Indian Journal of Ophthalmology

: 2000  |  Volume : 48  |  Issue : 2  |  Page : 83--92

Limbal stem cell deficiency : Concept, aetiology, clinical presentation, diagnosis and management.

HS Dua, JS Saini, A Azuara-Blanco, P Gupta 
 Department of Ophthalmology, Queens Medical Centre, Nottingham, UK

Correspondence Address:
H S Dua
Department of Ophthalmology, Queens Medical Centre, Nottingham, UK


Defects in renewal and repair of ocular surface as a result of limbal stem cell deficiency are now known to cause varying ocular surface morbidity including persistent photophobia, repeated and persistent surface breakdown and overt conjunctivalisation of the cornea. Ocular conditions with abnormalities of ocular surface repair include pterygium, limbal tumours, aniridia, severe scarring following burns, cicatricial pemphigoid and Stevens-Johnson Syndrome, sequelae of mustard gas exposure and Herpes simplex epithelial disease, radiation keratopathy, contact lens induced keratopathy, neuroparalytic keratitis and drug toxicity. Restoring ocular health in these eyes has traditionally been frustrating. An understanding of these intricate cell renewal and maintenance processes has spurred the evolution in recent years of new treatment methods for several blinding diseases of the anterior segment; many more exciting modalities are in the offing. However, there is inadequate awareness among ophthalmologists about the current principles of management of ocular surface disorders. The purpose of this article is to help elucidate the important principles and current treatment methods relevant to ocular surface disorders.

How to cite this article:
Dua H S, Saini J S, Azuara-Blanco A, Gupta P. Limbal stem cell deficiency : Concept, aetiology, clinical presentation, diagnosis and management. Indian J Ophthalmol 2000;48:83-92

How to cite this URL:
Dua H S, Saini J S, Azuara-Blanco A, Gupta P. Limbal stem cell deficiency : Concept, aetiology, clinical presentation, diagnosis and management. Indian J Ophthalmol [serial online] 2000 [cited 2021 Sep 20 ];48:83-92
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The first surface in the eye to interact with the environment is the epithelial lining covering the cornea, lids and scleral surface. In normal health this layer apparently heals and renews itself so fast and so completely that it was thought to be of no importance. However, we now know that very complex and intricate mechanisms help maintain the normal physiological health of this layer and the turnover of cells. A smooth and uninterrupted epithelium associated with a stable tear film, clear stroma with homogenous arrangement of collagen fibres, and functioning endothelium are essential for clear vision. The corneal epithelium also serves as a part of the ocular biodefense system. Its cells are tightly arranged without intercellular spaces, creating a barrier that is relatively impermeable to water and external substances. The corneal epithelium undergoes a constant process of cell renewal and regeneration. Cells in its uppermost layer are continuously desquamated and lost into the tear film and must be replaced by cell proliferation. Therefore it is endowed with a proliferative reserve in the form of multipotent stem cells located in the basal limbal epithelium.[1-4] The limbal stem cells serve as a proliferative barrier between corneal and conjunctival epithelia. Conditions that significantly damage the limbal stem cells can result in an invasion of conjunctival epithelium on to the corneal surface (conjunctivalisation). This process of conjunctivalisation results in a thickened, irregular, unstable epithelium, often with secondary neovascularisation and inflammatory cell infiltration. Epithelial defects are common in the conjunctivalised corneal surface and may lead to corneal ulceration, scarring, and loss of vision.

This article reviews the conditions that result in ocular surface disease from a loss of limbal stem cells, their diagnosis and management.

 The "Ocular Surface"

The term "ocular surface" describes the entire mucosal epithelial lining bordered by the skin at the superior and inferior eyelid margins.[5] This includes the epithelium from the mucocutaneous junction of the eyelid margin on to the back of the lids, into the and its reflection back over the globe, including that which covers the cornea[6] [Figure:1]. Histologically and physiologically, the epithelial surface has two major areas of cornea and conjunctiva separated by the limbus.[5]

The corneal epithelium is composed of nonkeratinised, stratified squamous epithelial cells. Its thickness is approximately 50 μm (five or six layers of cells).[7-10] The most superficial layer of the epithelium consists of flattened cells with microvilli. Coating these microvilli is a layer of glycocalyx that interacts with the mucous layer of the tear film and affords tear film stability. Below this surface there is a layer of polyhedral wing cells, two to three cells thick. The deepest layer of the epithelium, the basal layer, consists of a single layer of cylindrical cells anchored to the basement membrane and adjacent Bowman's zone.[10] The tear film protects the cornea from dehydration and maintains the smoothness of the epithelial surface. The tear film also serves as the source of the regulatory factors for the maintenance and repair of the corneal epithelium.[7-10] The conjunctival epithelium is 1-2 cell layers thick, with goblet cells intermixed with nongoblet epithelial cells.

At the corneo-scleral limbus there is a gradual transition from the stratified, nonkeratinised squamous epithelium of the cornea to the stratified, nonkeratinised columnar epithelium with mucin-secreting goblet cells of the conjunctiva. It has 7-10 layers of cells, which have attachments similar to the corneal cells. The architecture of the limbus demonstrates a palisade (of Vogt) arrangement.[11,12] The limbal stem cells probably reside in the basal layer of the palisades of Vogt.[13]

When there is a demand for tissue regeneration, the stem cells divide. When a stem cell divides, one of the daughter cells remains as a parent and serves to replenish the stem cell pool, whereas the other daughter cell is destined to divide and differentiate, acquiring features that characterise the corneal epithelium. Such daughter cells are called "transient amplifying cells" and are distributed in the corneal basal cell layer. The transient amplifying cells migrate centripetally and after a high but limited number of mitoses, they further differentiate to "post-mitotic cells" which do not divide any further. These post-mitotic cells ultimately differentiate into terminally differentiated cells which die after a certain time [Figure:2].[2,3,14] The stem cells are located exclusively in the limbal basal epithelium. The occurrence of transient amplifying cells is limited to the basal layer of limbal and corneal epithelia. Post-mitotic and terminally differentiated cells make up the superficial layers of limbal and corneal epithelia.[14]

Thoft and Friend[1] proposed a "X, Y, Z hypothesis of corneal epithelial maintenance" in which the desquamated cells (Z component) are continuously replaced not only by the basal cells (X) that divide but also by cells that migrate in from the periphery (Y). Thus, migration occurs centripetally and circumferentially from the limbus and vertically from the basal layer forwards. The kinetics of maintenance of corneal epithelium is confirmed in corneal epithelial wounding animal models.[14-16] These experimental studies produced a spectrum of corneal surface abnormalities characterised by conjunctival epithelial ingrowth (conjunctiva-lisation), vascularisation, and chronic inflammation, which indicate limbal stem cell dysfunction. The conjunctival source of the epithelial ingrowth was proved by immunofluoresccnt staining with monoclonal antibodies and by detection of goblet cells with impression cytology. Several observations indicate that these cell turnover kinetics are true in humans as well.[9-11] Support for the limbal location of corneal epithelial stem cells is also derived from the following observations: centripetal migration of epithelial cells during corneal epithelial wound healing,[17-23] circumferential migration of limbal epithelial cells during limbal wound healing,[24] abnormal corneal epithelial wound healing when the limbal epithelium is partially[25-26] or completely[14,27,28] removed, and by the evidence that limbal basal cells have a higher proliferative potential in culture.[29]

The differential expression of keratins provides direct evidence that basal limbal epithelium contains the unique and least differentiated cells of corneal epithelium.[3,30] Keratin 19 (CK 19) is expressed over the entire limbal and corneal epithelium in the human foetus but only in the basal limbal epithelium in the adult, suggesting the persistence of embryogenetically young (stem) cells at the limbus in adults.[31,32] Further evidence for limbal basal location of the less differentiated stem cells is provided by the distribution of keratin 3 (CK 3) which is the differentiation keratin, CK 3 is demonstrable in the entire corneal epithelium and suprabasal limbal epithelium but not in the basal epithelium of the limbus.[3] Evidence that the basal epithelium contains cells (stem cells) that proliferate has come from studies employing short-term labelling (such as tritiated thymidine[4]) and exposure to the tumour promoter TPA (12-O-tetradecanoylphorbol-l-3-acetate[33,34]) and the antimetabolite 5-FU.35 These studies confirm that the limbal epithelium contains a population of slow-cycling cells that display the characteristics of stem cells. Limbal stem cells are shown to be abundant in the superior and inferior limbus as compared with the temporal and nasal limbus.[36]

 Aetiology of Limbal Stem Cell Deficiency

Limbal stem-cell deficiency can be primary, related to an insufficient stromal microenvironment to support stem cell function, such as aniridia, congenital erythrokerato-dermia, keratitis associated with multiple endocrine deficiencies, neurotrophic (neural and ischaemic) keratopathy and chronic limbitis; or secondary (more common) related to external factors that destroy limbal stem cells such as chemical (most common) or thermal injuries, Stevens-Johnson Syndrome, ocular cicatricial pemphigoid (OCP), multiple surgeries or cryotherapies, contact lens wear, or extensive microbial infection.[2,14,16,37] Corneal stem-cell deficiency can be diffuse (total) or sectoral (partial). In the latter case conjunctivalisation of the corneal epithelium affects only part of the corneal surface. In some patients, limbal deficiency may be subclinical at the time of the insult, and may eventually progress to an overt stage of limbal deficiency as the stem cell population depletes further, over time.[15] If the conjunctival stem cells (presumed to be in the fornices)[38] are also depleted, which is rare because of the larger area they occupy (presumed to be the fornices),[39] the ocular surface eventually is covered by totally keratinised epithelium, which sometimes may be seen at the end stage of OCP or Stevens-Johnson syndrome.

 Clinical Presentation and Diagnosis of Limbal Stem Cell Deficiency

The symptoms of limbal deficiency may include decreased vision, photophobia, tearing, blepharospasm, and recurrent episodes of pain (epithelial breakdown), as well as a history of chronic inflammation with redness. The biomicroscopic findings at slitlamp examination may include a dull and irregular reflex of the corneal epithelium which varies in thickness and transparency. Severe malfunction of limbal stem cells may result in an ingrowth of thickened fibrovascular pannus, chronic keratitis, scarring and calcification. Conjunctivalised corneal surfaces are frequently stained abnormally by fluorescein because conjunctival epithelium is more permeable than corneal epithelium.[27] The conjunctivalised corneal surface always shows a stippled late staining pattern with fluorescein, is thinner than adjacent normal corneal epithelium, is irregular, prone to recurrent erosions and attracts new vessels.[15,39] In partial stem cell deficiency, a clear line of demarcation is often, but not always, visible between corneal and conjunctival phenotype of cells. At the line of contact of the two phenotypes, tiny "bud like projections" of normal corneal epithelium can be seen extending into the conjunctivalised area, and fluorescein dye tends to pool on the conjunctivalised side of the line of contact, because of its relative thinness.[24,25]Persistent epithelial defects, melting and perforation of the cornea can occur in patients with stem cell deficiency.

Corneal stem cell deficiency can be best confirmed histologically by the use of impression cytology[40] which can detect goblet cells containing conjunctival epithelium on the corneal surface. Immunohisto-chemically, the absence of a cornea-type differentiation (such as the absence of keratin CK3), and the presence of mucin in goblet cells, has been shown by monoclonal antibodies.[41] Diagnosis of limbal stem cell deficiency is crucial because patients with this abnormality generally are poor candidates for conventional corneal transplantation. Lamellar or penetrating keratoplasty provides only a temporary replacement of the host's corneal epithelium because the grafted epithelial cells have a limited proliferative capacity and lifespan.

 Management of Partial Stem Cell Deficiency

Asymptomatic patients with partial and peripheral conjunctivalisation of the corneal surface may not require intervention. Corneal and conjunctival epithelial cell phenotypes have been known to co-exist on the corneal surface for prolonged periods without significant extension of the conjunctivalised area or any transdifferentiation of conjunctival epithelium into cells of corneal phenotype.[15,39] If the visual axis or most of the corneal surface is covered with conjunctiva-like epithelium, mechanical debridement of conjunctival epithelium can allow adequate corneal epithelial healing to occur from the remaining intact limbal epithelium.[15,39] Scraping is done with a surgical blade under topical anaesthesia at the slitlamp. Any tendency of conjunctiva-like epithelium to re-encroach on to corneal surface is prevented by re-scraping.[15] This procedure can be employed to improve visual function and reduce symptoms even when as little as two clock hours of normal limbus and peripheral cornea remain. When visual improvement is the aim of treatment, the objective should be to achieve normal corneal epithelial cover over the visual axis. An attempt to achieve normal epithelial cover for the entire cornea, when only two clock hours or less of limbus are surviving, may stretch the capacity of the remaining limbus and could eventually lead to epithelial breakdown.

Mechanical debridement of conjunctiva-like epithelium and encouraging the denuded area to be resurfaced with corneal epithelial cells is a valid, simple and effective alternative to limbal transplantation in patients with partial limbal stem cell deficiency (see below).

Mechanical debridement can also be used to prevent migration of conjunctival epithelium on to the cornea in acute situations, with partial corneal and limbal epithelial loss. Close observation of patients with thermal, chemical or mechanical epithelial loss involving the limbus, will allow one to detect the advancing conjunctival epithelial sheet and prevent it from extending on to and beyond the limbus.[15] Tseng et al42 have successfully used amniotic membrane transplantation (AMT) and mechanical debridement and advocate its use to treat patients with partial stem cell deficiency. Their study showed that with partial or focal limbal stem cell deficiency, AMT improves both the corneal surface and the vision.

 Management of Total Stem Cell Deficiency

In patients with total limbal stem cell deficiency, limbal auto- or allo-transplantation are indicated for corneal surface reconstruction. This may be combined with or followed by keratoplasty. The current technique was proposed by Kenyon and Tseng,[43] but several modifications have been described. All these procedures aim to transplant a new source of epithelium for a diseased ocular surface and the removal of the host's altered corneal epithelium and pannus. After successful transplantation, the host's cornea (or grafted cornea) will be permanently covered by epithelium from the donor limbus.

Although all techniques used in stem cell transplantation are in principle similar, the source of. donor stem cells can vary. Donor tissue can be obtained from the fellow eye (limbal autograft) in cases of unilateral disease, or from a living related donor (usually gives a better tissue match), or from a cadaver donor (limbal allograft) when both eyes are affected. Limbal transplantation procedures also vary depending on the carrier tissue used for the transfer of the limbal stem cells. Carrier tissue is needed in limbal transplants because it is not possible to transfer limbal stem cells alone. Limbal transplant procedures have used either conjunctiva (conjunctival limbal graft) or cornea (keratolimbal graft) as a carrier tissue for limbal stem cells.[44,45] Tseng et al[42] used AMT associated with limbal transplantation in cases with total stem cell deficiency. In the past decade some authors have used conjunctival transplantation to treat corneal stem cell deficiency. This practice was (and perhaps is) supported by the belief that conjunctival epithelium "transdifferentiates" into cornea-like epithelium.[46,47] However, we believe that conjunctival epithelial transdifferentiation (that is, acquisition of morphologic, biochemical and physiologic transplantation properties of corneal epithelium) does not occur in humans and conjunctival transplantation to reconstruct the corneal surface in stem cell deficiency is inferior to limbal transplantation.[37] Conjunctival transplantation is, however, useful in other conditions, for example, to reconstruct the conjunctival surface in cases of symblepharon and to treat primary and recurrent pterygia.

Keratoepithelioplasty was proposed by Thoft[49] as another alternative to reconstruct the ocular surface in patients with conjunctivalisation of the corneal surface. In this technique lenticules of. peripheral corneal epithelium with superficial stroma, were grafted. A few years later the same author modified the technique to include limbal tissue, acknowledging the importance of stem cell transplantation in these conditions.[48,49] The use of cultured limbal epithelium is being explored in several laboratories, including our own, but is at present primarily investigative.[50]

Successful limbal transplantation can achieve rapid surface healing, stable ocular surface without recurrent erosions or persistent epithelial defects, regression of corneal vascularisation and restoration of a smooth and optically improved ocular surface, resulting in improved visual acuity and, probably, increased success for subsequent keratoplasty.

 Surgical Technique for the Auto-limbal Transplantation

In patients with unilateral total stem cell deficiency a limbal autograft transplantation is recommended. Partial removal of the limbus from the fellow eye is believed to be relatively safe, although some cases may have compromised donor surface after partial removal of the limbal zone. The risk of epithelial problems in the donor eye is low when less than four to six clock hours of limbal tissue and a moderate amount of conjunctiva are removed.

In our current technique for autologous limbal transplantation the donor tissue is prepared by harvesting two conjunctival-corneal-scleral specimens of approximately two clock hours each, in circumferential length, taken from the superior and inferior limbal zones of the donor eye. Each graft, approximately 150 μm thick (to include approximately 100 μm of stroma and 50 μm of epithelium), extends approximately 2 mm into bulbar conjunctiva, 1 mm into limbus and 2 mm into peripheral clear cornea.

To prepare the recipient eye, the abnormal corneal epithelium and the superficial vascularised scar is stripped off by blunt dissection. A conjunctival incision is done to expose the limbus and perilimbal sclera at the location where the donor tissue will be grafted. A bed, 100 μm deep, and corresponding in clock hours to the harvested donor tissue is prepared to receive the donor explants. The donor tissue is then sutured on to the recipient site with interrupted 10-0 nylon sutures at the corneal and scleral margin [Figure:3].

The donor site can either be allowed to remain open or covered with a bandage contact lens. Preservative-free topical antibiotics and corticosteroids, cycloplegics and unpreserved lubricants may be used as needed until re-epithelialisation is complete and postoperative inflamation resolved.

In the illustrative series by Kenyon and Tseng,[43] clinical results of 21 patients with follow up of 6 months or more showed improved visual acuity (17 cases), rapid surface healing (19 cases), stable epithelial adhesion without recurrent epithelial defect (20 cases), arrest or regression of corneal neovascularisation (15 cases) and probable increased success for lamellar or penetrating keratoplasty (8 cases). No adverse reactions developed in donor eyes. Results from other small series of limbal auto transplants in various clinical situations have also been reported.[51-54]

 Surgical technique for allo-limbal transplantation

When patients have bilateral total ocular surface disease, allograft transplantation becomes necessary. If living relatives are potential donors, an HLA-matched tissue is preferred. When cadaver donor tissue is used, "fresh" eyes are preferred because the success of the procedure depends on the transplantation of healthy limbal stem cells. Whole eyes are convenient because they provide better stabilisation during dissection of the lirnbal sclerocorneal rim. The donor eye is inflated with air (1 to 2 ml), injected through the stump of the optic nerve, to make the globe firm. The globe is wrapped around with a strip of wet gauze and heid on a Tudor Thomas stand. We use a vacuum trephine, with a diameter 3 mm smaller than the corneal diameter to trephine the donor central cornea into approximately one-fifth of the stromal depth. Superficial lamellar dissection of the peripheral cornea is then carried out with a bevel-up angled dissecting knife, and extended into the sclerocorneal junction and 1 mm beyond, into sclera. Approximately 1-2 mm of donor conjunctiva, if present, is maintained. The ring of limbal tissue thus harvested is cut open.The anterior edge of the "open ring" (with donor peripheral corneal tissue + limbus + episclera) is placed at the host limbus. Therefore, the donor limbus is slightly posterior to the host limbus, increasing the length of the circumference to be covered. The limbal tissue is sutured with interrupted 10-0 nylon sutures at the corneal and scleral margin. The corneal sutures are passed first. Scleral sutures are passed directly opposite the corneal sutures and tightened to take up any slack in the corneal sutures. This method invariably leaves a small gap (approximately 5 to 8 mm) between the two ends of the donor tissue ring. This can be filled with a "spacer" fashioned out of donor corneal stroma or a piece of donor limbal tissue, cut to size, harvested from the other eye of the same donor, if available [Figure:4].

Tseng et al[55] described an alternative technique employing a 360 cadaveric donor corneolimbal ring graft trephined from a corneoscleral button. This ring was secured to the surrounding conjunctival edge with 9-0 or 10-0 interrupted vicryl sutures and to the denuded corneal surface with a running 10-0 nylon suture.

Postoperative treatment consists of preservative-free topical antibiotic and prednisolone 0.5% four times daily for the first two weeks. Autologous serum eye drops probably help promote epithelial healing. We use them hourly until the epithelialisation is completed. To prepare serum eye drops, 50 ml of blood is obtained by venipuncture and centrifuged for 5 minutes at 1,500 rpm. A 20% solution of serum is then prepared with sterile saline within a laminar flow hood and placed into sterile vials, in 5 ml aliquots. The vials are kept frozen at -20? C and a fresh vial is thawed every day. Frequent preservative-free artificial tears and highly viscous methylcellulose four times daily are also used. Steroids are rapidly tapered in autologous limbal transplantation. In allo-limbal transplantation, longterm low-dose topical steroids are maintained. During the early postoperative period the limbal explant is carefully monitored for any areas of epithelial loss. Conjunctival epithelium can cross the explant at these sites and gain access to the corneal surface. This is particularly true if a stromal spacer is used. The spacer provides only a limited barrier to conjunctival encroachment. If conjunctival encroachment is apprehanded or observed, mechanical debridement of conjunctival cells from the explant, spacer or even the corneal surface should be promptly carried out.

In limbal allografts the surface disorder can recur if there is immunological destruction of the transplanted limbal stem cells. A high rate of immune reactions can be expected because of the high immunogenic stimulus of the limbal transplant, related to the relative abundance of Langerhans cells and HLA-DR antigens. They play an important role in the afferent arm of allograft rejection, and effective immunosuppression is considered essential for at least 12 months after surgery when non-HLA matched limbal allografts are used. In some instances permanent systemic immunosuppression may be needed. Oral cyclosporine A is the most commonly used agent.[55-57] In addition to oral cyclosporine, Tsubota et al[56] also used topical cyclosporine (0.05%) and high dose intravenous dexamethasone in their patients. The use of FK 506 (Tacrolimus, Fujisawa Ltd), a new immunosuppressive agent from the fermentation broth of Streptomyces tsukabaensis, for immunosuppression in limbal or corneal allografts has been recently reported by Dua et al.[58] This agent has immunosuppressive activities similar to and more potent than cyclosporine. All patients must undergo thoracic radiography, blood tests (renal function, full blood counts), trough levels of the drug, urine tests and blood pressure determination before and during the course treatment. The involvement of clinical immunologists in the management of these patients is highly desirable and recommended. The immunosuppressive dosage can be increased or decreased if it proves ineffective or causes adverse effects, respectively. Limbal rejection can be suspected with the development of inflammation and/ or acute or chronic severe surface abnormalities. Daya and Dua[59] recently reported the clinical features of limbal allograft rejection. In their study, the clinical featurses of limbal allograft rejection varied according to the presentation, whether acute or chronic, of the rejection episode. Acute rejection was associated with intense sector injection at the limbus, oedema and infiltration of the lenticule, punctate keratopathy and epithelial defects. In low-grade rejection there was mild diffuse or perilimbal injection, elevated perilimbal area, punctate epithelial keratopathy and epithelial irregularity

Tsubota et al[56] performed limbal allografts in nine patients (3 with chemical injury, 2 with moderate ocular cicatricial pemphigoid, 1 with traumatic limbal deficiency and 2 with limbal deficiency of unknown aetiology) and observed that corneal epithelium was completely reconstructed in all patients although two showed partial increased fluorescein permeability and two others required a second surgery. Epithelia of the five other patients remained clear at a mean follow up of 12.3 months, with two episodes of graft rejection which were controlled successfully by systemic steroids and oral cyclosporine A.

Tan et al[57] reported successful restoration of the ocular surface in seven of nine cases at a mean followup 14.7 months. One patient suffered graft failure related to microbial keratitis whereas one patient had an acute rejection episode after early cessation of oral cyclosporin.

 Amniotic Membrane Transplantation

Amniotic membrane transplantation (AMT) was first used by Kim and Tseng[60] for corneal surface reconstruction in a rabbit model of total limbal deficiency. Tsubota et al[61] later described use of amniotic membrane with limbal allograft transplantation in patients with ocular cicatricial pemphigoid and Stevensen-Johnson Syndrome. Amniotic membrane grafts have also been used as an alternative to conjunctival flaps in treating persistent and refractory corneal epithelial defects and ulceration.[62] The procedure has been used to create a limbal barrier in pterygium surgery[63] and for conjunctival surface reconstruction following excision of tumours, scars and symblepharon.[64]

The amniotic membrane is a thick basement membrane and avascular stromal matrix. Lee and Tseng[62] theorise that these features are crucial to successful transplantation. The basement membrane facilitates migration of epithelial cells,[65] reinforces adhesion of basal epithelial cells[66,67] and promotes epithelial differentiation.[68] The basement membrane is also important to prevent apoptosis.[55] As only the substrate without cells is employed, there is no adverse reaction of rejection.

Human amniotic membrane is prepared and preserved according to the method originally described by Lee and Tseng[62] and by Tseng and Prabhasawat et al.[55] Human placenta is obtained shortly after an elective caesarean section delivery from an individual pre screened for HIV, hepatitis, and syphillis. Placenta is cleaned of blood and washed with sterile phosphate buffered saline solution containing penicillin G 50 mg/ ml, streptomycin 50 mg/ml, neomycin 100 mg/ml and amphotericin B 2.5 mg/ml. The amnion is separated from the rest of the chorion by blunt dissection and flattened onto a nitro-cellulose paper with the epithelial basement membrane surface facing away from the paper. The paper with adherent amniotic membrane is then cut into 3x4 cm sheets and stored before transplantation at -800C in a sterile vial containing Dulbecco modified Eagle medium and glycol at a ratio of 1:1 (vol./vol.). The recipient eye is prepared the same way as for other surface reconstruction procedure that is peritomy, excision of perilimbal conjunctiva for 5-7 mm and superficial keratectomy. Preserved amniotic membrane is transferred to cover the recipient eye surface defect with the basement side surface facing up. The membrane is anchored to the surrounding tissues with 10-0 nylon sutures. It is then covered by a bandage contact lens. In a study employing amniotic membrane with and without limbal allograft transplants and penetrating keratoplasty, Tseng et al[55] demonstrated that in eyes with chemical burns (n=14); Stevensen Johnson Syndrome, toxic epidermal necrolysis or pseudopem-phigoid (n=5); contact lens induced keratopathy (n=3); aniridia (n=3); multiple surgical procedures (n=2); atopy (n=2); and unknown cause (n=2), all amniotic membrane covered eyes (except for two eyes with atopy) showed rapid epithelialisation (2-4 weeks) and reduced inflammation, vascularisation and scarring. For the mean follow up of 15.4 months, 25 of 30 eyes showed visual improvement ranging from 1 to 6 lines. Coneal graft rejection occurred in 9 of 14 eyes and reversible early limbal allograft rejection in 3 of 21 eyes. They concluded that AMT alone is sufficient for partial limbal deficiency with superficial involvement and is superior to allo-limbal transplantation (ALT) since it is not necessary to administer cyclosporine. ALT, however, is needed for total limbal deficiency, and AMT in this situation helps reconstruct the perilimbal stroma with reduced inflammation and vascularisation.

Lee and Tseng[62] performed AMT in 11 eyes for persistent epithelial defects with ulceration and obtained successful reepithelialization in 10 of 11 eyes. One patient's wound failed to heal because of preexisting corneal perforation pursuant to severe rheumatoid arthritis.

The judicious evaluation and management of ocular surface disorders is a challenging task. Currently available therapeutic options are able to rehabilitate the ocular surface in a reasonably large proportion of eyes. Ongoing research into the regulatory mechanism of limbal stem cells may open up exciting frontiers leading to an enhancement of our therapeutic armamentarium in successfully managing these disorders.


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