fernandes M,
• Thomas R.

PMID: 16490530 [PubMed - indexed for MEDLINE]

[Amniotic membrane utilization in ophthalmological surgical procedures]

• Waked N,
• El-Kazzi V.

Service d'ophtalmologie, Universite Saint-Joseph, Hotel-Dieu de France, Beyrouth, Liban. wakednaji@yahoo.com

The amniotic membrane, which is the innermost layer of the fetal membranes, is composed of a single layer of epithelial cells that lie on a basement membrane, and of a non-vascular collagenous stroma. These three components give the amniotic membrane its beneficial properties. The first therapeutic application of the amniotic membrane was in 1910, when it was used in skin transplantation. Thereafter, it was used in surgical procedures related to the abdomen, genitourinary tract and to the head and neck. In ophthalmology, De Roth, in 1940, was the first to use the amniotic membrane for conjunctival reconstruction. However, it was only in 1995 that publications on the subject started appearing again, when Tseng and many others began using the amniotic membrane again in the treatment of ocular surface (cornea and conjunctiva) diseases. In cases of total stem cell deficiency, amniotic membrane transplantation has been shown to be very useful when used in conjunction with limbal autografts or allografts. At this stage, however, further studies are needed to elucidate the real potential of the amniotic membrane in the treatment of different ocular surface (and other) disorders, and its exact mechanism(s) of action. This will help establish the applications of such treatment in medicine, in general, and in ophthalmology, in particular.

PMID: 16398211 [PubMed - indexed for MEDLINE]

Signaling-transduction pathways required for ex vivo expansion of human limbal explants on intact amniotic membrane.

• He H,
• Cho HT,
• Li W,
• Kawakita T,
• Jong L,
• Tseng SC.

TissueTech, Inc. and Ocular Surface Center, Miami, Florida 33173, USA.

PURPOSE: Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways. METHODS: The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis. RESULTS: In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached approximately 80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 microM LY294002 or 50 microM SR13668 and was significantly suppressed by 10 microM U0126, but was not affected by 10 microM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion. CONCLUSIONS: Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.

PMID: 16384957 [PubMed - indexed for MEDLINE]

[Moderate limbal deficiency in patients with congenital aniridia treated with amniotic membrane transplantation]

• Lopez-Garcia JS,
• Rivas L,
• Garcia-Lozano I.

Hospital Cruz Roja, Madrid, Spain. docsantilopez@hotmail.com

PURPOSE: To study the ocular surface evolution by impression cytology and limbal biopsy in patients with congenital aniridia treated with amniotic membrane transplantation (AMT). METHODS: We studied 14 eyes of patients with congenital aniridia and moderate limbal deficiency that were treated with AMT. The morphology of the limbocorneal epithelium and stroma was examined by impression cytology and limbal biopsy. Impression cytology was performed prior to surgery and 3, 6, 9, 12 and 24 months after treatment. Limbal biopsy was performed before surgery and 9 months after AMT. RESULTS: Visual acuity showed a mean improvement of 0.3 at 24 months of follow-up. Corneal reepithelialization was completed by 2 months in all patients, however, after 9 months some patients showed persistent epithelial defects and chronic ulceration. Corneal clarity and peripheral neovascularization was markedly improved after 2 months. Impression cytology showed an improvement in squamous metaplasia at 3 and 6 months, however after nine months, until as late as 24 months, a progressive worsening was seen in epithelial cell metaplasia then being similar in extent to that before surgery. Limbal biopsy nine months after surgery showed a nearly normal epithelium and limbocorneal stroma. CONCLUSIONS: AMT is a very effective procedure, although its effect is transitory, for restoring the ocular surface integrity in patients with congenital aniridia and moderate limbal deficiency.

PMID: 16193434 [PubMed - indexed for MEDLINE]

Mucous membrane pemphigoid: an update.

• Eschle-Meniconi ME,
• Ahmad SR,
• Foster CS.

Massachusetts Eye Research and Surgery Institute, Boston, Massachusetts, USA, and Medical School of Aga Khan University, Karachi, Pakistan.

PURPOSE OF REVIEW: To review articles on mucous membrane pemphigoid, published between June 2004-May 2005. RECENT FINDINGS: Decreased glycosylation of mucin was found in patients with ocular cicatricial pemphigoid. A unique antigen in oral mucous membrane pemphigoid has not yet been identified. Increased vascular cell adhesion molecule and intercellular adhesion molecule 1 expression was found in skin of patients affected by mucous membrane pemphigoid. Autoreactive T cells to an epitope of bullous pemphigoid antigen 180 kilodaltons were identified in the blood of some patients with mucous membrane pemphigoid. Circulating IgA against an antigen in mucous membrane pemphigoid was found in about 20% of patients, without prognostic significance. Enhanced sensitivity for direct immunofluorescence was reported if skin biopsy specimens were stored for 24 hours in saline. An enzyme-linked immunosorbent assay for detection of circulating autoantibodies against laminin-5 was developed. Sensitivity was higher than indirect immunofluorescence on salt-split skin and immunoblotting. Patients with younger onset (<60 years) of ocular cicatricial pemphigoid were found to have disease evolution similar to that of an older group (>70 years) but were visually impaired earlier in life. Intravenous immunoglobulin as treatment of ocular cicatricial pemphigoid was found to be superior to conventional immunosuppressants, with fewer side effects and better long-term outcome for halting disease activity. Intraoperative adjunction of mitomycin C during fornix reconstruction with amniotic membrane resulted in achieving a deeper fornix in 83% of patients with various cicatrizing conjunctivitis. Transplantation of cultured epithelial cells of oral mucosa in corneal limbal stem cell deficiency was successful in improving visual acuity and reestablishing corneal transparency in mid- to advanced ocular cicatricial pemphigoid. SUMMARY: Further advances have been achieved in the field of mucous membrane pemphigoid.

PMID: 16175044 [PubMed - indexed for MEDLINE]

Comment on:

Am J Ophthalmol. 2005 Aug;140(2):223-30.

Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency.

• Dogru M,
• Tsubota K.

PMID: 16086951 [PubMed - indexed for MEDLINE]

[Severe limbal deficiency treated by combined limbal allograft and amniotic membrane transplantation]

• Lopez-Garcia JS,
• Rivas L,
• Garcia-Lozano I.

Hospital Cruz Roja y Hospital Ramon y Cajal, Madrid, Spain. docsantilopez@hotmail.com

PURPOSE: To study the ocular surface evolution in patients with severe limbal deficiency treated with limbal allograft transplantation (LT) combined with amniotic membrane transplantation (AMT). METHODS: We studied 14 eyes with severe limbal deficiency, treated with LT combined with AMT (LT + AMT). We studied the morphology of the limbocorneal epithelium and stroma by impression cytology and limbal biopsy. RESULTS: Visual acuity showed a mean improvement of 0.4 at 24 months of follow-up. Corneal clarity and loss of neovascularization were markedly improved after 3 and 6 months, respectively. We did not find corneal conjuntivalization on cytology performed 6 months after surgery, except in a patient with chemical burn. Cytology and limbal biopsy performed nine months after surgery showed an epithelium and limbocorneal stroma near to normality. Squamous metaplasia was worse in patients with Stevens-Johnson syndrome and ocular cicatricial pemphigoid after 12 months, but was similar in patients with chemical burns and improved in patients with aniridia. CONCLUSIONS: LT + AMT is a very effective procedure for restoring the ocular surface integrity in patients with severe limbal deficiency. This combination improves the outcomes obtained with LT alone.

PMID: 16059817 [PubMed - indexed for MEDLINE]

Follow up of patients with ocular scarring secondary to LOC syndrome treated by amniotic membrane transplantation.

• Moore JE,
• Shah S,
• Kumar V,
• Ainsworth JR,
• Page AB,
• McLean WH.

Department of Ophthalmology, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA, Northern Ireland, UK. johnnymoore@doctors.org.uk

AIMS: To document and discuss the long term outcome of a new ophthalmic treatment for laryngo-onycho-cutaneous (LOC) syndrome. METHODS: Two children were treated by excision of ocular granulation tissue and ocular surface rehabilitation with frozen amniotic membrane (AM). The clinical course of both patients was followed and documented at 2 years and 4 years following the surgery. RESULTS: Patient 1 demonstrated limited recurrence of granulation tissue at 10 months. After 36 months, re-growth of granulation and scar tissue required a further three subsequent operations to the right eye in an attempt to keep the optical axis clear. 4 years postoperatively, neither eye has a clear visual axis. In contrast similar surgery for the right eye of patient 2 has been highly successful, with only very limited non-progressive recurrence after 2 years of follow up. The operation to the left eye has been similarly effective although the follow up is only 6 months. CONCLUSIONS: Ocular surface rehabilitation with AM is the first partially effective treatment for the eye complications of LOC syndrome. The surprising benefit from AM may stem from the primary pathology of the condition. LOC syndrome is caused by a genetic defect resulting in an unusual N-terminal deletion of the alpha3a chain of the basement membrane protein laminin 5. One mechanism through which AM transplantation may act to reduce ocular scarring in this disease is to supplement the abnormal secreted laminin 5 with healthy transplanted laminin. Despite its initial efficacy one episode of AM treatment does not guarantee long term control of the scarring process and variations in AM graft efficacy may be related to other complicating factors such as limbal stem cell deficiency or severity of the initial scarring process.

PMID: 16024838 [PubMed - indexed for MEDLINE]

Comment in:

Am J Ophthalmol. 2005 Aug;140(2):305-6.

Am J Ophthalmol. 2006 Mar;141(3):599-600; author reply 600.

Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency.

• Santos MS,
• Gomes JA,
• Hofling-Lima AL,
• Rizzo LV,
• Romano AC,
• Belfort R Jr.

Cornea and External Disease Service, Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), Sao Paulo, Brazil. myrna_serapiao@uol.com.br

PURPOSE: To evaluate the survival of conjunctival limbal grafts and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency (LSCD) and the influence of several parameters as cause of LSCD, dry eye, keratinization, eyelid abnormalities, HLA compatibility, systemic immunosuppression, and keratoplasty (PKP) on surgical outcome. DESIGN: Prospective, noncomparative, interventional case series. METHODS: Thirty-three eyes of 31 patients with total LSCD that underwent conjunctival limbal grafts and AMT at the Department of Ophthalmology, Federal University of Sao Paulo were included in this study. Cumulative graft survival as well as the influence of several variables on surgical outcome was analyzed. RESULTS: Ten eyes (30%) underwent conjunctival limbal autograft and 23 (70%) underwent conjunctival limbal allograft from living HLA-matched donor. Graft survival was seen in 13 eyes (40%) at 1 year and in 11 eyes (33.3%) at 2 years, with a cumulative survival of 33% after a mean follow-up time of 33 months. Increase in postoperative visual acuity was observed in 20 eyes (60.6%) during this period. Marked impact on graft survival was observed for patients with Stevens-Johnson syndrome, dry eye, keratinization, eyelid abnormalities, and allogeneic conjunctival limbal transplantation (independently of HLA compatibility) (P < .05). Preoperative dry eye was the most important prognostic parameter for surgical outcome (P < .001). CONCLUSIONS: Conjunctival limbal grafts associated with AMT are useful for restoring corneal epithelium phenotype in eyes with total LSCD. However, the cumulative survival declines substantially over a 2-year period. Considering all investigated variables, dry eye was the most important prognostic parameter.

PMID: 16023069 [PubMed - indexed for MEDLINE]

Erratum in:

Chin Med J (Engl). 2005 Sep 20;118(18):1563. Ma, Yang-ling [corrected to Ma, Yan-ling].

Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkaline burn.

• Song E,
• Yang W,
• Cui ZH,
• Dong Y,
• Sui DM,
• Guan XK,
• Ma YL.

Department of Ophthalmology, First Affiliated Hospital, Jilin University, Changchun 130021, China.

BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium. CONCLUSIONS: Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.

PMID: 15978194 [PubMed - indexed for MEDLINE]

New perspectives in ocular surface disorders. An integrated approach for diagnosis and management.

• Sangwan VS,
• Tseng SC.

Cornea service, L.V. Prasad Eye Institute, Hyderabad, India. vsangwan@lvpeye.stph.net

The cornea, conjuctiva and the limbus comprise the tissues at the ocular surface. All of them are covered by stratified, squamous, non-keratinizing epithelium and a stable tear film. The ocular surface health is ensured by intimate relationship between ocular surface epithelia and the preocular team film. There are two types of ocular surface failure. The first one is characterized by squamous metaplasia and loss of goblet cells and mucin expression. This is consistent with unstable tear film which is the hallmark of various dry-eye disorders. The second type of ocular surface failure is characterized by the replacement of the normal corneal epithelium in a process called limbal stem cell deficiency. It is essential to establish accurate diagnosis for appropriate management of complex ocular surface disorders. There has been considerable advancement in the understanding of the pathophysiology of ocular surface disease. Management has improved with introduction of the limbal stem cell concept and use of amniotic membrane transplantation.

PMID: 15887723 [PubMed - indexed for MEDLINE]

Detection of corneal epithelial defect through amniotic membrane patch by fluorescein.

• Kobayashi A,
• Ijiri S,
• Sugiyama K,
• Di Pascuale MA,
• Tseng SC.

Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan. kobaya@kenroku.kanazawa-u.ac.jp

PURPOSE: To demonstrate the usefulness of fluorescein dye to detect a corneal epithelial defect through an amniotic membrane patch in 4 cases. PATIENTS AND METHOD: Two patients with painful bullous keratopathy, 1 patient with total limbal deficiency, and 1 patient with acute chemical burn underwent surgeries in conjunction with amniotic membrane as patch. Postoperatively, slit-lamp biomicroscopic examination with and without fluorescein staining was performed. RESULTS: The status of epithelialization, ie, corneal epithelial defect on either denuded corneal stroma or amniotic membrane graft, was indiscernible by slit-lamp biomicroscopy through the amniotic membrane patch. Nevertheless, it could be detected by fluorescein staining, especially after saline rinse, in all 4 cases. CONCLUSIONS: Fluorescein staining permits clinicians to avoid prematurely removing the amniotic membrane patch during the treatment of a persistent corneal epithelial defect.

PMID: 15778616 [PubMed - indexed for MEDLINE]

Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction.

• Daya SM,
• Watson A,
• Sharpe JR,
• Giledi O,
• Rowe A,
• Martin R,
• James SE.

Corneo-Plastic Unit, Queen Victoria Hospital, East Grinstead, West Sussex, United Kingdom. sdaya@centreforsight.com

PURPOSE: To investigate the outcome of a new technique of ex vivo expanded stem cell allograft for limbal stem cell deficiency (LSCD), and to characterize the ocular surface genotype after surgery. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Ten eyes of 10 patients with profound LSCD arising from ectodermal dysplasia (3 eyes), Stevens-Johnson syndrome (3 eyes), chemical injury (2 eyes), thermal injury (1 eye), and rosacea blepharoconjunctivitis (1 eye). INTERVENTION: Allogeneic corneal limbal stem cells were cultured on plastic and transplanted to the recipient eye after removal of conjunctival pannus. Amniotic membrane was applied in a bandage capacity. The procedure was combined with other reconstructive surgery in 2 cases. Nine patients received systemic cyclosporin A immunosuppression, and the DNA genotype was investigated with surface impression cytology. MAIN OUTCOME MEASURES: Parameters of LSCD, including vascularization, conjunctivalization, inflammation, epithelial defect, photophobia, and pain. RESULTS: The mean follow-up period was 28 months (range, 12-50). Seven of 10 eyes (70%) had improved parameters of LSCD at final follow-up and were considered successes. Four (40%) had improved visual acuity, including 3 having had further procedures for visual rehabilitation. Three patients failed to improve-1 with a thermal burn and lid deformity, 1 with Stevens-Johnson syndrome and severe dry eye, and 1 with ectodermal dysplasia who developed an epithelial defect at 26 months. DNA analysis of the first 7 cases showed no ex vivo donor stem cell DNA present beyond 9 months. CONCLUSIONS: Ex vivo expanded stem cell allograft is a useful technique for restoring the ocular surface in profound LSCD. The absence of donor DNA beyond 9 months suggests that ongoing immunosuppression may be unnecessary and raises questions regarding the origin of the host corneal epithelium.

PMID: 15745776 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation for reconstruction of corneal epithelial surface in cases of partial limbal stem cell deficiency.

• Sangwan VS,
• Matalia HP,
• Vemuganti GK,
• Rao GN.

Sudhakar & Sreekanth Ravi Stem Cell Biology Laboratory, L V Prasad Eye Institute, Hyderabad, India. vsangwan@lvpei.org

PURPOSE: To assess the efficacy of amniotic membrane for treatment of partial limbal stem cell deficiency (LSCD). METHODS: Medical records of four patients with partial LSCD who underwent pannus resection and amniotic membrane transplantation (AMT) were reviewed for ocular surface stability and improvement in visual acuity. Clinico-histopathological correlation was done with the resected pannus tissue. RESULTS: All the eyes exhibited stable corneal epithelial surface by an average of 7 weeks postoperatively with improvement in subjective symptoms. Best corrected visual acuity improved from preoperative (range: 6/9p-6/120) to postoperative (range: 6/6p-6/15) by an average of 4.5 lines on Snellen visual acuity charts. Histopathological examination of excised tissue showed features of conjunctivalisation. CONCLUSION: Amniotic membrane transplantation appears to be an effective means of reconstructing the corneal epithelial surface and for visual rehabilitation of patients with partial limbal stem cell deficiency. It may be considered as an alternative primary procedure to limbal transplantation in these cases.

PMID: 15693318 [PubMed - indexed for MEDLINE]

Ocular manifestations of keratitis-ichthyosis-deafness (KID) syndrome.

• Messmer EM,
• Kenyon KR,
• Rittinger O,
• Janecke AR,
• Kampik A.

Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.

OBJECTIVE: Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital ectodermal dysplasia characterized by the association of hyperkeratotic skin lesions, moderate to profound sensorineural hearing loss and vascularizing keratitis. Mutations in the GJB2 gene coding for connexin 26, a component of gap junctions in epithelial cells, have been observed in several KID patients. Variable ocular manifestations of the disease in 3 patients with molecular genetically confirmed KID syndrome are reported. DESIGN: Retrospective case series. METHODS: Clinical examination and molecular genetic analysis for mutations in the GJB2 gene were performed in 3 patients with KID syndrome ages 5, 13, and 41 years. RESULTS: Visual acuity ranged from normal to severe visual loss. The ocular signs included loss of eyebrows and lashes, thickened and keratinized lids, trichiasis, recurrent corneal epithelial defects, superficial and deep corneal stromal vascularization with scarring, keratoconjunctivitis sicca, and, in one patient, presumed limbal insufficiency. Whereas ocular surface integrity could be maintained with artificial tears in one patient, and an epithelial defect healed under conservative treatment in the second patient, multiple surgical procedures including superficial keratectomies, limbal allograft transplantation with systemic immunosuppression, amniotic membrane transplantation, lateral tarsorrhaphies, and lamellar keratoplasty could not preserve useful vision in the third patient. CONCLUSIONS: KID syndrome may affect the ocular adnexae and surface with variable severity independent of the age of the patient. Lid abnormalities, corneal surface instability, limbal stem cell deficiency with resulting corneal complications, and dry eye are the main ocular manifestations.

PMID: 15691545 [PubMed - indexed for MEDLINE]

[Amniotic-membrane and limbic stem-cell transplantation in the management of ocular burns]

• Muraine M.

Service d'Ophtalmologie, CHU de Rouen, Hopital Charles Nicolle, boulevard Gambetta, 76031 Rouen cedex, France.

Severe ocular burns are potentially blinding injuries. During the first month, ocular surface destruction is caused by a vicious circle of complications that may result in the pathological state of total limbal stem-cell deficiency. Amniotic membrane transplantation can be considered an early, if not immediate, surgical procedure to promote epithelialization and calm inflammation so that scarring-induced sequelae can be prevented in the chronic stage. However, when partial or total limbal stem-cell deficiency involves one or both eyes, corneal surface reconstruction relies on transplantation of autologous limbal stem cells, an allogenic source of limbal stem cells or corneal epithelial stem-cell transplantation. In this late stage, amniotic-membrane transplantation may improve prognosis of keratolimbal allograft and is useful in the treatment of symblepharons.

PMID: 15687934 [PubMed - indexed for MEDLINE]

Ophthalmic applications of preserved human amniotic membrane: a review of current indications.

• Burman S,
• Tejwani S,
• Vemuganti GK,
• Gopinathan U,
• Sangwan VS.

Ocular Immunology and Uveitis Services, L.V. Prasad Eye Institute, Hyderabad, India.

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.

PMID: 15509905 [PubMed - indexed for MEDLINE]

Amniotic membrane graft: histopathological findings in five cases.

• Tosi GM,
• Traversi C,
• Schuerfeld K,
• Mittica V,
• Massaro-Giordano M,
• Tilanus MA,
• Caporossi A,
• Toti P.

Department of Ophthalmology and Neurosurgery, University of Siena, Siena, Italy.

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. 2004 Wiley-Liss, Inc.

PMID: 15481059 [PubMed - indexed for MEDLINE]

Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency.

• Harkin DG,
• Barnard Z,
• Gillies P,
• Ainscough SL,
• Apel AJ.

Tissue BioRegeneration and Integration Program, Institute for Health and Biomedical Innovation, Queensland University of Technology, 2 George Street, Brisbane 4000, Australia. d.harkin@qut.edu.au

AIM: To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells. METHODS: A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button. RESULTS: Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane. CONCLUSIONS: Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium.

PMID: 15317707 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation in acute chemical burns.

• Arora R,
• Mehta D,
• Jain V.

Cornea Services, Guru Nanak Eye Center, New Delhi 110002, India. aroraj@del3.vsnl.net.in

PURPOSE: To evaluate the outcome of fresh amniotic membrane transplantation (AMT) for ocular surface reconstruction in acute chemical burns. METHODS: A prospective study of 15 consecutive eyes with acute chemical burns was performed. In all, 10 eyes had lime burns and five eyes had acid burns. There were three eyes of grade II, four eyes of grade III and eight eyes of grade IV burns. AMT was performed within 3 weeks of injury. RESULTS: Patients were followed up for 10.14 +/- 4.41 months. All patients had immediate relief of pain postoperatively. Of 15 eyes, nine (60%) showed epithelialization within 1-4 weeks (15.33 +/- 9.91 days). The final visual acuity improved in 10 of 15 eyes (66.66%). Eyes with burns of grade II and III showed more visual improvement than those with grade IV burns. None of the eyes showed perforation. Symblepharon was seen in nine of 15 eyes (60%). Of 15 eyes, 12 (80%) experienced limbal stem cell deficiency and showed superficial corneal vascularization. CONCLUSIONS: Amniotic membrane transplantation with fresh amniotic membrane increases patient comfort and reduces inflammation. In mild burns, AMT alone restores corneal and conjunctival surfaces. In moderate to severe burns, it probably reduces conjunctival scarring sequelae, but does not prevent the sequelae of limbal stem cell deficiency that requires further limbal stem cell transplantation. In the acute stage, amniotic membrane transplantation probably has a protective role against the progressive melting and perforation.

PMID: 15286672 [PubMed - indexed for MEDLINE]

Application of Preserved Human Amniotic Membrane for Corneal Surface Reconstruction.

• Prabhasawat P,
• Kosrirukvongs P,
• Booranapong W,
• Vajaradul Y.

Department of Ophthalmology; The Bangkok Biomaterial Center, Faculty of Medicine, Siriraj Hospital, Madhidol University, Bangkok 10700, Thailand; Department of Ophthalmology, Siriraj Hospital, Faculty of Medicine, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand (Tel.: (662)411-2006; Fax: (662)411-1906; E-mail: sippb@mahidol.ac.th)

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.

PMID: 15256948 [PubMed - as supplied by publisher]

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

• Rama P,
• Giannini R,
• Bruni A,
• Gatto C,
• Tiso R,
• Ponzin D.

Hospital "SS Giovanni e Paolo', Division of Ophthalmology, Venezia, Italy.

Objective: To define the best conditions for amniotic membrane preparation, storage and banking in its use for corneal reconstruction. Methods: Amniotic membrane pieces were prepared under sterile conditions from placentas selected on the basis of donor medical and social history, serology, microbiological tests and histology. The pieces were kept at -140 degrees C but before grafting they were thawed and stored at 4 degrees C in RPMI medium, to have a preparation usable within 72 h. This procedure was validated by testing its therapeutic effectiveness in 25 patients 13 of which had corneal ulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 band keratopathy and 6 corneal stem cell deficiency due to chemical or thermal burns. Results: The preparation showed appreciable anti-inflammatory and analgesic effects. In the absence of corneal stem cell deficiency a stable re-epithelialisation was achieved in 15 out of 19 patients. When the limbus was lesioned, the amniotic membrane decreased vascularization and increased the number of corneal epithelial cells only in 1 of the 6 patients. No adverse reactions attributable to the tissue were recorded. Conclusions: A ready-to-use amniotic membrane preparation stored at 4 degrees C after cryopreservation has been tested in corneal reconstruction. Like the amniotic membrane thawed immediately before grafting, this preparation displayed full therapeutic effect in epithelial defects with stromal ulceration but without severe limbal stem cell deficiency. In two years banking activity 463 pieces of the preparation were successfully distributed to 90 Italian hospitals.

PMID: 15256913 [PubMed - as supplied by publisher]

[First experience with amniotic membrane transplantation]

• Trittibach P,
• Goldblum D,
• Frueh BE.

Department of Ophthalmology, University of Bern, Inselspital, Bern, Switzerland. peter.trittibach@insel.ch

BACKGROUND: The purpose of this retrospective study was to analyze the outcome of amniotic membrane transplantation (AMT) performed at the University Eye Clinic Bern during the last 12 months. PATIENTS AND METHODS: Nine men (62.4 +/- 16.7 yrs.) and four women (78.3 +/- 22.3 yrs.) were treated with an AMT and grouped according to the ophthalmologic diagnosis: Group A, chronic corneal surface defect without limbal stem cell deficiency (n = 8); Group B, conjunctival fornix reconstruction (n = 7); Group C, filtering bleb defect (n = 2). RESULTS: 11/17 (65 %) AMT's performed in 14 eyes of 13 patients showed a favorable postoperative result after a mean follow-up time of 8.7 (+/- 2.9) months. In Group A (chronic corneal surface defect) 4/8, in Group B (conjunctival fornix defect) 7/7 and in Group C (filtering bleb defect) 0/2 showed an improvement of the basic ocular problem. 4/8 patients from Group A and 7/7 patients of Group B showed postoperatively a strong reduction of the ocular inflammation. CONCLUSIONS: In the present small study, favorable results were achieved in patients with chronic corneal surface defects without limbal stem cell deficiency and conjunctival fornix defects following AMT. In patients with fornix defects, the AMT seemed to be a valuable alternative to the more complicated transplantation of mouth- or nose mucous membrane. The two eyes with filtering defects failed.

PMID: 15162267 [PubMed - indexed for MEDLINE]

[Limbal stem cell transplantation from HLA-compatible living donors. Long term observation]

• Wylegala E,
• Tarnawska D,
• Wroblewska EM.

Oddzialu Okulistyki Okregowego Szpitala Kolejowego w Katowicach.

PURPOSE: The assessment of clinical outcome in patients after Ir-CLAL procedure. MATERIALS AND METHODS: Retrospective, interventional case series including 11 patients (12 eyes), all with non-ambulatory visual acuity (from light perception to hand movements). Mean age was 39.4 years (ranging from 18 to 76 years). In 7 eyes Limbal Stem Cells Deficiency (LSCD) resulted from chemical burn, in 2 eyes from ocular cicatricial pemphigoid, in one eye from Stevens-Johnson syndrome (SJS). In one eye congenital LSCD was diagnosed and in another one post-inflammatory LSCD. Mean follow-up was 16.2 months (from 10 to 32). Among donors prospective class I HLA matching was performed. Maximal two mismatches were accepted. General immunosuppressive therapy was switched-on in all subjects. Surgical excision of corneal pannus with clearing of limbal area was performed. Five clock hours stem cells tissue grafts from living related donors were harvested and transplanted to the recipient eye. Three penetrating keratoplasty, one deep lamellar keratoplasty and seven amniotic membrane transplantations were additionally performed. Main outcome measures were graft survival as a restoration of corneal epithelium, visual acuity improvement and complication in donors and recipients. Kaplan-Meyer survival curve and generalized Peto test were used for comparison. RESULTS: The graft survival was 83.6% in mean 20.3 months follow-up (from 10 to 32 months). Two cases of graft failure concern eye with SJS and eye with PKP regrafting after chemical burn. Visual improvement in 7 cases (58.3%), the same visual acuity in 4 cases (33.3%) and deterioration one case (8.3%) were noted. Ambulatory vision in 50% of patients was achieved. Graft rejection syndrome in four eyes was noticed also when ideal matching and CsA serum level 220 ng/ml. Two cases were treated with success and another two grafts failed. No complications in donors eyes were noticed. Impression cytology in eyes with successful grafts not related to pathological changes. CONCLUSIONS: Lr-CLAL are effective in ocular surface restoration. Living-related donors are suitable source of stem cells for cultivation in vitro. The prognosis for stem cells grafting is definitely worse when eyelid anomaly and adhesions exist.

PMID: 15049259 [PubMed - indexed for MEDLINE]

Correlation of long term phenotypic and clinical outcomes following limbal epithelial transplantation cultivated on amniotic membrane in rabbits.

• Ti SE,
• Grueterich M,
• Espana EM,
• Touhami A,
• Anderson DF,
• Tseng SC.

TissueTech, Inc, and Ocular Surface Center, Miami, FL, USA.

AIM: To determine the epithelial phenotype in rabbits with total limbal stem cell deficiency (LSCD) after reconstruction with autologous limbal epithelial stem cells ex vivo expanded on rabbit amniotic membrane (AM). METHODS: Left eyes of 52 rabbits were rendered total LSCD, verified by impression cytology. The fibrovascular pannus of each cornea was removed. Group I (n = 10) received rabbit AM transplantation alone, while groups II-IV (n = 42) underwent transplantation of LSC cultured on rabbit AM (LSC-AM) from a small limbal biopsy taken from the right eye. Clinical outcome was graded as "success," "partial success," or "failure" depending on the corneal smoothness and avascularity. Epithelial phenotype was determined by immunostaining and graded as "corneal (K)," "conjunctival (J)," or "mixed (M)" depending on expression of K3 and Muc5AC. RESULTS: After 1 year follow up, group I showed 100% failure and groups II-IV showed 26% success (p<0.001). Clinical failure correlated with J phenotype p = 0.001), while clinical success correlated with K phenotype p = 0.01). When the phenotypic outcome was used for comparison, J phenotype was significantly high in group I (p = 0.003), while K phenotype was significantly high in groups II-IV (p<0.05). CONCLUSION: There is a strong correlation between clinical success and resultant corneal epithelial phenotype. Ex vivo expanded LSC can successfully reconstruct corneal surfaces with unilateral total LSCD.

PMID: 14977781 [PubMed - indexed for MEDLINE]

Stepwise surgical approach for in vivo expansion of epithelial stem cells to treating severe acute chemical burns with total limbal deficiency.

• Park GS,
• Je J,
• Kim JC.

Department of Ophthalmology, College of Medicine, Chung-Ang University, Seoul, Korea.

To describe the clinical outcome of a new surgical treatment for the acute stages of severe corneal burn injury and its complications, a prospective study of five acute corneal burn patients with severe limbal damage was performed. Amniotic membrane transplantation (AMT) and conjunctival limbal autograft (CLAU) was performed at the acute stage of corneal burn injury to reconstruct the damaged ocular surface (step I). Three to six months later, the opaque central part of the amniotic membrane containing in vivo grown corneal stem cells were removed and retransplanted to the defect created after the removal of pseudopterygium (step II). All injured eyes were successfully treated, but in one eye with marked stromal lysis, three-layered AMT and penetrating keratoplasty with retransplantation of in vivo grown corneal stem cells was performed. In the former cases, visual acuity was greatly improved more than three lines (ranging from 3 to 12 lines). In short, retransplantation of in vivo grown corneal stem cells after AMT and CLAU is a recommendable modality for restoring a stable corneal epithelium of a severely burned ocular surface in the acute stage and can be considered a preventative measure for avoiding late onset complications.

PMID: 14717484 [PubMed - indexed for MEDLINE]

Functional reconstruction of rabbit corneal epithelium by human limbal cells cultured on amniotic membrane.

• Du Y,
• Chen J,
• Funderburgh JL,
• Zhu X,
• Li L.

Peking University Stem Cell Research Center, Beijing China. yiqindu@pitt.edu

PURPOSE: To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model. METHODS: Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively. RESULTS: Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium. CONCLUSIONS: During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.

PMID: 14685149 [PubMed - indexed for MEDLINE]

Comment in:

Br J Ophthalmol. 2003 Dec;87(12):1437-9.

Corneal stromal changes following reconstruction by ex vivo expanded limbal epithelial cells in rabbits with total limbal stem cell deficiency.

• Espana EM,
• Ti SE,
• Grueterich M,
• Touhami A,
• Tseng SC.

TissueTech, Inc. and Ocular Surface Center, Miami, FL 33173, USA.

AIM: To study corneal stromal changes and the presence of myofibroblasts after transplantation of ex vivo expanded limbal epithelium. METHODS: A state of limbal deficiency was induced in 16 rabbits. After transplantation with autologous ex vivo expanded limbal epithelium on amniotic membrane (AM), their clinical outcomes were classified as success, partial success or failure according to surface smoothness, stromal clarity, and vascularisation. Clinical outcomes were correlated with phenotypic outcomes of corneal, conjunctival, or mixed epithelium, defined by expression of K3 keratin or MUC5AC. Immunostaining was performed with antibodies against collagen IV, fibronectin, and alpha-smooth muscle actin (alpha-SMA) to assess stromal wound remodelling. RESULTS: Rabbits were sacrificed after a mean follow up of 10 (SD 3.3) months. Collagen IV, expressed in the basement membrane of all three groups, was found in the stroma of the partial success, but not in that of the success or the failure. Fibronectin was absent in the success and the failure, but expressed in the stroma of the partial success. Alpha-SMA was expressed in superficial stroma of the partial success, but suppressed in areas with AM remnants. CONCLUSION: Restoration of a clear and transparent cornea is associated with a normal corneal epithelium and complete wound remodelling. In contrast, wound healing remains active and incomplete in conjunctivalised corneas, which remain opaque with myofibroblasts.

PMID: 14660463 [PubMed - indexed for MEDLINE]

Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche.

• Grueterich M,
• Espana EM,
• Tseng SC.

Department of Ophthalmology, Ludwig Maximilians University, Munich, Germany.

Identification, maintenance, and expansion of stem cells for subsequent transplantation has become a new strategy for treating many diseases in most medical subspecialties. The stem cells of the corneal epithelium are located in the limbal basal layer and are the ultimate source for constant corneal epithelial renewal. Like those in other tissues, limbal stem cells are supported by a unique stromal microenvironment called the stem cell niche, which consists of certain extracellular matrix components, cell membrane-associated molecules, and cytokine dialogues. Destructive loss of limbal stem cells or dysfunction of their stromal environment renders many corneas with a clinical entity called limbal stem cell deficiency, which is characterized by variable extents of conjunctival ingrowth depending on the severity of limbal damage. A new strategy of treating limbal stem cell deficiency is to transplant a bio-engineered graft by expanding limbal epithelial stem cells ex vivo on amniotic membrane. This review summarizes the published literature data collectively explaining how amniotic membrane is an ideal biological substrate that can help maintain and support the expansion of limbal epithelial stem cells.

PMID: 14609709 [PubMed - indexed for MEDLINE]

Amniotic membrane with living related corneal limbal/conjunctival allograft for ocular surface reconstruction in Stevens-Johnson syndrome.

• Gomes JA,
• Santos MS,
• Ventura AS,
• Donato WB,
• Cunha MC,
• Hofling-Lima AL.

Department of Ophthalmology, Paulista School of Medicine/Federal University of Sao Paulo, Sao Paulo, Brazil. japgomes@uol.com.br

OBJECTIVE: To report the use of preserved amniotic membrane and living related corneal limbal/conjunctival transplantation in total limbal stem cell and conjunctival deficiency secondary to severe Stevens-Johnson syndrome. DESIGN: Prospective, noncomparative, interventional case series.Patients and METHODS: Ten eyes of 10 patients with total limbal stem cell and conjunctival deficiency secondary to Stevens-Johnson syndrome underwent excision of cicatricial tissue followed by amniotic membrane and living related corneal limbal/conjunctival transplantation. MAIN OUTCOME MEASURES: Reconstruction of corneal epithelium (clear appearance without epithelial defect, normal fluorescein permeability, and the absence of conjunctiva-derived goblet cells on impression cytologic testing), decrease in corneal vascularization, and improvement in visual acuity. RESULTS: During a mean follow-up of 16.7 months, satisfactory ocular surface reconstruction was obtained in 2 eyes (20%), with reduced inflammation and vascularization and a mean epithelialization time of 3 weeks. Surgical failure was observed in 4 cases (40%) and complications (infection) in 4 cases (40%). Visual acuity improved in 4 eyes (40%), remained stable in 5 eyes (50%), and decreased in 1 eye (10%). CONCLUSIONS: Amniotic membrane and living related corneal limbal/conjunctival transplantation were successful in 20% of severe cases of total limbal stem cell and conjunctival deficiency secondary to Stevens-Johnson syndrome. A high proportion of postoperative complications, in particular, infection, seemed to jeopardize a favorable outcome.

PMID: 14557171 [PubMed - indexed for MEDLINE]

Ex vivo expansion of corneal limbal epithelial/stem cells for corneal surface reconstruction.

• Ramaesh K,
• Dhillon B.

Princess Alexandra Eye Pavilion, Edinburgh, UK. k.ramaesh@talk21.com

PURPOSE: The management of severe ocular surface disease due to limbal stem cell deficiency has changed dramatically. The concept of limbal stem cells, as the source of corneal epithelium revolutionised the therapeutic approach of ocular surface reconstruction. Deficiency of limbal stem cells results in blinding ocular surface diseases. Grafting viable limbal tissue, from either fellow healthy eye or a donor eye, with the resident stem cell population may replenish limbal stem cells and can restore the corneal surface to normality. Transplanting the limbal tissue can be achieved through a variety of procedures that include cadaveric keratolimbal allograft (KLAL), live or living related conjunctival limbal allograft (Ir-CLAL) and limbal autograft. Advances in tissue engineering techniques have offered a viable alternative to overcome the limitation of limbal tissue available for transplantation. Epithelial stem cells harvested from a small limbal biopsy can be expanded in vitro on a suitable carrier and then transplanted to the diseased cornea to successfully restore the corneal surface. This article is a chronological review of the important steps that brought ex vivo expanded stem cell transplantation in ocular, particularly corneal surface reconstruction. METHODS: The MEDLINE data base was searched for the years 1966-2002, using key words cornea, cell culture, ex-vivo expansion, limbus, stem cell, ocular surface and transplantation. Several articles that were not found by MEDLINE search were taken from references from other articles. Inclusion or exclusion of article was based on the relevance to the subject. CONCLUSIONS: Corneal epithelial reconstruction with ex vivo expanded limbal cells is a potential tool in ocular surface reconstruction, although the technique is currently investigational. Strategies to achieve conjunctival epithelial restoration and tear film replenishment will allow ophthalmic surgeons to truly reconstruct the ocular surface.

PMID: 12948308 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation with conjunctival limbal autograft for total limbal stem cell deficiency.

• Meallet MA,
• Espana EM,
• Grueterich M,
• Ti SE,
• Goto E,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA.

PURPOSE: To evaluate the outcomes of corneal surface reconstruction with conjunctival limbal autograft when combined with amniotic membrane transplantation on both the donor and recipient eyes. DESIGN: Retrospective, noncomparative, interventional small case series. PARTICIPANTS: Five eyes of five patients with total limbal stem cell deficiency (LSCD) resulting from pseudopemphigoid (n = 1), chemical burns (n = 3), and extensive removal of conjunctival intraepithelial neoplasia (n = 1) were operated on by one surgeon (SCGT). INTERVENTION: After the removal of fibrovascular pannus from the corneal surface, two conjunctival limbal free grafts were harvested from the fellow eyes in all five patients with unilateral LSCD. Amniotic membrane, with the basement membrane side up, was grafted onto the defect created at the donor site and onto the recipient corneal and limbal sclera before placement of conjunctival limbal grafts. MAIN OUTCOME MEASURES: Symptomatic relief, improvement in visual acuity, fornix deepening, and rapid healing and restoration of normal cornea and limbus in the recipient and donor eyes were assessed. RESULTS: During the mean follow-up of 22 months (range, 11-48 months), all eyes experienced symptomatic relief. All recipient eyes had a mean improvement in visual acuity of nine lines (range, 7-12). The three eyes with stromal vascularization showed regression, and all recipient eyes had marked improvement in corneal clarity. Three eyes receiving simultaneous symblepharon lysis and fornix reconstruction successfully regained deep, stable fornices. The donor eyes showed rapid healing and restoration of the normal limbal landmark, even in one eye where nearly the entire limbus was removed. CONCLUSIONS: Limbal conjunctival transplantation is an effective procedure for restoring the corneal surface integrity in eyes with total LSCD. The additional use of amniotic membrane may contribute to a higher rate of success in the recipient eye and a lower rate of complications in the donor eye, as well as allow the simultaneous correction of concomitant cicatricial abnormalities.

PMID: 12917178 [PubMed - indexed for MEDLINE]

Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury: a case report of unique application.

• Sangwan VS,
• Vemuganti GK,
• Iftekhar G,
• Bansal AK,
• Rao GN.

Cornea and Anterior Segment Service, L.V. Prasad Eye Institute, Banjara Hills, Hyderabad, India. vsangwan@lvpeye.stph.net

PURPOSE: Reconstruction of the ocular surface in a case of severe bilateral partial limbal stem cell deficiency (LSCD) with extensive symblephara using autologous cultured conjunctival and limbal epithelium. CASE REPORT: A 31-year-old woman presented with severe bilateral ocular surface disease with partial limbal stem cell deficiency, symblephara, lid and facial scarring, with a vision of 20/400 and counting fingers at 1 m in both eyes. Limbal and conjunctival tissue was harvested from the healthy-appearing left eye and used to generate two sheets of composite epithelium consisting of central limbal and peripheral conjunctival cells. The limbal tissues were explanted in the central region while the conjunctival tissues were explanted on the periphery of the deepithelialized human amniotic membrane (HAM) and nurtured using human corneal epithelial cell medium. After successful generation of a monolayer from both tissues had been confirmed, the composite of cultivated limbal and conjunctival epithelium with HAM was transplanted in each eye after excision of fibrous tissue and release of symblephara. One year postoperatively, the patient had a best spectacle-corrected visual acuity of 20/40 in the right eye (preoperative acuity 20/400) and counting fingers at 1 m in the left eye (same as preoperative) with a stable ocular surface. CONCLUSIONS: Autologous cultured epithelial transplantation is as an excellent option in selected patients with bilateral partial LSCD with small area(s) of healthy limbus in either eye and avoids the attendant risk of rejection and cost and potential toxicity of immunosuppression in allogeneic tissue transplantation. This case also highlights the feasibility of generating a composite culture of limbal and conjunctival epithelium using a single amniotic membrane.

PMID: 12827056 [PubMed - indexed for MEDLINE]

Does amniotic membrane transplantation improve the outcome of autologous limbal transplantation?

• Marinho D,
• Hofling-Lima AL,
• Kwitko S,
• Tseng SC.

Opthalmology Service, Hospital de Clinicas de Porto Alegre, Federal University of Rio Grande do Sul, Brazil. diane@portoweb.com.br

PURPOSE: To evaluate the additional benefits of amniotic membrane transplantation (AMT) when combined with conjunctival limbal autograft (CLAU) in the treatment of chronic chemical burns. METHODS: Thirty eyes of 30 rabbits underwent a chemical burn to create limbal deficiency. Forty-five days later, the animals were randomized into three groups of 10 rabbits each. Eyes from group 1 were treated with CLAU, group 2 underwent CLAU and AMT, and group 3 served as control without surgery. Corneal vascularization and opacity were documented with external photographs at postoperative days 30, 60, and 90. The rabbits were killed 3 months after surgery, and their corneas divided into two halves. One half was prepared for hematoxylin-eosin and periodic acid-Schiff staining and the other for frozen sections and immunostaining with AM3 (to MUC 5AC mucin) and AE5 (to K3 keratin). Final clinical outcome was also scored using external photographs at the last follow-up examination. RESULTS: After chemical burn, all groups showed similar degrees of conjunctivalization. After transplantation, corneal vascularization was worse in controls at 60 and 90 days (p < 0.001). At 30 days, eyes from group 1 had less corneal opacity (p < 0.05). At 90 days, corneal opacity was worse in controls but the same in groups 1 and 2 (p < 0.05). At the same time, limbal deficiency was significantly worse in controls (p < 0.05) but similar between groups 1 and 2. Corneal phenotype was present in 70% of the eyes in group 1, 50% in group 2, but in 10% of the controls. Clinical success with clear corneas was significantly more common in groups 1 and 2 when compared with controls (p < 0.001). CONCLUSIONS: CLAU is effective in treating limbal deficiency. The concurrent AMT does not add benefits in this rabbit model of chemical burns.

PMID: 12792477 [PubMed - indexed for MEDLINE]

Corneal calcification after amniotic membrane transplantation.

• Anderson SB,
• de Souza RF,
• Hofmann-Rummelt C,
• Seitz B.

Liverpool Royal University Hospital, Liverpool, UK Department of Ophthalmology, University of Erlangen-Nurnberg, Germany. asarah@hotmail.com

BACKGROUND:/aims: Amniotic membrane transplantation (AMT) has become well established as a treatment for chronic epithelial defects, conjunctival reconstruction, and partial limbal cell deficiency. The aim of this study was to describe cases of corneal calcification following AMT and to search for risk factors that might predispose to this unusual finding. METHODS: Details of 117 AMTs on 93 corneas of 91 patients with a follow up period of at least 1 month performed since 1999 were collected prospectively. In those with calcification clinical photographs were studied and the medical records retrospectively examined. RESULTS: 15 calcifications in 117 AMTs (12.8%) were identified, occurring 3-17 (median 6.1) weeks after AMT, during a follow up period of 4-151 (median 25) weeks. Overall epithelial healing rate was 83%. Calcification covered a surface area between 0.7-40.5 mm(2) maximum size with varied morphology. The primary diagnosis was diverse. Risk factors included the use of phosphate eye drops and pre-existing calcification in the operative or other eye. No patient with a "patch" AMT developed calcification. CONCLUSIONS: Corneal calcification occurs after some cases of AMT. A common risk factor was the postoperative use of phosphate containing eye drops.

PMID: 12714401 [PubMed - indexed for MEDLINE]

Phenotypic study of a case receiving a keratolimbal allograft and amniotic membrane for total limbal stem cell deficiency.

• Espana EM,
• Grueterich M,
• Ti SE,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL 33176, USA.

PURPOSE: To report the expression pattern of key molecules by the reconstructed corneal epithelium after a keratolimbal allograft (KLAL) and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency. DESIGN: Interventional case report. METHOD: A 50-year-old woman with severe chemical burns in both eyes received an AMT as a temporary patch at the acute stage, and a KLAL with AMT as a graft at the chronic stage for total limbal stem cell deficiency. The corneal button removed during subsequent corneal transplantation was submitted for immunofluorescence staining with monoclonal antibodies against keratin K3, MUC5AC, connexin 43, integrins alpha3beta1 and alpha6beta4, and laminin 5 for comparison with a normal cornea. RESULTS: Histologically, a normal stratified corneal epithelium has five to six cell layers that lay on the thick amniotic membrane basement membrane. The phenotype was of a corneal origin, based on expression of positive keratin K3, negative MUC5AC, and positive connexin 43. Furthermore, intact basement membrane complexes were present, evidenced by positive staining to integrins alpha3beta1 and alpha6beta4 and to laminin 5. CONCLUSIONS: A normal corneal epithelial phenotype with normal basement membrane complexes was restored after a KLAL and AMT in a case with total limbal stem cell deficiency.

PMID: 12623808 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation for partial and total limbal stem cell deficiency secondary to chemical burn.

• Gomes JA,
• dos Santos MS,
• Cunha MC,
• Mascaro VL,
• Barros Jde N,
• de Sousa LB.

Cornea and External Disease Service, Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), R. Sabara, 566 Cjuto. 212, 01239-010 Sao Paulo/SP, Brazil.

PURPOSE: To evaluate the surgical outcome of preserved amniotic membrane transplantation (AMT) for ocular surface reconstruction in chemical burn with limbal stem cell deficiency. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Twenty eyes of 20 consecutive patients with limbal stem cell deficiency secondary to ocular chemical injury. INTERVENTION: AMT with or without adjunctive limbal transplantation using limbal tissue from either the healthy contralateral eye (CLAU) or a living related donor (lr-CLAL). MAIN OUTCOME MEASURES: Reconstruction of corneal epithelium (clear appearance without epithelial defect, normal fluorescein permeability and the absence of conjunctiva-derived goblet cells on impression cytology), decrease in corneal vascularization and improvement in visual acuity. RESULTS: With a mean follow-up time of 19 months (range, 8-27 months), satisfactory ocular surface reconstruction was obtained in 15 eyes (75%), with reduced inflammation and vascularization of the ocular surface and a mean epithelialization time of 3.3 weeks. Success was observed in all cases of partial limbal stem cell deficiency (PLD) and in 68.75% (11 eyes) of cases of total limbal stem cell deficiency (TLD). Surgical failure was observed in five severe cases (31.25%). A significant visual improvement was observed in all cases after surgery, except for 2 eyes that maintained preoperative visual acuity. CONCLUSIONS: AMT seems to be an efficient adjunct for ocular surface reconstruction in chemical burns with PLD. When performed in conjunction with limbal stem cell transplantation, it is also effective in most cases of TLD.

PMID: 12623806 [PubMed - indexed for MEDLINE]

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane for treating corneal diseases with total limbal stem cell deficiency.

• Tseng SC,
• Meller D,
• Anderson DF,
• Touhami A,
• Pires RT,
• Gruterich M,
• Solomon A,
• Espana E,
• Sandoval H,
• Ti SE,
• Goto E.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA.

PMID: 12614074 [PubMed - indexed for MEDLINE]

[Amniotic membrane transplantation with limbal stem cell transplantation as a combined procedure for corneal surface reconstruction after severe thermal or chemical burns]

• Stoiber J,
• Ruckhofer J,
• Muss W,
• Grabner G.

Landesklinik fur Augenheilkunde und Optometrie, Landeskliniken Salzburg, Germany. j.stoiber@lks.at

BACKGROUND: Severe thermal and chemical burns may result in limbal deficiency leading to persistent epithelial defects, complete conjunctival epithelial ingrowth and vascularisation of the cornea. If sufficiently severe, these burns may lead to very significant visual impairment. Amniotic membrane transplantation with limbal transplantation has recently been proposed as a new method for corneal surface reconstruction. PATIENTS AND METHODS: A total of 14 patients (age 18-62 years, mean age 42 years) with limbal deficiency resulting from thermal ( n=1) or chemical burns ( n=13) underwent surgery. The corneal pannus was completely removed and the amniotic membrane was grafted onto the cornea. Limbal transplantation using autografts obtained from contralateral eyes was performed simultaneously in seven cases. Allografts from a donor were transplanted in seven cases with bilateral involvement. These patients received oral cyclosporin A postoperatively. The mean follow-up time was 18 months. RESULTS: In all cases of limbal autografts the corneal surface showed a complete and stable epithelialisation within a few weeks. Out of seven patients with limbal allografts three displayed recurrent epithelial defects in the long term. The initially semitransparent amniotic membrane became more translucent and biomicroscopically invisible within several months after surgery. There was an increase in visual acuity in most cases, limited mostly by irregular astigmatism due to the initial stromal loss. CONCLUSIONS: Amniotic membrane transplantation with limbal transplantation allows reconstruction ocular surfaces severely damaged by chemical or thermal burns. In most cases, however, additional surgical procedures such as lamellar or penetrating keratoplasty are required for adequate visual rehabilitation.

PMID: 12430036 [PubMed - indexed for MEDLINE]

Phenotypic study of a case with successful transplantation of ex vivo expanded human limbal epithelium for unilateral total limbal stem cell deficiency.

• Grueterich M,
• Espana EM,
• Touhami A,
• Ti SE,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida, USA.

OBJECTIVE: To minimize the risk to the donor eye when a conjunctival limbal autograft is performed for unilateral total limbal stem cell deficiency (LSCD), a new approach has been reported of expanding limbal epithelial progenitor cells from a small limbal biopsy cultured on amniotic membrane (AM). Herein, we present for the first time the morphologic and phenotypic outcome of one such patient. DESIGN: Interventional case report. METHODS: A 31-year-old male with a severe acid burn to his left eye received AM transplantation at the acute stage and a keratolimbal allograft (KLAL) at the chronic stage for total LSCD. As an alternative to combat the failed KLAL, the above-mentioned new surgical procedure was performed. The corneal button, obtained after a penetrating keratoplasty performed 5.5 months later, and a normal corneal button as a control were submitted to hematoxylin-eosin and immunofluorescence staining for keratin K3, connexin 43, goblet-cell mucin MUC 5AC, laminin 5, and integrins alpha3beta1 and alpha6beta4. MAIN OUTCOME MEASURES: Clinical and immunohistologic features. RESULTS: The resultant epithelium was stratified with five to six cell layers and anchored to laminin 5 of the amniotic basement membrane via integrins alpha3beta1 and alpha6beta4 in a manner similar to the normal corneal epithelium. Intriguingly, the epithelial phenotype was limbal and not corneal, based on the negative expression of keratin K3 and connexin 43 of the basal epithelium. CONCLUSIONS: The technique described ensures the preservation of amniotic basement membrane, which allows formation of adhesion complexes and maintains normal corneal architecture. The preservation of a limbal epithelial phenotype on the reconstructed corneal surface indicates that AM provides a unique stromal environment conducive to the preservation and expansion of limbal epithelial progenitor cells.

PMID: 12153809 [PubMed - indexed for MEDLINE]

Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.

• Ti SE,
• Anderson D,
• Touhami A,
• Kim C,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA.

PURPOSE: To determine factors affecting the outcome of corneal surface reconstruction in rabbits with total limbal stem cell deficiency (LSCD), by using autologous limbal epithelial stem cells (LSC) ex vivo, expanded on rabbit amniotic membrane (AM). METHODS: Left eyes of 52 rabbits were rendered totally limbal stem cell deficient by n-heptanol debridement of the entire corneal epithelium followed by surgical removal of 360 degrees of limbal rim. After cytologic verification of LSCD, the fibrovascular pannus of each cornea was removed. Group I (n = 10) received a rabbit AM transplant, whereas groups II, III, and IV (n = 42) underwent transplantation of LSCs cultured on rabbit AM (LSC-AM graft) derived from a small limbal biopsy specimen from the right eye. Clinical outcome was graded as a success if a smooth, avascular corneal surface was restored, a partial success if more than two quadrants of corneal surface were smooth, or a failure if the corneal surface was revascularized and irregular. RESULTS: A long-term follow-up of more than 1 year was achieved. Compared with the 100% failure rate in group I, inclusion of expanded LSCs resulted in variable success rates in groups II, III, and IV (all P < 0.001). Kaplan-Meier survival analysis showed that different suturing techniques, subconjunctival injection of long-acting steroid, and tarsorrhaphy used in groups II (n = 17) and III (n = 13) did not significantly alter the outcome (P = 0.89). However, the use of a larger graft and human AM as a temporary patch with the explant retained for 1 week in group IV (n = 12) significantly improved the success rate to 83% (P = 0.002). Among eyes showing clinical failure, there was a significant correlation between the logarithm of the first day when an epithelial defect was noted and the time of graft failure (r(2) = 0.60, P < 0.001). Furthermore, the presence of severe lid deformity was borderline significant when correlated with failure cases in all four groups (P = 0.069). CONCLUSIONS: Ex vivo expansion of LSCs can be achieved by using rabbit AM culture. Such expanded LSCs can successfully reconstruct corneal surfaces affected by total LSCD. This animal model is useful to investigate culturing variables affecting epithelial stemness so that surgical reconstruction of corneas with total LSCD can be successfully performed. Furthermore, this model can be used to test the feasibility of gene therapies targeting LSCD in the future.

PMID: 12147589 [PubMed - indexed for MEDLINE]

[Surgical approaches for limbal stem cell deficiency]

• Gruterich M,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Fl, USA.

BACKGROUND: Stem cells are the ultimate source of the rapidly self-renewing corneal epithelium and are located in the basal layer of the limbal epithelium. A variety of diseases can compromise the stem cell pool, causing an entity called limbal stem cell deficiency. Several therapeutic strategies should be taken into account. METHODS AND RESULTS: Ocular surface defence should be restored prior to any kind of stem cell transplantation to assure a sufficient and stable tearfilm. Surgical strategies comprise superficial keratectomy with or without amniotic membrane transplantation, conjunctival-limbal autograft and keratolimbal allograft transplantation. A damaged limbal and corneal stroma can be reconstructed by amniotic membrane as a biological substrate. First encouraging results have been reported using tissue bio-engineered limbal epithelium on amniotic membrane for corneal surface reconstruction. CONCLUSION: The correct diagnosis and a restored ocular surface defence are a prerequisite for subsequent transplantation of limbal stem cells. Optimal patient selection for the respective surgical approach and systemic immunosuppression protocols to improve allograft survival need to be further investigated.

PMID: 12094314 [PubMed - indexed for MEDLINE]

Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders.

• Shimazaki J,
• Aiba M,
• Goto E,
• Kato N,
• Shimmura S,
• Tsubota K.

Department of Ophthalmology, Tokyo Dental College, 5-11-13 Sugano, Ichikawa-shi, Chiba 272-8513, Japan.

PURPOSE: To study the short-term clinical results of transplanting of cultivated corneal/limbal epithelial cells on human amniotic membrane (AM) for limbal deficiency. DESIGN: Noncomparative, retrospective interventional case series. PARTICIPANTS: Thirteen eyes of 13 patients with severe limbal deficiency (Stevens-Johnson syndrome in eight eyes, ocular cicatricial pemphigoid in three eyes, and chemical burns in two eyes) were treated at the department of Ophthalmology, Tokyo Dental College, Japan. INTERVENTION: Cultivated allo-limbal epithelium was transplanted onto the ocular surface of patients with severe limbal deficiency. MAIN OUTCOME MEASURES: Ocular surface reconstruction with corneal epithelialization, changes in visual acuity, and postoperative complications were studied. Histologic examinations were also performed on cultivated epithelium. RESULTS: Cultivated corneal epithelium on AM formed two to three layers with the formation of basement membrane-like structures. After the surgery, the epithelium regenerated and covered the ocular surface in eight eyes (61.5%). However, three of the eight eyes developed partial conjunctival invasion, and two eyes later developed epithelial defects. At last examination, corneal epithelialization was achieved in six eyes (46.2%). Five eyes had conjunctivalization, one eye had dermal epithelialization, and one eye was not epithelialized. Complications were corneal perforation in four eyes and infectious keratitis in two eyes. CONCLUSIONS: This study demonstrates that the success rate for transplanting cultivated allo-limbal epithelium on the AM is not different from the conventional limbal and AM transplantation for the treatment of severe limbal stem cell dysfunction.

PMID: 12093651 [PubMed - indexed for MEDLINE]

Histopathology of human corneas after amniotic membrane and limbal stem cell transplantation for severe chemical burn.

• Stoiber J,
• Muss WH,
• Pohla-Gubo G,
• Ruckhofer J,
• Grabner G.

Department of Ophthalmology and Optometry, St. Johanns-Spital, Landeskliniken Salzburg, Muellner Hauptstrasse 48, 5020 Salzburg, Austria. j.stoiber@lks.at

PURPOSE: To describe the histopathologic changes in the cornea following amniotic membrane transplantation (AMT) combined with limbal transplantation. METHODS: Four eyes with complete limbal stem cell deficiency after severe chemical burn underwent AMT with either a living-related conjunctival limbal allograft (lr-CLAL) (three eyes) or a conjunctival limbal autograft (CLAU) (one eye) for ocular surface reconstruction. Penetrating keratoplasty was performed several months after the initial procedure for further visual rehabilitation. Mean follow up time was 20 months. Light and transmission electron microscopy (TEM) and indirect immunofluorescence microscopy of the excised corneal buttons were performed. RESULTS: All specimens displayed a multilayered epithelium without conjunctival goblet cells over the entire corneal surface. Basal epithelial cells demonstrated a firm connection to the remnants of the transplanted amniotic membrane (AM), which at some places appeared to be in a state of "modification" or "remodeling" in the collagen layers. The basement membrane zone displayed a positive staining when using antibodies against collagen IV and VII, integrin alpha6 and beta4, laminin 5, and bullous pemphigoid antigen 2. Remnants of the AM in the specimen showed staining of collagen IV, which was found also in cross-sections of cryopreserved AM. The recipients Bowman's membranes that were only partially present after the initial trauma were significantly disturbed. CONCLUSION: Within the time frame studied, the transplanted AM apparently survives and integrates into the host tissue being modified or remodeled by recipient cells. AMT in combination with a CLAU or lr-CLAL is a useful technique in promoting a rapid and stable reepithelialization of a corneal surface following severe chemical or thermal damage.

PMID: 12072723 [PubMed - indexed for MEDLINE]

Human limbal progenitor cells expanded on intact amniotic membrane ex vivo.

• Grueterich M,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, FL, USA.

BACKGROUND: The transplantation of human limbal epithelium on amniotic membrane as a substrate is a new treatment for limbal stem cell deficiency. Limbal epithelial stem cells are characterized by a slow cell cycle and the lack of K3 keratin and connexin 43 (Cx43), a gap junction protein. We investigated Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of limbal epithelium expanded on amniotic membrane. METHODS: Connexin 43 expression and bromodeoxyuridine (BrdU) incorporation were determined by immunohistology. The GJIC was investigated by a scrape-loading dye transfer assay. Expression of Cx43 and K3 keratin as well as BrdU-retaining nuclei were also analyzed after xenotransplantation in nude mice. RESULTS: Limbal epithelium showed mean +/- SD 12.4% +/- 14.5% positive units of Cx43 expression and a low BrdU labeling index of 2.4% +/- 0.9% (n = 5), of which the latter was due to slow cycling, as proved by its increase to 62.0% +/- 9.5% after continuous BrdU labeling for 5 days. Most of the expanded epithelium did not show GJIC (83%), significantly more than that grown on plastic (6%; P<.002). Basal cells of the stratified epithelium after xenotransplantation did not express Cx43 and K3 keratin, but their nuclei retained BrdU. CONCLUSION: These results support the hypothesis that intact amniotic membrane preferentially preserves and expands Cx43-negative, keratin K3-negative, and GJIC-deficient limbal epithelium, a phenotype resembling that of stem cell-containing limbal basal epithelial cells in vivo. CLINICAL RELEVANCE: Intact amniotic membrane is a suitable substrate for bioengineering limbal epithelia for ocular surface reconstruction.

PMID: 12049584 [PubMed - indexed for MEDLINE]

Comment in:

Ophthalmology. 2003 Oct;110(10):2071; author reply 2071-2.

Long-term outcome of keratolimbal allograft with or without penetrating keratoplasty for total limbal stem cell deficiency.

• Solomon A,
• Ellies P,
• Anderson DF,
• Touhami A,
• Grueterich M,
• Espana EM,
• Ti SE,
• Goto E,
• Feuer WJ,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, USA.

PURPOSE: To evaluate the long-term outcome of ocular surface reconstruction, including keratolimbal allograft (KLAL) and amniotic membrane transplantation (AMT) with or without penetrating keratoplasty (PKP), in patients with nonambulatory vision secondary to total limbal stem cell deficiency (LSCD). DESIGN: Retrospective, non-comparative interventional case series. PARTICIPANTS: Thirty-nine eyes in 31 consecutive patients with total LSCD, as defined by impression cytology, who had a preoperative best-corrected visual acuity of less than 20/200 and a minimum follow-up of 12 months. Patients were divided into three groups: group 1 (16 eyes) with chemical burns, group 2 (9 eyes) with Stevens-Johnson syndrome (SJS), and group 3 (14 eyes) with other causes of LSCD, including ocular cicatricial pemphigoid, atopic keratoconjunctivitis, and aniridia. INTERVENTION: All patients underwent KLAL and AMT by one surgeon (SCGT). If needed, PKP was performed at the same surgical setting using tissue from the same donor. MAIN OUTCOME MEASURES: Cumulative rates of survival of ambulatory vision (> or = 20/200), survival of KLAL, survival of PKP, and incidence of complications. RESULTS: Fifty-three KLAL with AMT procedures were performed in 39 eyes, of which 23 eyes received simultaneous PKP at the time of the first KLAL. The mean follow-up was 34.0 +/- 21.5 months (range, 12-117.6). The mean period of ambulatory vision was 23.9 +/- 20.9 months (range, 0-104). The overall survival of ambulatory vision was 53.6% at 3 years and 44.6% at 5 years. The survival of ambulatory vision was significantly worse in SJS compared with other causes (67%, 81%, and 92% for groups 1, 2, and 3, respectively; P = 0.06 for group 1 versus 2, P = 0.0008 for group 1 versus 3). KLAL performed alone resulted in higher survival of ambulatory vision at 2 years (86.1% +/- 9.1%) compared with KLAL with PKP (46.9% +/- 10.6%, P = 0.100). The survival of PKP was significantly worse in SJS compared with the other causes (20.0% +/- 17.9% compared with 55.6% +/- 11.7%, respectively, P = 0.028). After 2 years, the survival of the second KLAL was better than that of the first: 68.2% +/- 15.4% compared with 27.3% +/- 13.4%, respectively (P = 0.041). CONCLUSIONS: Ambulatory vision for a period of more than 2 years can be achieved by KLAL with or without PKP in eyes with severe ocular surface disorders caused by total LSCD. However, a progressive decline of the visual outcome and graft survival is evident with time. Performing PKP simultaneously with KLAL may be associated with a less favorable outcome. The failure of KLAL is associated with the loss of donor cells in the recipient. Augmentation of ocular surface defense is essential in securing the success of KLAL and PKP. Future modifications of the surgical procedure and of the immune suppressive protocols may improve survival of the allogeneic grafts and the final visual outcome.

PMID: 12045060 [PubMed - indexed for MEDLINE]

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

• Meller D,
• Pires RT,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, USA.

BACKGROUND/AIM: Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. METHODS: The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. RESULTS: Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. CONCLUSIONS: These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.

PMID: 11914219 [PubMed - indexed for MEDLINE]

Limbal stem cells in health and disease.

• Sangwan VS.

Cornea and Anterior Segment Disease Service, L. V. Prasad Eye Institute, L. V. Prasad Marg, Hyderabad (AP), India. vsangwan@lvpeye.stph.net

Stem cells are present in all self-reviewing tissues and have unique properties. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. These epithelia are stratified, squamous and non-keratinized. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. The stem cells for the cornea are located at the limbus. The microenvironment of the limbus is considered to be important in maintaining stemness of the stem cells. They also act as a "barrier" to conjunctival epithelial cells and prevent them from migrating on to the corneal surface. In certain pathologic conditions, however, the limbal stem cells may be destroyed partially or completely resulting in varying degrees of stem cell deficiency with its characteristic clinical features. These include "conjunctivalization" of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. The stem cell deficiency can be managed with auto or allotransplantation of these cells. With the latter option, systemic immunosuppression is required. The stem cells can be expanded ex vivo on a processed human amniotic membrane and transplanted back to ocular surface with stem cell deficiency without the need of immunosuppression.

PMID: 11900318 [PubMed - indexed for MEDLINE]

Nonpreserved human amniotic membrane transplantation in acute and chronic chemical eye injuries.

• Ucakhan OO,
• Koklu G,
• Firat E.

Department of Ophthalmology, Ankara University School of Medicine, Cinnah Caddesi 9/7, Cankaya 06680, Ankara, Turkey. omuru@yahoo.com

PURPOSE: To evaluate the safety and efficacy of nonpreserved amniotic membrane transplantation (AMT) with or without limbal autograft transplantation (LAT) in management of acute and chronic chemical eye injuries. METHODS: Amniotic membrane transplantation or AMT + LAT was performed on nine eyes of seven consecutive patients, five eyes with acute chemical burn and four eyes with limbal stem cell deficiency secondary to previous chemical burn. Nonpreserved amniotic membrane was used in all procedures. RESULTS: Five patients (71.5%) were men and two (28.5%) were women. The average age at the time of surgery was 32.7 +/- 10.9 years (range, 20-45). Mean follow-up after last surgery was 8.9 +/- 3.2 months (range, 6-14). The average epithelial healing time was 24.6 +/- 17.3 days (range, 3-45). At the end of the follow-up period, visual acuity improved in all eyes, inflammation subsided, and the subjective complaints decreased remarkably. CONCLUSION: AMT with nonpreserved amniotic membrane promoted epithelial healing, reduced surface inflammation, increased patient comfort, and decreased the extent and severity of vascularization when used in patients with acute chemical burns. When used in limbal stem cell deficiency owing to past chemical burns, AMT alone or in combination with LAT aided in ocular surface reconstruction. Infectious, inflammatory, or toxic/allergic reactions were not encountered in any patient owing to the use of nonpreserved amniotic membrane. Further studies are required to establish the safety and efficacy of preserved and nonpreserved AMT in ocular surface reconstruction.

PMID: 11862088 [PubMed - indexed for MEDLINE]

Comment in:

Br J Ophthalmol. 2001 Dec;85(12):1400-1.

Single and multilayer amniotic membrane transplantation for persistent corneal epithelial defect with and without stromal thinning and perforation.

• Prabhasawat P,
• Tesavibul N,
• Komolsuradej W.

Department of Ophthalmology, Faculty of Medicine, Siriraj Hospital Mahidol University, Bangkok, Thailand. sippb@mahidol.ac.th

AIMS: To evaluate the efficacy of amniotic membrane transplantation (AMT) in persistent corneal epithelial defect with or without stromal thinning and corneal perforation. METHODS: 28 patients (28 eyes) with persistent corneal epithelial defect unresponsive to medical treatment were given preserved human amniotic membrane transplants. The patients were divided into three groups: group A, persistent corneal epithelial defect 10 eyes; group B, epithelial defect with stromal thinning 13 eyes; and group C, corneal perforation five eyes. AMT was performed using one layer in group A and multilayers in group B and C. The causes of persistent epithelial defect were neurotrophic keratopathy (24 eyes), limbal deficiency (six eyes), exposure keratopathy (four eyes), and Mooren's ulcer (one eye). RESULTS: Success was noted in 82.1% (23/28 eyes) in all groups, with 80% (8/10 eyes), 84.6% (11/13 eyes), and 80% (4/5 eyes) in groups A, B, and C respectively, with a mean follow up of 10.9 months (1-30 months). The mean epithelialisation time after AMT was 2.1 weeks. The healing times of groups B and C are also significantly shorter than group A (p=0.017 and 0.018, respectively). Corneal stromal thickness was significantly increased in all cases in groups B and C (p=0.006). Those with corneal perforation in group C were completely healed by multilayer AMT. There was no difference in the epithelialisation time between successful cases treated by a single operation (17 eyes) or repeated operation (six eyes). Vision improved in 18.9% (8/28 eyes) and worsened as a result of cataract formation in 2.3% (1/28 eyes). Failure was noted in 17.9% (5/28 eyes), because of corneal infection (two eyes), neurotrophic keratopathy with and without limbal deficiency (two eyes), and intractable corneal perforation (one eye). No patient developed major immediate postoperative complications or graft rejection. CONCLUSION: Amniotic membrane can successfully treat refractory corneal epithelial defect by promoting epithelial healing and thus prevent corneal perforation. It can be used as a treatment for corneal perforation by restoring corneal stromal thickness so that emergency penetrating keratoplasty can be avoided.

PMID: 11734521 [PubMed - indexed for MEDLINE]

[Limbus transplantation for reconstruction of the ocular surface]

• Kruse FE,
• Reinhard T.

Augenklinik der Universitat Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg. Friedrich_kruse@med.uni-heidelberg.de

Proliferation of the corneal epithelium originates in undifferentiated, long-lived stem cells that are located in the basal limbal epithelium. Stem cells are important for corneal epithelial regeneration and wound healing. Depletion of stem cells due to accidents as well as malfunctions of stem cells due to inborn or inflammatory diseases result in limbal stem cell deficiency. Limbal deficiency is characterized by conjunctivalization of the cornea with vascularization and opacification. Partial limbal deficiency can be treated by removing ingrown conjunctival epithelium thus allowing normal limbal epithelium to repopulate the cornea. Unilateral limbus-derived stem cell disease requires either limbal autograft transplantation from the healthy partner eye or kerato-limbal allograft transplantation. Several modifications of the latter technique have been performed including large kerato-limbal lamellar grafts and central penetrating kerato-limbal allografts. All homologous procedures render a very high risk of immunological reactions that require long term systemic immunosuppression. The use of amniotic membrane, better pharmacological drugs for immunosuppression and improvements in the HLA-matching of limbal allografts as well as ex vivo expansion of corneal stem cells should allow for better reconstruction of the ocular surface in limbal deficiency.

PMID: 11594221 [PubMed - indexed for MEDLINE]

[Amniotic membrane transplantation for reconstruction of the ocular surface]

• Kruse FE,
• Meller D.

Augenklinik der Universitat Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg. Friedrich_Kruse@med.uni-heidelberg.de

Amniotic membrane transplantation has been recently gained wide-spread attention as a new method for reconstruction of the ocular surface. However long term prognosis of the surgical intervention as well as action mechanism are poorly defined. Therefore, we review the current literature concerning the application of amniotic membrane in ophthalmology and the outcome of the surgery. Amniotic membrane is used as graft or patch to promote epithelial healing of persistent epithelial defects, corneal ulcers and following penetrating keratoplasty. It is also used to seal corneal perforations and to treat bullous keratopathy. Amniotic membrane patches facilitate epithelial healing and reduce the number of complications after chemical burns. For partial limbal deficiency, amniotic membrane alone can be sufficient, while total limbal deficiency requires combination with stem-cell transplantation. Furthermore amniotic membrane grafts restore conjunctival surfaces following removal of lesions such as pterygium, tumor, scar, symblepharon, and conjunctivochalasis. The prognosis of amniotic membrane transplantation depends on the underlying disease and the quality of the ocular surface and is inversely related to the degree of inflammation. Recent investigations of the action mechanisms describe intrinsic antiinflammatory properties of the amniotic membrane.

PMID: 11594219 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation for ocular surface reconstruction.

• Prabhasawat P,
• Kosrirukvongs P,
• Booranapong W,
• Vajaradul Y.

Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

OBJECTIVE: To study the efficacy of amniotic membrane transplantation in various indications for ocular surface reconstruction. METHOD: Amniotic membrane transplantations were performed in 140 eyes (130 patients) for ocular surface reconstruction. The indications for the corneal group were limbal stem cell deficiency, bullous keratopathy, persistent epithelial defect, band keratopathy, prosthesis, corneal ulcer and acute chemical burn. The indications for the conjunctival group were grafts for pterygium, conjunctival tumors, symblepharon, and covering the scleral graft. RESULTS: Success was noted in 75.7 per cent (106/140) eyes, partial success in 17.9 per cent (25/140) eyes, and failure in 6.4 per cent (9/140) eyes for a mean follow-up of 6.6 months (1-19 months). The success and partial success rate were 80.6 per cent (54/67), 14.9 per cent (10/67) in the corneal group and 71.2 per cent (52/73), 20.6 per cent (15/73) in the conjunctival group. CONCLUSION: Amniotic membrane transplantation can solve some difficult ocular surface problems, and can be used to promote epithelial healing, reduce inflammation and scarring.

PMID: 11560222 [PubMed - indexed for MEDLINE]

Ipsilateral limbal translocation for treatment of partial limbal deficiency secondary to ocular alkali burn.

• Nishiwaki-Dantas MC,
• Dantas PE,
• Reggi JR.

Department of Ophthalmology, Santa Casa of Sao Paulo, Corneal and External Disease Service, Ocular Emergency Service, Sao Paulo, Brazil. speyecare@originet.com.br

BACKGROUND/AIM: Chemical ocular burn, especially by alkali, may result in damage to the limbal stem cells, fundamental in maintaining the integrity of ocular surface. Clinically, patients manifest abnormal conjunctival-corneal healing with peripheral neovessels, chronic epithelial defect, severe stromal inflammation, and conjunctivalisation of the corneal, which is characteristic of limbal deficiency. Penetrating keratoplasty has a poor prognosis in these cases. Available therapeutic options included limbal autograft and allograft transplantation associated or not with human amniotic membrane transplant. The authors propose an alternative treatment with ipsilateral autologous limbal transplantation, transferring the healthy portion of the limbus from the partially burned eye to the deficient area in patients with partial limbal deficiency secondary to alkali burn. METHODS: Ipsilateral limbal autografts were performed in five patients with partial limbal deficiency secondary to alkali burn, transferring the healthy portion of the limbus to the burned area of the same eye, without intervening in the contralateral eye. RESULTS: All cases had regression of the limbal deficiency with improvement of visual acuity to a minimum of 20/60-20/20 (partial), without complications during the follow up period (range 7-11 months). CONCLUSION: Ipsilateral autologous limbal autograft is a therapeutic option for partial limbal deficient eyes.

PMID: 11520748 [PubMed - indexed for MEDLINE]

Amniotic membrane surgery.

• Sippel KC,
• Ma JJ,
• Foster CS.

Cornea Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA.

Human amniotic membrane (AM) is composed of three layers: a single epithelial layer, a thick basement membrane, and an avascular stroma. Amniotic membrane has anti-adhesive properties and is felt to promote epithelialization and decrease inflammation, neovascularization, and fibrosis. Amniotic membrane transplantation (AMT) is currently being used for a continuously widening spectrum of ophthalmic indications. Amniotic membrane transplantation has been shown to be effective in the reconstruction of the corneal surface in the setting of persistent epithelial defects, sterile corneal ulcerations, and partial limbal stem cell (LSC) deficiency states, including those secondary to chemical or thermal burns. Amniotic membrane transplantation also has been used in conjunction with limbal stem cell transplantation (LSCT) both in a concurrent fashion as well as in preparation for LSCT. Amniotic membrane transplantation also has been used in place of conjunctival autografting after pterygium excision and to reconstruct the conjunctival surface after removal of conjunctival lesions. Most recently, ex vivo cultivation and expansion of limbal epithelial cells has been performed utilizing AM as a matrix. However, the superiority of AMT over other treatment modalities in many of these settings needs to be substantiated by controlled clinical trials.

PMID: 11507340 [PubMed - indexed for MEDLINE]

Reconstruction of ocular surface with heterologous limbal epithelium and amniotic membrane in a rabbit model.

• Avila M,
• Espana M,
• Moreno C,
• Pena C.

Departamento de Oftalmologia, Facultad de Medicina, Hospital San Juan de Dios, Universidad Nacional de Colombia, Colombia. mavila@mail.med.upenn.edu

PURPOSE: To report in vivo reconstruction of the ocular surface using amniotic membrane and heterologous transplants of epithelial limbal cells in rabbits with chemical burns. METHODS: After severe damage to the ocular surface with n-heptanol and keratectomy, 15 rabbits developed total limbal deficiency with conjunctival epithelialization, vascularization, and chronic inflammation. One month later, a complete keratectomy was performed in all eyes: 12 received additional transplantation of human amniotic membrane and heterologous limbal epithelial cells in a double amniotic membrane layer, 2 received amniotic membrane only, and 1 control eye received no procedure. RESULTS: After 1 month of follow-up, corneas in eight of the operated eyes presented minimal vascularization, without signs of rejection. Corneal surface reconstruction was demonstrated with the growth of new corneal-like epithelial phenotype and integration of amniotic membrane to the basal corneal surface. A superficial amniotic membrane (with the amnion side up as a dressing) peeled off after 7 to 10 days. The epithelialization with heterologous limbal epithelial cells was evident underneath. The other four operated eyes were followed for 6 months; the ocular surface was also stable with a corneal-like epithelial phenotype. CONCLUSION: Simultaneous transplantation of amniotic membrane and heterologous limbal epithelial cells in severe ocular surface disorders could restore ocular surface and may be useful in patients with severe bilateral limbal epithelial loss, giving new perspectives for the treatment of severe ocular surface disorders.

PMID: 11333332 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation after the primary surgical management of band keratopathy.

• Anderson DF,
• Prabhasawat P,
• Alfonso E,
• Tseng SC.

Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida 33136, U.S.A.

PURPOSE: To determine the safety and efficacy of amniotic membrane transplantation to restore and maintain a stable corneal epithelium and reduce ocular surface pain after surgical removal of band keratopathy arising from ocular causes. METHODS: Fifteen patients (16 eyes) from two centers with band keratopathy secondary to ocular causes underwent amniotic membrane transplantation as a graft after surgical removal of calcific deposits with or without the use of ethylenediaminetetraacetic acid. In a prospective, consecutive, uncontrolled case series, the rate of corneal epithelialization and resultant surface stability were recorded over a mean follow-up period of 14.6 months. RESULTS: Pain from ocular surface instability was the presenting complaint in 14 of 15 (93.3%) patients and resolved in all cases after the procedure even for those who experienced a recurrence of the calcific deposit. Fifteen of 16 eyes (93.7%) achieved epithelialization with a mean time to epithelial healing of 15.2 days. The only eye that failed to heal was subsequently diagnosed with total limbal stem cell deficiency. Visual acuity improved in five of nine (44%) sighted eyes and remained unchanged in four of nine (56%). No patient experienced any major surgical or medical complication after the procedure. CONCLUSION: Amniotic membrane transplantation represents a safe and effective method to restore a stable corneal epithelium in eyes after primary surgical removal of band keratopathy arising from ocular causes.

PMID: 11333320 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation for partial limbal stem cell deficiency.

• Anderson DF,
• Ellies P,
• Pires RT,
• Tseng SC.

Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida 33136, USA.

AIM: To examine the efficacy, safety, and long term outcomes of amniotic membrane transplantation for corneal surface reconstruction in cases of partial limbal stem cell deficiency. METHODS: 17 eyes of 15 patients with partial limbal stem cell deficiency underwent superficial keratectomy of the conjunctivalised corneal surface followed by amniotic membrane transplantation. Cases were followed up for at least a year. RESULTS: All eyes exhibited a stable, intact corneal epithelial surface after a mean follow up period of 25.8 months with no eyes developing recurrent erosion or persistent epithelial defect. The mean time to re-epithelialisation was 22.8 days. Overall improvement in visual acuity was observed in 92.9% of 14 eyes with visual potential. Of those, five eyes gained six or more lines, two eyes gained between four and five lines, six eyes gained between one and three lines, and one eye lost three lines of Snellen acuity. Pain and photophobia were abolished in 86% of cases and substantially reduced in 14%, with all eyes exhibiting decreased vascularisation and inflammation at final follow up. CONCLUSIONS: Amniotic membrane transplantation appears to be a safe and effective method of restoring a stable corneal epithelium for cases of partial limbal stem cell deficiency and can be considered as an alternative to limbal autograft or allograft.

PMID: 11316719 [PubMed - indexed for MEDLINE]

Use of autologous limbal epithelial cells cultured on amniotic membranes for unilateral stem cell deficiency.

• Cohen EJ.

Wills Eye Hospital, Philadelphia, PA 19105, USA. ejcohen@hslc.edu

PMID: 11198702 [PubMed - indexed for MEDLINE]

[Limbal stem cell deficiency]

• Gatinel D,
• Hoang-Xuan T.

Fondation ophtalmologique Adolphe de Rothschild, Service d'Ophtalmologie, 25 rue Manin, 75019 Paris, France.

PMID: 10992070 [PubMed - indexed for MEDLINE]

Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease.

• Schwab IR,
• Reyes M,
• Isseroff RR.

University of California, Davis Medical School, Department of Ophthalmology, USA.

PURPOSE: To bioengineer a corneal surface replacement using ex vivo expanded, cultured corneal epithelial stem cells seeded on a matrix derived from amniotic membrane and use this bioengineered graft to manage difficult ocular surface disease. METHODS: Fourteen patients with ocular surface disease unresponsive to standard medical and surgical treatments, including seven patients with presumed limbal stem cell deficiency were chosen for transplantation of a bioengineered composite corneal surface in eye each. Presumed corneal stem cells were harvested from either the patient's or related donor's limbus, expanded ex vivo, and cultivated on a carrier of modified human amniotic membrane. The resulting composite cultured tissue was transplanted to the ocular surface of the diseased eye, from which the abnormal tissue had been surgically removed. Ten patients received autologous grafts, and four received allogeneic grafts. RESULTS: A successful outcome, defined as restoration or improvement of vision, along with maintenance of corneal re-epithelialization and absence or recurrence of surface disease was obtained in 6 of the 10 patients with autologous procedures and in all 4 allogeneic transplants. Follow-up ranged 6-19 months with a mean of 13 months. CONCLUSIONS: This novel technique documents that presumed corneal epithelial stem cells can be harvested safely from the limbus, expanded successfully in vitro, and grown on denuded amniotic membrane. The resultant composite cultured tissue can be transplanted and appears to successfully manage eyes with difficult ocular surface disease, including those with stem cell deficiency. This technique minimizes the threat of damage or depletion to the contralateral or donor limbus.

PMID: 10928750 [PubMed - indexed for MEDLINE]

Comment in:

N Engl J Med. 2000 Jul 13;343(2):136-8.

Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells.

• Tsai RJ,
• Li LM,
• Chen JK.

Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. raytsai@ms4.hinet.net

BACKGROUND: Stevens-Johnson syndrome, ocular pemphigoid, and thermal or chemical burns can cause scarring and opacification of the cornea and loss of vision. Transplantation of epithelial cells from the limbus of the contralateral cornea can restore useful vision. However, this procedure requires a large limbal graft from the healthy eye and is not possible in patients who have bilateral lesions. METHODS: We took specimens of limbal epithelial cells from the healthy contralateral eyes of six patients with severe unilateral corneal disease. The epithelial cells were cultured and expanded on amniotic membrane. The amniotic membrane, together with the sheet of limbal epithelial cells, was transplanted to the denuded corneal surface of the damaged eye after superficial keratectomy to remove fibrovascular ingrowth. The mean (+/-SD) follow-up period was 15+/-2 months. RESULTS: Complete reepithelialization of the corneal surface occurred within two to four days of transplantation in all six eyes receiving transplants. By one month, the ocular surface was covered with corneal epithelium, and the clarity of the cornea was improved. In five of the six eyes receiving transplants (83 percent), the mean visual acuity improved from 20/112 to 20/45. In one patient with a chemical burn who had total opacification of the cornea, the acuity improved from the ability to count fingers at 40 cm to 20/200. No patient had recurrent neovascularization or inflammation in the transplanted area during the follow-up period. CONCLUSIONS: Transplantation of autologous limbal epithelial cells cultured on amniotic membrane is a simple and effective method of reconstructing the corneal surface and restoring useful vision in patients with unilateral deficiency of limbal epithelial cells.

PMID: 10891515 [PubMed - indexed for MEDLINE]

Application of the amniotic membrane in ocular surface pathology.

• Gris O,
• Guell JL,
• Lopez-Navidad A,
• Caballero F,
• Del Campo Z.

Department of Ophthalmology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

The amnion is a fine semi-transparent membrane that has been used in clinical practice to encourage epithelization in burns, in skin ulcers, or as a skin graft. Application in ocular surface disorders first took place in 1940. We carried out the membrane amniotic implantation on 11 patients with different pathologies: three cases of limbal stem cell deficiency (caustication with failure of prior keratoplasty, congenital aniridia and post-radiotherapy keratopathy), one case with persistent neurotrophic corneal ulcer after prior keratoplasty, four cases with epithelial defect of long evolution, one case of extensive Salzmann's degeneration of the cornea, and two cases after the resection of conjunctival tumour. The follow-up period varied between 2 and 6.5 months (mean = 4 months). Amniotic membrane was obtained by elective Caesarean, and it was preserved at -80 degrees C. In all transplanted patients the reabsorption of the amniotic membrane took place between the third and the fifth week. In the cases of resection of conjunctival tumour the epithelialization was completed between the first and the second post-operative week, with minimal residual scarring. In the other cases, with affliction of the corneal epithelium, the complete epithelialization, together with a marked reduction in the inflammatory response, occurred in all except 2 cases. In conclusion, the implantation of preserved human amniotic membrane can favour the recovery of a normal ocular surface in different pathologies, both in corneal and conjunctival lesions.

PMID: 10853789 [PubMed - indexed for MEDLINE]

Comment in:

Cornea. 2001 Oct;20(7):773-4.

Use of nonpreserved human amniotic membrane for the reconstruction of the ocular surface.

• Mejia LF,
• Acosta C,
• Santamaria JP.

Instituto de Ciencias de la Salud-CES, Medellin, Colombia. lfmejia@epm.net.co

PURPOSE: To describe the use of nonpreserved human amniotic membrane (NP-AMT) as an alternative to preserved human amniotic membrane (AMT) for the reconstruction of the ocular surface in several diseases. METHODS: NP-AMT was used in the treatment of five patients with the following diseases: noninvasive conjunctival squamous cell carcinoma, corneal persistent epithelial defect, severe alkali burn, near total limbal deficiency secondary to multiple surgeries, and ocular cicatricial pemphigoid. In some cases, a limbal autograft or allograft was employed simultaneously, sutured on top of the NP-AMT. All sutures were made with 10-0 Nylon and were removed at two weeks. RESULTS: Ocular surface was satisfactorily reconstructed, eyes were quiet, and patients were comfortable despite prolonged deepithelialization in some cases. There was a case of a limbal autograft ischemia--in the burned patient--that caused partial corneal conjunctivalization. Initially, the NP-AMT looks thickened but thins around the fifth day and looks similar to AMT. CONCLUSION: Results using NP-AMT are similar to those of AMT. It is a good alternative and it is easily obtained in places were AMT is not available or is too expensive to procure.

PMID: 10832685 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation or conjunctival limbal autograft for limbal stem cell deficiency induced by 5-fluorouracil in glaucoma surgeries.

• Pires RT,
• Chokshi A,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida 33136, USA.

PURPOSE: To determine if human amniotic membrane transplantation or limbal stem cell transplantation is effective to restore the corneal surface with partial or total limbal stem cell deficiency, respectively, caused by 5-fluorouracil (5-FU) toxicity after glaucoma surgeries. METHODS: Partial and total limbal stem cell deficiency was confirmed by impression cytology as the cause of reduced vision and corneal surface breakdown in a 69-year-old man and a 67-year-old man, respectively, who both had received a total of 105 mg 5-FU injections. Amniotic membrane transplantation or conjunctival limbal autograft was performed for corneal surface reconstruction, respectively. RESULTS: For a period of 15 months of follow-up, the visual acuity improved, and their corneal surfaces remained avascular, smooth, and without recurrence of limbal stem cell deficiency. CONCLUSION: Limbal stem cell deficiency can occur as a late complication for patients receiving 5-FU after glaucoma filtering surgeries. Partial limbal stem cell deficiency can be treated with amniotic membrane transplantation alone, whereas limbal transplantation must be considered as an alternative for total limbal stem cell deficiency to restore the corneal surface integrity and vision.

PMID: 10832684 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation for acute chemical or thermal burns.

• Meller D,
• Pires RT,
• Mack RJ,
• Figueiredo F,
• Heiligenhaus A,
• Park WC,
• Prabhasawat P,
• John T,
• McLeod SD,
• Steuhl KP,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida 33136, USA.

PURPOSE: To determine whether preserved human amniotic membrane (AM) can be used to treat ocular burns in the acute stage. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Thirteen eyes from 11 patients with acute burns, 10 eyes with chemical burns and 3 with thermal burns of grades II-III (7 eyes) and grade IV (6 eyes), treated at 7 different facilities. METHODS: Patients received amniotic membrane transplantation (AMT) within 2 weeks after the injury. MAIN OUTCOME MEASURES: Integrity of ocular surface epithelium and visual acuity during 9 months of follow-up. RESULTS: Ten patients were male and one patient was female; most were young (38.2 +/- 10.6 years). For a follow-up of 8.8 + 4.7 months, 11 of 13 eyes (84.63%) showed epithelialization within 2 to 5 weeks (23.7 +/- 9.8 days), and final visual acuity improved > or = 6 lines (6 eyes), 4 to 5 lines (2 eyes), and 1 to 3 lines (2 eyes); only one eye experienced a symblepharon. Eyes with burns of grade II to III showed more visual improvement (7.3 +/- 3 lines) than those with burns of grade IV (2.3 +/- 3.0 lines; P < 0.05, unpaired t test). In the group with grade II or III burns, none had limbal stem cell deficiency. All eyes in the group with grade IV burns did experience limbal stem cell deficiency. CONCLUSIONS: Amniotic membrane transplantation is effective in promoting re-epithelialization and reducing inflammation, thus preventing scarring sequelae in the late stage. In mild to moderate burns, AMT alone rapidly restores both corneal and conjunctival surfaces. In severe burns, however, it restores the conjunctival ocular surface without debilitating symblepharon and reduces limbal stromal inflammation, but does not prevent limbal stem cell deficiency, which requires further limbal stem cell transplantation. These results underscore the importance of immediate intervention in the acute stage of eyes with severely damaged ocular surface. Further prospective randomized studies including a control group are required to determine the effectiveness of AMT in acute chemical and thermal burns of the eye.

PMID: 10811094 [PubMed - indexed for MEDLINE]

[Amniotic membrane transplantation with or without limbal allografts in corneal surface reconstruction in limbal deficiency]

• Meller D,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, USA.

PURPOSE: We examined whether amniotic membrane transplantation (AMT) is useful in preparing the perilimbal stroma to enhance the success of allograft limbal transplantation (ALT). METHODS: Forty-seven eyes of 42 consecutive patients with cytologically proven limbal deficiency (LD) were included in this prospective study. Based on the severity of LD, group A (mild) with 18 eyes received AMT alone, group B (moderate) with 13 eyes received AMT and ALT, and group C (severe) with 16 eyes received AMT, ALT and penetrating keratoplasty. All except for group A received continuous systemic cyclosporin A. RESULTS: Except for the two eyes with atopy, all amniotic membrane-covered surfaces showed rapid epithelialization in 2-4 weeks, reduced inflammation, vascularization and scarring, and became smooth. For the mean follow-up period of 23 months, 38 eyes (82.6%) showed visual improvement, consisting of > or = 6 lines (15 eyes), 4-5 lines (10 eyes), 1-3 lines (13 eyes). Visual improvement was noted in 16/18 eyes (88.9%) in group A, in 10/13 eyes (77%) in group B, and in 12/16 eyes (75%) in group C. In group C corneal graft rejection occurred in 12 of 16 eyes (75%). In group B and C, early reversible limbal allograft rejection was noted in 3 of 29 eyes (10.3%) and a recurrent limbal deficiency was observed in 8 of 29 eyes (27.6%). CONCLUSION: For partial LD with superficial involvement, AMT alone is sufficient and hence superior to ALT because of no need for using cyclosporin A. For total LD, additional ALT is needed and AMT helps reconstruct the perilimbal stroma with reduced inflammation and vascularization, which collectively may enhance ALT success.

PMID: 10734735 [PubMed - indexed for MEDLINE]

Limbal stem cells of the corneal epithelium.

• Dua HS,
• Azuara-Blanco A.

Queen's Medical Centre, University of Nottingham, Nottingham, UK. harminder.dua@nottingham.ac.uk

Stem cells have certain unique characteristics, which include longevity, high capacity of self-renewal with a long cell cycle time and a short S-phase duration, increased potential for error-free proliferation, and poor differentiation. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. Stem cells for the cornea reside at the corneoscleral limbus. The limbal palisades of Vogt and the interpalisade rete ridges are believed to be repositories of stem cells. The microenvironment of the limbus is considered to be important in maintaining the stemness of stem cells. Limbal stem cells also act as a "barrier" to conjunctival epithelial cells and normally prevent them from migrating on to the corneal surface. Under certain conditions, however, the limbal stem cells may be partially or totally depleted, resulting in varying degrees of stem cell deficiency with resulting abnormalities in the corneal surface. Such deficiency of limbal stem cells leads to "conjunctivalization" of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. This results in ocular discomfort and reduced vision. Partial stem cell deficiency can be managed by removing the abnormal epithelium and allowing the denuded cornea, especially the visual axis, to resurface with cells derived from the remaining intact limbal epithelium. In total stem cell deficiency, autologous limbus from the opposite normal eye or homologous limbus from living related or cadaveric donors can be transplanted on to the affected eye. With the latter option, systemic immunosuppression is required. Amniotic membrane transplantation is a useful adjunct to the above procedures in some instances.

PMID: 10734241 [PubMed - indexed for MEDLINE]

[Limbal autograft transplantation, eight consecutive cases]

• Muraine M,
• Salessy P,
• Watt L,
• Retout A,
• Brasseur G.

Service d'Ophtalmologie, Hopital Charles Nicolle, Boulevard Gambetta, 76031 Rouen Cedex.

BACKGROUND: Limbal autograft transplantation is the procedure of choice in the management of ocular surface disorders secondary to stem cells deficiency. The aim of our study was to investigate the indications, results and limits of this infrequent surgery. METHODS: Limbal autograft transplantation was performed in 8 patients and the mean follow-up period was 11 months. Limbal stem cell deficiency was due to chemical burns in 4 patients, history of prior surgery extending to the limbus in 2 patients, chronic limbitis with dystichiasis in one patient and persistent corneal epithelial defect after keratoplasty in one case. RESULTS: In 6 out of 8 cases, the stability of the ocular surface normalized and comfort significantly improved. Four of these patients had increased visual acuity after surgery. Two patients who presented with severe alkali burn did not respond well to limbal autotransplantation. CONCLUSION: Limbal autograft transplantation is a reliable and effective procedure in limbal stem cells deficiencies. New associate procedures such as amniotic membrane transplantation will however be necessary to improve the prognosis of very severe corneal surface disorders.

PMID: 10705110 [PubMed - indexed for MEDLINE]

[Reconstruction of the conjunctival and corneal surface. Transplantation of amnionic membrane]

• Meller D,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, USA.

The ocular surface epithelia with the tear film as a unit provide clear vision and comfort and serve as the first line the defense. It is important to understand how ocular surface health is maintained and how ocular surface failure occurs. Furthermore, it is timely to summarize new information concerning action mechanisms and clinical uses of amniotic membrane transplantation for ocular surface reconstruction. When appropriately processed and preserved, amniotic membrane as a native matrix can be used as a graft to restore conjunctival surfaces following removal of lesions such as pterygium, tumor, scar, symblepharon, and conjunctivochalasis. It can also be used as a graft to restore corneal surfaces suffering from limbal stem-cell deficiency. For partial limbal deficiency, amniotic membrane alone is frequently sufficient, while for total limbal deficiency it needs to be combined with limbal stem-cell transplantation with or without corneal transplantation. When used as a graft or patch, amniotic membrane can facilitate healing of persistent corneal ulcers and recurrent corneal erosion and reduce corneal haze following keratectomy. Reported data indicate that amniotic membrane transplantation facilitates rapid healing with recovery of a normal epithelial phenotype in the epithelium and reduces inflammation, vascularization, and scarring in the stroma.

PMID: 10025141 [PubMed - indexed for MEDLINE]

Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency.

• Tseng SC,
• Prabhasawat P,
• Barton K,
• Gray T,
• Meller D.

Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Fla 33136, USA. stseng@bpei.med.miami.edu

OBJECTIVE: To examine whether amniotic membrane transplantation (AMT), in preparing the perilimbal stroma, enhances the success of allograft limbal transplantation (ALT). METHODS: Thirty-one eyes of 26 consecutive patients had cytologically proven limbal deficiency resulting from chemical burns (14 eyes); Stevens-Johnson syndrome, toxic epidermal necrolysis, or pseudopemphigoid (5 eyes); contact lens-induced keratopathy (3 eyes); aniridia (3 eyes); multiple surgical procedures (2 eyes); atopy (2 eyes); or an unknown cause (2 eyes). Based on the severity of limbal deficiency, group A (mild), comprising 10 eyes, received AMT alone; group B (moderate), comprising 7 eyes, received AMT and ALT; and group C (severe), comprising 14 eyes, received AMT, ALT, and penetrating keratoplasty. All patients except those in group A received continuous oral cyclosporine. RESULTS: Except for the 2 eyes with atopy, all amniotic membrane-covered surfaces showed rapid epithelialization (in 2 to 4 weeks) and reduced inflammation, vascularization, and scarring, and the surfaces became smooth and wettable. For the mean follow-up period of 15.4 months, 25 (83%) of 30 eyes showed visual improvement, consisting of 6 or more lines (13 eyes), 4 to 5 lines (6 eyes), or 1 to 3 lines (6 eyes). Visual improvement decreased with the severity of limbal deficiency from 8 (100%) of 8 eyes in group A to 5 (71%) of 7 eyes in group B and 11 (79%) of 14 eyes in group C. In group C, corneal graft rejection occurred in 9 (64%) of 14 eyes, and reversible early limbal allograft rejection was noted in 3 (14%) of 21 eyes of groups B and C. CONCLUSIONS: For partial limbal deficiency with superficial involvement, AMT alone is sufficient and hence superior to ALT because there is no need to administer cyclosporine. For total limbal deficiency, additional ALT is needed, and AMT helps reconstruct the perilimbal stroma, with reduced inflammation and vascularization, which collectively may enhance the success of ALT.

PMID: 9565039 [PubMed - indexed for MEDLINE]

Impression cytology study of epithelial phenotype of ocular surface reconstructed by preserved human amniotic membrane.

• Prabhasawat P,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Fla., USA.

OBJECTIVE: To determine the epithelial phenotype of the ocular surface reconstructed by preserved human amniotic membrane. METHODS: Impression cytology was performed in 6 patients who received a large patch of amniotic membrane for conjunctival surface reconstruction during removal of acquired melanosis, conjunctival intraepithelial neoplasia, or bilateral inferior conjunctival chalasis, or for corneal surface reconstruction during removal of pannus associated with limbal deficiency caused by aniridia, toxic epidermal necrolysis, or chemical burn. RESULTS: The nongoblet epithelial cells covering the amniotic membrane were uniformly smaller and the cell density was almost twice that of age- and sex-matched normal control eyes at the corresponding site, and the goblet cell density was almost 10 times that of the control (both P < .05; Student paired t test) (N = 7 eyes). Furthermore, the conjunctival epithelial phenotype with goblet cells was found on corneal surfaces of all 3 patients with limbal deficiency. CONCLUSIONS: The success of conjunctival surface reconstruction correlated well with recovery of the conjunctival epithelial phenotype. The lack of corneal epithelial phenotype even on an avascular corneal stroma supports the concept that conjunctival transdifferentiation does not occur in vivo, and indicates that additional limbal stem cell transplantation is needed for effective corneal surface reconstruction in patients with limbal deficiency.

PMID: 9366664 [PubMed - indexed for MEDLINE]

Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas.

• Kim JC,
• Tseng SC.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida 33101, USA.

After n-heptanol removal of the total corneal epithelium and a limbal lamellar keratectomy, 23 rabbit eyes developed features of limbal stem cell deficiency including conjunctival epithelial ingrowth, vascularization and chronic inflammation. One month later, 10 control eyes received a total keratectomy, and 13 experimental eyes received additional transplantation of glycerin-preserved human amniotic membrane. In 3 months of follow-up, all control corneas were revascularized to the center with granuloma and retained a conjunctival epithelial phenotype. In contrast, five corneas in the experimental group became clear with either minimal or no vascularization; the rest had either mid peripheral (n = 5) or total (n = 3) vascularization and cloudier stroma. The success of corneal surface reconstruction correlated with the return of a cornea-like epithelial phenotype and the preservation of amniotic membrane, whereas the failure maintained a conjunctival epithelial phenotype and the amniotic membrane was either partially degraded or covered by host fibrovascular stroma. These results suggest that measures taken to facilitate epithelialization without allowing host fibrovascular ingrowth onto the amniotic membrane might prove this procedure clinically useful for ocular surface reconstruction.

PMID: 8536460 [PubMed - indexed for MEDLINE]

The effects on inhibition of corneal neovascularization after human amniotic membrane transplantation in severely damaged rabbit corneas.

• Kim JC,
• Tseng SC.

Department of Ophthalmology, Chung-Ang University Hospital, Seoul, Korea.

Human amniotic membrane isolated from the placenta contained basement membrane components such as type IV collagen, laminin, and 6 and 4 integrins, all of which remained detectable while preserved in glycerin for one week. One month after the n-heptanol removal of the total corneal epithelium and the limbal lamellar keratectomy, all rabbit eyes carried features of limbal deficiency, including conjunctival epithelial ingrowth, vascularization and chronic inflammation. Ten control eyes then received a total keratectomy, and 13 experimental eyes received an additional amniotic membrane transplantation. Three-month follow-ups revealed that all control corneas were revascularized to the center with granuloma and retained a conjunctival phenotype. In contrast, in the experimental groups, 5 corneas became clear with either minimal or no vascularization; the rest had either mild peripheral (5) or total (3) vascularization and more cloudy stroma. Using monoclonal antibodies for epithelial markers and matrix components, we concluded that the success correlated with the return of a cornea-like epithelial phenotype and the preservation of the amniotic membrane, whereas the failure maintained a conjunctival epithelial phenotype and the amniotic membrane was either partially degraded or covered by host fibrovascular stroma. Measures taken to facilitate the former might prove this procedure clinically useful for ocular surface reconstruction.

PMID: 7674551 [PubMed - indexed for MEDLINE]