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

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 S?o 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.     

Unusual intermediate-term outcome in three cases of limbal autograft transplantation.

OBJECTIVE: To report an unusual intermediate-term outcome after limbal autograft transplantation for unilateral, severe chemical burns. DESIGN: Case reports of three consecutive cases. PARTICIPANTS: Three patients with a history of chemical burns. The fellow eye was clinically uninvolved in all patients. Surgery was indicated for conjunctivalization (cases 1 and 3) and persistent epithelial defect (case 2). INTERVENTION: Three (cases 2 and 3) or four (case 1) 1.25-clock-hour-wide limbal grafts were harvested from the fellow eye and transplanted to the limbus in the affected eye. Surface re-epithelialization, improvement in visual acuity, and maintenance of surface stability were monitored. RESULTS: Limbal grafts were effective in re-epithelialization of the corneal surface within 4 weeks in all cases. Symptomatic relief and improvement in visual acuity were noted during early follow-up. However, a progressive conjunctival ingrowth that tended to override the grafts and encroach the visual axis was noted over the course of a year in all three recipient eyes. CONCLUSIONS: Limbal autograft transplantation is an effective method for surface re-epithelialization in strictly unilateral chemical burns. Caution should be exercised in case selection. The authors' experience suggests a variable degree of attenuation in viability of transplanted limbus. Close follow-up of operated eyes is necessary to determine the long-term efficacy of limbal transplants as a source of corneal phenotypic cells.     

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

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.     

眼烧伤后重度睑球粘连眼表重建的临床研究

目的探讨新鲜供体带角膜缘板层角膜移植（LKP）联合带角膜缘干细胞自体球结膜移植和羊膜移植治疗眼烧伤后重度睑球粘连的眼表重建效果。方法选择1998年2月至2003年2月我院收治的眼烧伤后重度睑球粘连患者26例（26只眼）。根据视功能受损程度将睑球粘连分为4度．其中Ⅲ度19只眼、Ⅳ度7只眼。手术方法：（1）分离睑球粘连,保留假性胬肉;（2）利用新鲜供体行带角巩膜缘的部分LKP（19只眼）或全LKP（7只眼）;（3）取带角膜缘组织自体球结膜瓣移植于粘连最重的创面;（4）假性胬肉后徙,部分替代球结膜,联合羊膜移植重建结膜囊。术后随访8～36个月,观察结膜囊情况、眼球活动、角膜植片免疫排斥反应情况以及羊膜和假性胬肉的转归。结果除1只眼因睑裂闭合不全行永久性睑裂缝合术外,其余25只眼的眼表情况均得到不同程度改善。（1）睑球粘连：17只眼（65％）第一次手术后粘连完全分离,复视消失;8只眼（31％）在单纯羊膜覆盖的创面区复发条带状睑球粘连,其中5只眼经再次手术粘连完全解除,3只眼残余Ⅰ度粘连。（2）羊膜在术后1周开始自溶,3个月时完全消失;保留的假性胬肉术后1～3个月充血消退,逐渐变薄,相应区域无睑球粘连复发。（3）角膜情况：6只眼角膜植片发生免疫排斥反应,其中3只眼治疗后植片恢复透明,2只眼因植片混浊行二次LKP。结论新鲜供体带角膜缘LKP联合带角膜缘干细胞自体球结膜移植和羊膜移植是治疗眼烧伤后重度睑球粘连的有效方法,假性胬肉可部分替代球结膜重建结膜囊,羊膜移植在重度睑球粘连的治疗中仅发挥暂时支持创面促进眼表恢复的作用。（中华眼科杂志,2005,41：791-795）     

角膜缘移植联合羊膜移植治疗角膜缘缺陷症

目的 探讨角膜缘移植联合羊膜移植术在治疗角膜缘缺陷症中的作用。方法 以自体或异体角膜缘移植手术治疗复发性翼状胬肉、眼化学烧伤、热烧伤等角膜缘缺陷症患者52例59眼，术中随访5－24月。结果 随访期内植片存活，角膜上皮重建，角膜透明性增另，新生血管减退。仅2眼翼状胬肉复发。结论 角膜缘移植联合羊膜移植是一种较好的治疗角膜缘缺陷症的手术方法。     

活体异体结膜缘和羊膜移植治疗化学性眼外伤造成的角膜缘干细胞缺失(英文)

目的:评价利用活体异体结膜缘和羊膜移植治疗化学性眼外伤造成的角膜缘干细胞缺失的临床效果。方法:从2005-07/2007-12,本研究包括了9名男性化学性眼外伤患者(10眼)。所有患者接受了亲属活体异体结膜缘和羊膜移植,2例眼接受了睑缝术。用环孢菌素和泼尼松龙进行全身性免疫抑制。结果:在3例眼中观察到完全角膜上皮化(30%),其中1例在术后1.5mo出现免疫排斥,角膜溶解引起穿孔,加大全身性免疫抑制剂量来控制病情。3例眼中植片无法在角膜表面重新形成上皮,被定为原发性失败。其余4眼有部分上皮形成,但上皮细胞无法完全覆盖角膜表面。术前最佳矫正视力从手动到1m处数指,术后最佳矫正视力从光感到20/80。有5眼视力得到改进,不需其他治疗。手术失败的主要原因为干眼症和持续性炎症。结论:对于能控制泪量和眼部炎症的病例,亲属活体异体角膜缘和羊膜移植是治疗化学性眼外伤造成的角膜缘干细胞缺失最佳方法之一。     

新鲜羊膜联合带活性角膜缘的全板层角膜移植治疗早期严重眼烧伤

目的：探讨新鲜羊膜联合带活性角膜缘的全板层角膜移植治疗早期严重眼烧伤疗效。方法：对早期严重眼烧伤14例15眼施行新鲜羊膜联合带活性角膜缘的全板层角膜移植术,术后观察移植物贴附、生长及眼表情况,监测重建的穹窿深度,了解是否有睑球粘连的发生。结果：移植的羊膜、角膜缘及角膜全部成活,重建的眼表面很快上皮化并保持基本稳定。除6眼因白内障及角膜持续水肿外,余视力均有提高。4眼发生睑球粘连,余形成足够的穹窿深度,眼球运动基本正常。术后3眼移植角膜缘发生排斥反应,局部用药控制。结论：治疗早期严重眼烧伤,新鲜羊膜移植能够有效防止睑球粘连,异体角膜缘及全板层角膜移植可恢复角膜结构并提供足够的干细胞,获得稳定眼表面。积极防治移植角膜缘排斥反应十分重要。     

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

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.     

Cultivated limbal and oral mucosal epithelial transplantation

Stem cells located at the limbus are the ultimate source for regeneration of the corneal epithelium in normal and traumatized states. When limbal stem cells are dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. Its surgical management depends on laterality and severity of corneal-limbal involvement. Conventional methods of stem cell transplantation are conjunctival-limbal autograft (CLAU), conjunctival-limbal allograft (CLAL), and kerato-limbal allograft (KLAL) surgeries. Cultivated limbal epithelial transplantation (CLET) and cultivated oral mucosal epithelial transplantation (COMET) on a carrier such as amniotic membrane are current surgical alternatives. These new surgical procedures are effective in stabilizing the ocular surface. The theoretical advantage of ex-vivo expansions over conventional methods is that only a small limbal or mucosal biopsy is needed, thus minimizing the risk to the donor eye; there is also a lower risk of rejection. They can be used in cases with unilateral or bilateral total stem cell deficiency. In the unilateral cases, the source for CLET is a healthy fellow eye and in bilateral cases the source can be living-related or cadaveric eyes. The oral explants do not have limbal stem cells, but they seem to be a source of limbal stem cell equivalents that are able to generate cornea-like epithelium under the proper culture conditions. The main advantage of COMET is that patients with bilateral LSCD can be treated with grafts derived from their own autologous oral mucosal cells. The long-term outcomes of COMET have to be elucidated.     

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

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.     

Sutureless amniotic membrane transplantation for partial limbal stem cell deficiency

PURPOSE: To evaluate the results of sutureless amniotic membrane (AM) transplantation using fibrin glue for reconstructing corneal surfaces with partial limbal stem cell deficiency (LSCD). DESIGN: Retrospective noncomparative interventional case series. METHODS: Eleven eyes of nine patients that had LSCD with 120 degrees to almost 360 degrees of limbal involvement underwent superficial keratectomy to remove the conjunctivalized pannus followed by AM transplantation using fibrin glue. Additional sutureless AM patch (ProKera; Bio-Tissue, Inc, Miami, Florida, USA) was used in seven patients, and mitomycin C was applied on the cornea in four eyes and during fornix reconstruction in seven eyes. The surgery was repeated in three eyes for residual pannus. RESULTS: During a mean follow-up of 14.2 +/- 7.7 months (range, six to 26 months), all eyes maintained a smooth and stable corneal epithelial surface without recurrent erosion or persistent epithelial defect, and showed less stromal cloudiness and vascularization. Best-corrected visual acuity improved in nine eyes (81.8%). Corneal epithelialization proceeded by epithelial growth over AM (n = 4), accompanied by dissolution of AM (n = 4) or a combination of both (n = 3). No complication was noted regarding initial or repeated uses of fibrin glue. CONCLUSION: AM transplantation using fibrin glue appears to be a safe and effective method of restoring a stable corneal epithelium for cases with partial LSCD. This approach avoids the need of transplanting limbal epithelial stem cells.     

自体角膜缘联合羊膜移植术与联合结膜移植术治疗复发性翼状胬肉比较

目的 比较自体角膜缘联合羊膜移植术和自体角膜缘联合结膜移植术治疗复发性翼状胬肉的疗效，探讨自体角膜缘联合羊膜移植术能否代替自体角膜缘联合结膜移植术治疗复发性翼状胬肉。方法 36例42眼复发性翼状胬肉患者随机分成A、B2组，A组18例22眼行自体角膜缘联合羊膜移植术；B组18例20眼行自体角膜缘联合结膜移植术。结果 A组20眼术后效果满意，2眼术后5个月复发，复发率9．09％；B组19眼术后效果满意，1眼术后3个月复发，复发率5．00％．2组术后复发率在统计学上无显著差异。结论 自体角膜缘联合羊膜移植术可代替自体角膜缘联合结膜移植术治疗复发性翼状胬肉。     

Clinical outcome of autologous cultivated limbal epithelium transplantation

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.     

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

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.     

Amniotic membrane graft in ocular surface disease. Prospective study with 31 cases

INTRODUCTION: Amniotic membrane's unique combination of properties including the facilitation of migration of epithelial cells, the reinforcement of basal cellular adhesion and the encouragement of epithelial differentiation [6] together with its ability to modulate stromal scarring and its anti-inflammatory and anti-bacterial activity has led to its use in the treatment of ocular surface pathology as well as an adjunct to stem cell grafts of the corneal limbus [6-4]. We report a prospective study of 30 patients so treated. MATERIAL AND METHODS: We studied 31 eyes of 30 patients subjected to amniotic membrane grafts between September 1999 and May 2000. There were 25 men and 5 women with an average age of 60.1 (range 25-86) years who were followed for a mean of 7.7 (range 4-11) months. 5 groups (A to D) were observed: A: 6 eyes. Small chronic ulcers without limbal involvement. B: 4 eyes. Ulcers of at least 75% corneal area or occupying 75% of the limbus. C: 9 eyes. Corneal burns. D: 8 eyes. Painful bullous corneal dystrophies unresponsive to other treatment. E: 4 eyes. Symblepharons. Amniotic membrane was placed on the corneal lesion, epithelial surface externally [6, 15], trimmed and sutured with interrupted 10/0 nylon, removed at one month. In two patients (11, 12) inflamed conjunctiva was recessed and amnion sutured to the recessed margin. For the bullous dystrophies we removed all the corneal epithelium and either sutured the amnion to peri-limbal conjunctiva (4 eyes) or to the limbus (4 eyes). For the symblepharons the conjunctiva was dissected to reform the fornix which was lined with amniotic membrane, sutured with 8/0 vicryl. Patients were reviewed regularity. RESULTS: Group A: All healed within 15 days, in most with dissolution of the amnion over 2-3 months although some persisted, covered with corneal epithelium. An eye with a Descemetocoele and one with a microperforation both healed. Vision improved more than two lines in 4 of 6 eyes. Group B: 2 of 4 eyes healed, one despite detachment of the membrane after 15 days. One eye was salvaged by tarsorrhaphy over a fresh keratoplasty after perforation of a neuroparalytic ulcer on failure of three successive amnion grafts. The final cornea vascularised despite an amnion graft for a meta-herpetic ulcer. Group C: 2 of 9 eyes had limbal damage in one quadrant but 7 had vessels in at least three-quarters of the circumference. One (15) also had a limbal autograft. 3 of 9 eyes healed satisfactorily with more than 2/10 improvement in acuity in each case. 2 showed further neovascularisation despite surface healing. One old chemical burn healed satisfactorily but vascularisation remained 5 eyes failed to heal with lysis of the graft, the patient who had a limbal autograft developed a vascular pannus, and in 4 eyes neovascularisation progressed to cover the entire cornea. Group D: 3 eyes settled with loss of symptoms but in 5 the graft detached within 15 days. All eyes where the membrane had been sutured to the conjunctiva beyond the limbus failed whilst 3 of 4 in which it had been sutured anterior to the limbus succeeded, leaving a persistent whitish membrane under the epithelium. Group E: We were able to reconstruct the cul de sac in 3 out of 4 eyes. In one patient with recurrent pterygium good ocular movement was restored, previously limited by scarring. One with associated ocular surface damage from a thermal burn failed by scarring of the cul de sac a month after surgery. DISCUSSION: Our best results were in persistent trophic ulcers of the cornea (Groups A and B) with a success rate of 80%, comparable to those of others [49, 37, 38]. The ready availability of amniotic membrane in our facility makes amniotic membrane transplantation the main secondary treatment for such lesions, especially because of the visual improvement we obtained. Because we did not observe any improvement in corneal thickness after this treatment we advise its early use before significant stromal lysis. The technique was not sufficient to control the effect of corneal anaesthesia in two eyes [40] or in chemical burns suggesting that amniotic membrane alone is insufficient to promote corneal healing in the absence of limbal stem cells. Nevertheless, three eyes did benefit. It has been suggested [13] that the anti-apoptotic function of amnion may prevent stem cell loss in such eyes [42], thus it appears logical to offer an amniotic membrane graft first, before stem cell transplantation, which may entrain complications in the donor eye if autografted [43] or because of the rejection risk of an allograft. It may be that an amniotic membrane graft simply becomes a holding procedure allowing time to settle the eye so as to allow secondary procedures to address the underlying cause of further damage. Our treatment of bullous dystrophy only succeeded on confining the graft to within the limbus, 3 out of 4 eyes becoming comfortable. By contrast we found amniotic membrane helpful in reconstructing symblepharons in the absence of local inflammation. CONCLUSION: Amniotic membrane grafting is a simple and straightforward surgical technique which should form part of the therapeutic arsenal for the treatment of ocular surface disease. Indications for the technique need further clarification for it is evident that it cannot correct all secondary pathology associated with limbal destruction. It is certainly preferable to conjunctival advancement and has proved useful in the reconstruction of the cul-de-sac.     

Contemporary limbal stem cell transplantation – a review

Conjunctivalization of the cornea is the hallmark of limbal stem cell deficiency (LSCD). This is often associated with persistent corneal epithelial defects and a fibrovascular pannus. LSCD can be unilateral or bilateral and partial or total. In partial LSCD involving the visual axis sequential sector conjunctival epitheliectomy (SSCE) is a useful option. In total LSCD, transplantation of limbal tissue or of ex vivo expanded sheets is the mainstay. In unilateral cases autolimbal transplant is the procedure of choice. In bilateral cases living (related) and cadaver donors are considered. The former has the advantage of being fresh and can be human leucocyte antigen matched. Procedures for harvesting limbal tissue from living donors are identical. Different strategies are required for harvesting tissue from cadaver whole globes or sclero‐corneal rims. Recipient eye preparation requires removal of the fibrovascular tissue. Donor explants are generally sutured directly on the denuded recipient surface without the preparation of a ‘bed’ to fit the explant. It is imperative that inflammation is meticulously controlled before limbal transplantation especially if tissue from living donors is used. Limbal transplantation, with the exception of a corneal graft, should be the last surgical intervention planned. Meticulous postoperative care and treatment with antibiotics, steroids, artificial tears and autologous serum are required. With allografts long‐term immunosuppression is necessary. Limbal transplantation is contraindicated in the presence of severe dry eye. Despite its complexities limbal transplantation does significantly improve vision related quality of life. Autografts give the best results and living related donor grafts are next best. Majority of cadaver grafts fail in 5 years.     

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits

PURPOSE: To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. METHODS: An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. RESULTS: A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. CONCLUSION: Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.     

Long-term outcomes of donor eyes after conjunctival limbal autograft and allograft harvesting

ObjectiveTo investigate the ocular surface stability of donor eyes after conjunctival limbal autograft (CLAU) or conjunctival limbal allograft (CLAL).DesignRetrospective interventional case series.ParticipantsPatients diagnosed with limbal stem cell deficiency (LSCD) who underwent ocular surface stem cell transplantation with postoperative follow-up of at least 3 months.MethodsDonor eye data collected included preoperative and postoperative Snellen best-corrected visual acuity (BCVA), intraoperative and postoperative complications, re-epithelialization times, and ocular surface stability at the last follow-up.ResultsSixteen donor eyes of 16 patients were included, the mean age at the time of surgery was 59.3 years, and the mean follow-up period was 53 months. The most common injury etiology was chemical injury, followed by iatrogenic causes and Stevens-Johnson syndrome, then congenital aniridia. Eleven patients underwent CLAU, 3 underwent living-related CLAL, and 2 underwent CLAL combined with keratolimbal allograft. Preoperative mean donor eye BCVA was 0.22 ± 0.32 logMAR (Snellen equivalent ≈ 20/33), and mean BCVA at the last follow-up was 0.18 ± 0.24 logMAR (Snellen equivalent ≈ 20/30) (p = 0.4). All donor eyes had a stable ocular surface at the last follow-up, with no signs of iatrogenic LSCD or delayed corneal epithelial healing. There were no intraoperative or postoperative complications such as infections, persistent epithelial defects, corneal neovascularization, or chronic inflammation.ConclusionThis study provides additional evidence for the long-term safety of donor eyes when providing limbal stem cell tissue for CLAU or CLAL.     

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

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.     

Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series

Purpose: Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal‐derived products and non‐human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. Methods: We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. Results: Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11‐ to 28‐month follow‐up). Conclusions: Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal‐derived products and non‐human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter‐species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.