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

Sangwan, Virender S; Matalia, Himanshu P; Vemuganti, Geeta K; Rao, Gullapalli N

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Indian Journal of Ophthalmology: Oct–Dec 2004 - Volume 52 - Issue 4 - p 281-285
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Corneal epithelial stem cells, responsible for the renewal of the corneal epithelium are located at the corneoscleral limbus.[12] These stem cells are essential for the maintenance of a healthy corneal surface. Diseases that destroy these stem cells can lead to limbal stem cell deficiency (LSCD).[3] Loss of limbal stem cells can be partial or total. Patients suffering from LSCD complain of decreased vision, pain, photophobia and watering. Clinically they may present with recurrent or persistent epithelial defect, conjunctivalisation of cornea, corneal neovascularisation and loss of limbal palisades of Vogt.[45] Histopathologically, LSCD is characterised by progressive invasion of conjunctival epithelial cells on to the cornea, superficial vascularisation, squamous metaplasia of corneal epithelial cells, presence of goblet cells and destruction of the corneal basement membrane with fibrous tissue deposition and chronic inflammatory cells infitration.[6] The diagnosis of LSCD is important as these patients are poor candidates for conventional corneal transplantation.

In cases of unilateral LSCD or partial LSCD, autologous limbus or conjunctiva can be transplanted from the fellow eye or the normal area of same eye.[7] A major concern related to the autograft is the possibility of LSCD at the donor site.[8] Amniotic membrane transplant (AMT) has been reported with or without limbal transplant as a modality for LSCD.[910] Herein we report the results of AMT for partial LSCD.

Material and Methods


Case records of four patients who underwent AMT for partial limbal stem cell deficiency at our institute between December 1998 and August 2001 were retrospectively reviewed for age, gender, laterality, cause of partial LSCD, degrees of LSCD, any previous surgery, other interventions, preoperative and postoperative visual acuity, detailed slitlamp examination, details of surgical procedures and postoperative follow-ups.

Following the Ethics Committee approval, a written informed consent was obtained from each patient or attendant after explaining to them the nature of surgical procedure, risk involved and possible adverse consequences. All the surgeries were done by a single surgeon (VSS) at L V Prasad Eye Institute, Hyderabad, India. The clinical diagnosis of LSCD was based on the presence of conjunctivalisation, and loss of palisades of Vogt with an increased permeability to fluorescein.

Processing of amniotic membrane

Placenta was obtained from donors following an elective Caesarian section, after screening for HIV seronegativity, hepatitis B virus, hepatitis C virus and syphilis. Amniotic membrane was processed under sterile conditions by the methods described by Lee and Tseng.[11] The processed amniotic membrane was flattened on a sterile nitrocellulose paper with the epithelial/basement membrane surface facing up and stored at -80°C in a sterile vial containing Dulbeco’s modified Eagles medium and glycerol (1:1, v/v).

Surgical procedure

Three patients were operated under peribulbar anaesthetia and one received general anaesthesia. A conjunctival peritomy was performed 2 mm from the limbus in the area of conjunctivalisation. A surgical plane was identified and the fibrovascular pannus was peeled off and dissected from the cornea. The tissue was sent for histopathological examination. Preserved amniotic membrane was thawed at room temperature, peeled from the nitrocellulose paper and placed on the cornea with epithelial/basement membrane surface up, covering the whole cornea except in patient 2 in whom only the area of LSCD was covered. The amniotic membrane was sutured into place with six circumferential interrupted sutures with 10-0 monofilament nylon sutures and all the knots were buried. The peripheral edge of the membrane was sutured to the conjunctiva with 8-0 polyglactin interrupted sutures. The excess membrane was trimmed. A bandage contact lens was put in after surgery in patient 3. All the eyes were patched after surgery. Postoperatively patients were treated with gentamicin 0.3% eye drops four times a day until the corneal epithelial defect healed and prednisolone acetate 1% eye drops six times a day, tapered over a period of 6 weeks and then stopped. Case 3 developed postoperative reactivation of viral keratouveitis after one year of resolution and was started on oral acyclovir 200mg twice daily and topical acyclovir 3% five times a day. The latter was discontinued after 2 weeks but the tablet was continued. All the patients were regularly followed with proper clinical and photographic documentation with special attention to the conjunctivali-sation and inflammation of the affected area.

Case Report

A 20-year-old male presented to us on January 2, 1998 with history of pain, decreased vision, redness and watering with a white spot in his left eye for 6 months. He denied any history of trauma, previous surgery or use of any indigenous medicine. He gave a history of a skin disease 6 years ago, the details of which were not available. At the time of presentation he was using ciprofloxacin hydrochloride 0.3% eye drops and polyvinyl alcohol-povidone lubricant eye drops four times a day in both eyes. On examination his visual acuity was 6/6 in the right eye and 6/9p in the left eye. Anterior segment and dilated fundus examination of right eye were unremarkable except for mild blepharitis in both eyes. The left eye showed 1+ conjunctival congestion, particularly in the nasal area. The cornea showed tongue-shaped scarring with hyperplastic epithelium nasally measuring 2.5mm X 5.5mm. The rest of the anterior segment and dilated fundus examination was within normal limits. The patient was treated with betamethasone sodium phosphate 0.1% eye drops and chloramphenicol 0.5% eye drops with oral doxycycline for four weeks for the bilateral blepharitis and the resolving blepharokeratitis in the left eye. Over the next 11 months, the pannus progressed in the left eye, threatening the visual axis [Figure 1a]. A diagnosis of focal stem cell deficiency in left eye was made, based on the clinical findings of loss of palisades of Vogt and progressive conjunctivalisation. We removed the pannus under peribulbar anaesthesia following the surgical technique described above. The pannus was sent for histopathology. Postoperatively he received prednisolone acetate 1% eye drops and gentamicin sulphate 0.3% eye drops. The corneal sutures were removed at four weeks. After seven weeks postoperative, the patient was comfortable, the ocular surface was stable and the patient regained visual acuity of 6/6p in the left eye [Figure 1b]. He had no recurrence [Figure 1c] and was comfortable 30 months after the surgery (he was last seen on June 7, 2001). The excised pannus from the cornea showed chronic inflammatory cells with the goblet cells indicating conjunctivalisation.


A total of four eyes of 4 patients received AMT for partial LSCD with a mean follow up period of 17.6 ± 11.4 months. Three patients were male, and the mean age was 32 ± 24.9 years ranging from (range 4 to 66 years) [Table - 1]. The extent of the LSCD was from 40° to 330° [Figure 2a]. The surgery was uneventful in all cases, with no postoperative complications. Postoperatively, all the eyes showed less inflammation and vascularisation [Figure 2b], with all four patients improving symptomatically. None of the patients showed recurrence of conjunctivalisation, persistent epithelial defect or recurrent epithelial erosions and all eyes maintained a smooth and stable corneal epithelial surface at the last follow up [Figure 2c]. The best-corrected visual acuity improved from preoperative (6/9p-6/120 range to postoperative 6/6p-6/15 range in all four cases. The mean improvement was of 4.5 ± 2.5 lines on the Snellen visual acuity chart. Postoperative best-corrected visual acuity in all the patients was better than 6/15. Resected pannus was sent for histopathological examination in all four cases; this showed presence of conjunctival epithelium with goblet cells suggestive of conjunctivalisation.

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We have demonstrated that using AMT, the corneal epithelial surface can be reconstructed and a stable ocular surface can be achieved with definite improvement in vision in cases of partial LSCD. Tseng et al[9] and Anderson et al[10] have reported encouraging results of AMT for partial LSCD. In our study all four patients improved symptomatically and the visual acuity improved by an average of 4.5 lines. In our cases the degree of LSCD ranged from 40° to 330°. As earlier reported by Anderson et al,[10] the surgery was beneficial in our case of near-total LSCD too (case no.4).

Our results also support the hypothesis of Tseng et al[9] that the AMT helps in preservation and expansion of residual limbal epithelial cells in cases of partial LSCD. In our study the histopathological analysis of all the resected tissues showed conjunctival phenotype with goblet cells and squamous metaplasia, confirming our clinical diagnosis of LSCD.[4] Partial LSCD can be treated by conjunctival or limbal autografts from same eye or the contralateral normal eye, but there are chances of the donor site developing LSCD as reported by Basti and Mathur.[8] The other option described by Dua[12] of repeated mechanical scraping is relatively simple but the longterm outcome is not known and treated cases were not very severe. A randomised controlled trial is required to answer these questions. Considering these issues, AMT could be a safer and easier alternative. [910] The amniotic membrane serves as a “transplanted basement membrane” and acts as a new healthy surface for proper re-epithelialisation,[13] as it is known that the basement membrane supports and reinforces basal epithelial cell adhesion, promotes its differentiation, and migration.[14] It also prevents epithelial apoptosis.[15] Additionally the stromal matrix of the membrane can also prevent inflammatory cells,[16] produces various growth factors[17] and several forms of protease inhibitors.[18] Collectively, these actions of AMT contribute to the creation of a healthy stromal bed and a basement membrane that may subsequently support limbal epithelial stem cells, reduce stromal inflammation and promote epithelialisation.

In summary, amniotic membrane transplantation is a simple, safe and effective treatment to restore corneal epithelial surface and visually rehabilitate patients with partial limbal stem cell deficiency. Possible complications of limbal allografts such as rejection and immunesuppression or those of autografts such as donor site stem cell deficiency can be avoided by this procedure.

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Partial limbal stem cell deficiency; ocular surface reconstruction; amniotic membrane transplantation

© 2004 Indian Journal of Ophthalmology | Published by Wolters Kluwer – Medknow