Induction of Ocular Surface Regeneration 20

The ocular surface consists of the cornea, the con- junctiva, and the intervening transition area (the limbus) (Figure 20.1). The avascular cornea is con- tinuous with the sclera, forming together the outer envelop of the eyeball [Figure 20.1 and 20.2 (see color section)]. The transparent cornea is the gateway for the entrance of images into the eye, and accounts for more than two thirds of the total refractive power of the eye. The cornea consists of five layers: The epithelium, Bowman’s layer, the stroma, Descemet’s membrane, and the endothe- lium. The corneal transparency is essentially maintained by its avascularity, an intact epithe- lium, and a normal morphology and function of its other layers. These components of the ocular surface are essential for vision, the integrity of the eye, and for preventing ocular infections.

Injury to the ocular surface may be caused by physical or chemical agents, infectious, oculocu- taneous disorders, drugs, or systemic disorders.

A variety of physical agents may induce tissue damage: Thermal burns, microwaves, lasers, ionizing radiation. Chemical agents are a com- mon cause for severe ocular surface injury:

Acids tend to precipitate tissue proteins and cause coagulation and necrosis, thus creating a barrier against deeper penetration, and damag- ing mainly the external eye; in alkali burns, the hydroxyl ions saponify lipids in the corneal epithelium, denature proteins, and cause tissue melting and may penetrate into the deeper lay- ers. Various microorganisms may be associ- ated with damage to the ocular surface, such as herpes zoster virus that may cause a chronic

conjunctivitis with submucosal scarring, hypo- esthesia, and lid impairment caused by cica- trization. Chlamydia trachomatis (serotypes A, B, Ba, and C) is a major cause for blindness in developing countries because of the infection of conjunctival cells that initiates an inflammatory response with fibrosis of the subconjunctival tis- sue, and cicatrizing process of the external eye.

A wide range of dermatologic conditions are associated with ocular surface injury. They include mainly ocular cicatricial pemphigoid and Stevens-Johnson syndrome. In ocular cica- tricial pemphigoid, a condition with an autoim- mune origin, chronic conjunctival inflammation is progressive with exacerbations; the disease is usually bilateral and can lead to severe scarring of the conjunctiva and adherence between bul- bar and palpebral conjunctiva (symblepharon) with limitation of ocular motility and to vascu- larization of the cornea, which may progress to blindness. The ocular manifestations of Stevens- Johnson syndrome are a pseudomembranous conjunctivitis in the acute stage, and in the later phase, conjunctival cicatrization with involve- ment of the limbus and the cornea are predomi- nant. Moreover, genetic diseases such as aniridia also result in disruption of the normal ocular surface.

Ocular surface reconstruction (OSR) has recently become a common methodology in the regenerative treatment of severe ocular surface disease. The challenge in this field was moti- vated by the necessity to find a cure for patients as mentioned above, affected by severe and difficult

20

Induction of Ocular Surface Regeneration

Irina S. Barequet

281

to treat diseases that damage the integrity of the ocular surface.

The most important breakthrough in OSR came when the limbus was identified as the anatomic location of the corneal epithelial stem cells, which led to the development of various effective techniques of limbal stem cell trans- plantation. These developments further bene- fited from the realization that the tears are vital for ocular surface integrity in addition to their lubrication and optical functions, and the use of amniotic membrane (AM) as a basement mem- brane substitute and other physiologic func- tions.¹ This review will describe the various current methodologies for induction of ocular surface regeneration and its reconstruction and those in process of clinical development.

The Corneal Epithelium

The role of the corneal epithelium, which forms 10% of the total corneal thickness, is to absorb

nutrients and oxygen while protecting the eye by acting as a barrier to fluid loss and pathogen entrance. The corneal epithelium is composed of basal cells, wing cells, and stratified nonkera- tinizing squamous cells. The corneal epithelium is constantly renewing itself and regenerating.

The cells in the most superficial layer of the epithelium are continuously desquamated from the surface and replaced by proliferating cells.

The proliferation of the cells is probably lim- ited to the basal layer of the epithelium.² The basal cells rest on a basement membrane and are connected through an adhesion complex (hemidesmosomes) to the underlying connec- tive tissue stroma. Only cells that are in contact with the basement membrane have the ability for mitotic cell division, whereas cells that are displaced into the suprabasal layers become postmitotic and lose their capability for cell division.³

The kinetics of the maintenance of the corneal epithelial mass consists of a vertical movement caused by the proliferative pressure of the basal

Bulbar conjunctiva

Iris

Cornea Anterior chamber Corneo-scleral border

Zonules

Rectus Ms

Choroid

Sclera Optic nerve

Dural sheath Central retinal

artery and vein Lamina

cribrosa Rectus Ms

Posterior chamber

Lens

Ciliary body

Ora serrata

Patellar fossa

Corona

ciliaris Orbicularis ciliaris

Cloquet's canal

Retina Fovea Area of

martegiani

Vitreous

Figure 20.1.Limbal autografts, early postoperative and the donor eye. [From Basti S, Rao SK. Current status of limbal conjunctival autograft. Curr Opin Ophthalmol 2000;11:224-232.]

cell layer, and a horizontal movement from the periphery of the cornea toward its center. Also, transient amplifying cells that are found in the basal layers and are generated by stem cells (see below) have a role in corneal epithelial wound healing.³

The Limbus

The corneal limbus is the transition zone between the corneal and neighboring con- junctival epithelium. Davanger and Evensen⁴ proposed that the corneal epithelium is renewed from a source of cells located at the limbus. They observed that pigment in the epithelium in heavily pigmented eyes migrated in lines from the limbus to the central cornea in healed eccentric corneal epithelial defects.

Melanin pigmentation of the limbus pro- vides the resident cells with protection from potential damage by ultraviolet light, a sen- sible strategy for an area thought to harbor stem cells although this does not occur in Caucasians. Cotsarelis et al.⁵reported the exis- tence of slow-cycling limbal epithelial basal cells that retained tritiated thymidine label for long time periods.

Adult stem cells are defined as clonogenic, self-renewing progenitor cells that can generate one or more specialized types of cells. However, major obstacles in this field have been a lack of molecular markers to identify stem cells (most available markers identify proliferating cells rather than stem cells) and uncertainty regard- ing the precise location of the putative stem cells in vivo.

Although the stem cell itself is yet to be fully characterized, circumstantial evidence in favor of the limbal location of stem cells has been reported in the literature, and they were identi- fied as the source of corneal epithelial cells.^6,7 These stem cells are responsible for repopula- tion of the corneal epithelial cells, and maintain high capacity of self-renewal throughout the adult’s life. The stem cells are a small subpopu- lation of the total epithelial tissue and con- sist between 0.5% and 10% of the total cell population.

There is more evidence supporting the loca- tion of corneal epithelial stem cells at the limbus: In culture, limbal basal cells have the highest proliferative capacity^8–11 and surgical removal of the limbus results in delayed heal-

ing taking place by noncorneal epithelium.^12–15 It is widely believed that the maintenance of stem cells is controlled by their particular microenvironments (or “niches”),which are best thought of as clusters of environmental cues affecting the state and behavior of the cell.¹⁶

Limbal Stem Cell Deficiency

There are both primary and acquired causes of limbal stem cell deficiency in the cornea, which can be focal or diffuse, depending on the extent of limbal involvement with underlying disease processes.

Another approach to the classification of lim- bal deficiency is based on its pathogenic nature of the limbal involvement.³Category I is char- acterized by a clear pathogenic cause responsi- ble for the destruction of the limbal stem cell population. This includes chemical or thermal injury, radiation injury, Stevens-Johnson syn- drome (in which severe conjunctivitis and ker- atitis are common complications of stem cell deficiency), multiple surgeries in the limbal area, or cryotherapy applied at the limbus region. Rare causes are contact lens-induced keratopathy, lens-wearing injuries, or toxic effects from lens-cleaning solutions. Category II includes diverse causes with a dysfunction of stromal microenvironment of limbal stem cells, such as aniridia, chronic limbitis, neurotrophic keratopathy, and pterygium/pseudopterygium, or immunologic conditions, such as ocular cica- tricial pemphigoid. In this category, a milder form of corneal diseases, in which limbal stem cell dysfunction is not the result of the total loss of limbal stem cells, but rather is associated with either a gradual loss of stem cell popula- tion or poor transient amplifying cell genera- tion and amplification. Because it has not resulted from traumatic loss, the underlying pathogenesis might stem from deficient microenvironment support for limbal stem cells or transient amplifying cells, or a poor reg- ulatory mechanism. In the case of aniridia (a heritable disease in which the eye develops with a vestigial iris only), in which the stem cells are dysfunctional, such poor regulation is probably associated with microenvironmental alteration caused by the anomalous development of the adjacent angle-iris structures. Poor nutritional supply of endocrine factors and of trophic

cytokines might be the basis for the develop- ment of limbal stem cell deficiency in keratitis associated with multiple endocrine deficiency and neurotrophic keratopathy derived from a primary neuronal or ischemic component, respectively. The introduction of adverse unde- sirable cytokines secreted by chronic inflamma- tion of various etiologies might inhibit or antagonize normal regulators and create a state of limbal stem cell dysfunction. These mecha- nisms might explain the poor support of stem cell function in clinical examples of chronic limbitis and pterygium or pseudopterygium.

In each of the limbal deficiency conditions mentioned above, patients may experience severe photophobia, pain, reduced visual acu- ity, and even blindness. The common pathology in these diverse diseases is depletion of the corneal stem cell population in the limbus.

When limbal stem cell deficiency occurs, the neighboring conjunctival epithelium, which is normally prevented from encroaching on the corneal surface by the limbal cells, migrates over the corneal stroma. This process is known as conjunctivalization. This may seem to be a useful strategy to protect the stroma, except that the conjunctival epithelial cells do not share the same phenotypic properties as corneal epithelial cells. The conjunctival cells are not able to fully transdifferentiate into corneal epithelial cells and they express differ- ent proteins and keratins.^17–19 In addition, mucin-producing goblet cells of conjunctival origin are present in the epithelium covering the corneal stroma. The result is corneal opac- ity and therefore very poor vision, vasculariza- tion,¹⁹ an unstable surface prone to epithelial breakdown, irregular corneal epithelium, and patient discomfort.

These eyes are poor candidates for the conventional treatment of corneal opacities – corneal transplantation. After a standard penetrating keratoplasty in such cases, the transplanted corneal button is invariably replaced by invading vascularized tissue, which is further complicated by immunologic rejection and secondary glaucoma. This occurs because of the fact that the limbal stem cells are not part of the graft, and the corneal button contains only transient amplifying cells. Also, the preexisting corneal vascularization and inflammation increases the risk of allograft rejection, and the loss of stem cell function leads to recurrent con- junctivalization.

Ocular Surface Reconstruction

Over the past few years, OSR has become a wide- spread method in the treatment of severe ocular surface disease. A number of therapeutic strate- gies have been adopted to treat limbal stem cell deficiency, using several techniques with the same aim of restoring stem cell function. Limbal stem cell transplantation aims to replace the absent or damaged cells that are incapable of differentiating into normal corneal epithelium, in order to regenerate corneal-like epithelium.²⁰ It is required to restore the ocular surface of patients with stem cell deficiency covering the whole cornea.²¹ Penetrating keratoplasty and lamellar keratoplasty in these eyes have recently regained their status as a surgical tool to provide the essential clarity of the cornea, only in con- junction with limbal transplantation.

The major breakthroughs in OSR were: 1) the identification of the limbus as the anatomic loca- tion of corneal epithelial stem cells²²; 2) the real- ization that tears are not only for lubrication but that many physiologic components of tears are vital for ocular surface integrity^23,24; and 3) the use of AM as a basement membrane substitute,²⁵as well as provider of other physiologic functions.

Moreover, restoration of the adnexal anatom- ical and functional integrity also has an impor- tant role in the long-term reconstruction of the ocular surface. Thus, lid morphology and the proper alignment of the lid margin are impor- tant to the tear meniscus.

Initially, the term OSR was used synony- mously with limbal, or stem cell, transplantation.

The cases presented in the literature varied greatly, ranging from mild forms of stem cell deficiency, such as aniridia, to severe cases in the form of Stevens-Johnson syndrome. Early attempts were made to restore changes in the ocular surface by limbal transplantation.²⁶After years of follow-up, certain factors that had to be addressed came to the attention of anterior seg- ment surgeons. There was a need to standardize terminology used to describe OSR techniques.

Holland and Schwartz²⁷proposed a classification of the surgical techniques in order to standardize the nomenclature. This classification is based not only on the carrier tissue of the limbal stem cells (conjunctiva or cornea) but also on the origin of the tissue (autograft or allograft). They thus sep- arated limbal transplantation into the following four categories: Conjunctival limbal autograft,

living-relative conjunctival limbal allograft, cadaveric conjunctival limbal allograft, and cadaveric keratolimbal allograft (Table 20.1).

Amniotic Membrane Transplantation

The amniotic membrane (AM) constitutes the inner wall of the fetal membranes, and consists of a single layer of epithelium with an underlying stroma rich in extracellular matrix and collagens.

Although first used in the 1940s, this method was abandoned, and then reintroduced for ophthalmic use in 1995 by Kim and Tseng.²⁵In a rabbit model, 40% of corneas with total limbal deficiency could be reconstructed by replacing the conjunctivalized surface with a preserved human AM. Since then, AM has proven to be an integral part of OSR. AM can easily be obtained from seronegative mothers undergoing routine Cesarean delivery. The AM is usually used after cryopreservation, but fresh AM seems to work as well.²⁸

The AMT can be used for several indications, either as a graft to replace the damaged ocular surface stromal matrix or as a patch (dressing) to prevent unwanted inflammatory insults from gaining access to the damaged ocular surface, or a combination of both. Reports indicated that potential action mechanisms might include reduction of inflammation, vascularization and scarring, and facilitation of epithelialization.

Compositionally, the basement membrane of the AM resembles that of the conjunctiva. The basement side of the membrane can act as a sub- strate for supporting the growth of epithelial

progenitor cells by prolonging their lifespan and maintaining their clonogenicity. This may sup- port the idea of using AM transplantation (AMT) to expand the remaining limbal stem cells and corneal transient amplifying cells during the treatment of partial limbal deficiency²⁹ and to facilitate epithelialization for persistent corneal epithelial defects with stromal ulceration.^30–32In tissue cultures, AM supports epithelial cell grown from explant cultures^33–35 or other cul- tures,^36,37and maintains their normal epithelial morphology and differentiation.^34,35The result- ant epithelial cells–AM can be transplanted back to reconstruct the damaged corneal surface in humans³⁸and in rabbits.³⁶The AM can also be used to promote non-goblet cell differentiation of the conjunctival epithelium.³⁴

The stromal side of the membrane contains a unique matrix component that suppresses transforming growth factor β signaling, and proliferation and myofibroblast differentiation of normal human corneal and limbal fibrob- lasts³⁷ and of normal conjunctival fibroblasts and pterygium body fibroblasts.³⁹ This may explain the scar reduction during conjunctival surface reconstruction,^40,41 recurrent scarring prevention after pterygium removal,^37–46 and corneal haze reduction after phototherapeutic keratectomy and photorefractive keratec- tomy.^47,48 Although such an action is more potent when fibroblasts are in contact with the stromal matrix, a lesser effect is also noted when fibroblasts are separated from the membrane by a distance,³⁷suggesting that some diffusible fac- tors might also be involved besides the insoluble matrix components in the membrane.

Table 20.1. Classification of surgical procedures for the management of severe ocular surface disease

Procedure Abbrev Donor Transplanted tissue

Conjunctival transplantation

Conjunctival autograft CAU Fellow eye Conjunctiva

Living-related conjunctival allograft lr-CAL Living relative Conjunctiva Limbal transplantation

Conjunctival limbal autograft CLAU Fellow eye Limbus/conjunctiva

Cadaveric conjunctival limbal allograft c-CLAL Cadaveric whole globe Limbus/conjunctiva Living related conjunctival limbal allograft lr-CAL Living relative Limbus/conjunctiva

Keratolimbal allograft KLAL Cadaverie stored tissue Limbus/cornea

Ex-vivo expanded limbal autograft EVELAU Fellow eye Ex-vivo expanded limbal cells Living related ex-vivo expanded limbal allograft lr-EVELAL Living-relative Ex-vivo expanded limbal cells Amniotic membrane transplantation AMT Stored human amniotic membrane Human amniotic membrane Source: Holland EJ, Schwartz GS. The Paton lecture: ocular surface transplantation: 10 years’ experience. Cornea 2004;23:425-431.

Several growth factors have been identified in the AM.⁴⁹The stromal matrix of the membrane can also exclude inflammatory cells by stimulat- ing them into rapid apoptosis⁴⁸ and contains various forms of protease inhibitors.⁵⁰This can explain the reduction of stromal inflammation after AMT^29,30and corneal neovascularization,⁵¹ actions important for preparing the stroma for supporting limbal stem cells to be transplanted either at the same time or later.29,44,52–55

AMT can facilitate epithelialization, maintain normal conjunctival epithelium phenotype (with goblet cells when performed on conjunc- tiva⁵⁶), and reduce inflammation, vasculariza- tion, and scarring. Based on these therapeutic effects, one can envision that AMT can be used for conjunctival surface reconstruction to restore normal stroma and provide a healthy basement membrane for renewed epithelial pro- liferation and differentiation. Several reports showed that AMT can be used to reconstruct the conjunctival surface as an alternative to con- junctival graft after removal of large conjuncti- val lesions such as pterygium,^42–46conjunctival intraepithelial neoplasia and tumors,⁴⁰scars and symblepharon,^43–45 and conjunctivochalasis.⁵⁷ These results indicate that the reconstructed area can be very large so long as the underlying bed is not ischemic and the bordered conjunc- tiva has a normal conjunctival epithelium and subconjunctival stroma.

AM acts as a basement membrane allowing the migration of epithelial cells over areas of bare sclera, and can avert impending perfora- tion of the cornea. A report by Chen et al.³¹ shows the efficacy of AMT as a substrate in the treatment of neurotrophic ulcers of the cornea.

More than 70% of patients in this series healed by AMT, after a mean follow-up of 18.8 months.

The significance of focusing on neurotrophic ulcers is the fact that these patients present one of the most difficult cases to manage. The suc- cess of AMT in neurotrophic ulcers leads one to speculate that humoral factors of AM origin may also be involved in the healing process.

Segments of AM can also be used as a filling in localized stromal deficiencies, even when accompanied by perforation.³² Small segments of AM in this case are stuffed under an overlying layer of AM that acts as a basement membrane.

This procedure can also be done with the use of surgical adhesion glue.^58,59 The possibility of patching a perforation with AM can save the eye in many ways. Institutions without the immedi-

ate availability of donor tissue can buy time before performing a therapeutic keratoplasty.³²

The AM has been used as a graft in adjunction to limbal stem cell transplantation,^20,26,29 intended to restore the damaged limbal stromal environment, as a support to restoration of the limbal stem cell population. Reported clinical experience showed that this combined approach is effective in treating various extents of limbal deficiency according to the following parame- ters: The extent of limbal deficiency, presence or absence of the central corneal transient amplify- ing cells, and depth of central corneal involve- ment.⁴¹AMT is an important adjunct in limbal transplantation for both transplanted limbal stem cells to expand on the recipient eye and the residual stem cells to expand in the donor eye.

Partial limbal deficiency can be reconstructed by AMT without the use of limbal transplanta- tion.⁶⁰This result first observed in rabbit exper- iments at the time when no explanation was available,²⁵ indicates that partial limbal defi- ciency can be treated without long-term use of immunosuppression with oral cyclosporin.

AM can also be applied to treat corneal sur- face diseases as a graft. When used as a graft or a patch, AM can promote healing of persistent corneal ulcers from different causes including neurotrophic keratopathy caused by various underlying etiologies,^30,31,44and band keratopa- thy.^61,62This approach is superior to conjuncti- val flaps or tarsorrhaphy because it preserves a cosmetically more acceptable appearance.

AM can also be used as a patch in a temporary or prolonged manner. Experimentally, when used as a patch on a temporary basis, this mem- brane has been shown to reduce corneal haze after photorefractive keratectomy or photother- apeutic keratectomy,^46,62 an effect verified in human patients.^48,64As a temporary patch, AM can reduce inflammation, facilitate epithelializa- tion, and prevent scarring caused by acute chemical burns in a rabbit model⁴⁴ and in human patients.^65,66AM as a patch was also used successfully in the acute stage of Stevens- Johnson syndrome⁵² and to suppress refrac- tory inflammation in various ocular surface disorders.⁶⁷

The fact that the AM can help preserve and expand limbal epithelial stem cells indicates that it can also be used as a carrier to expand them in in vitro culture. This new approach is applicable to those patients with limited limbal reserve or who are concerned about having a large part of

the healthy limbus removed from the fellow eye or from a living-related donor. In this case, a small limbal biopsy will be performed and the sample will be placed on the AM and appropri- ately cultured. Within 3 to 4 weeks, such an ex vivo expanded culture together with the AM can then be transplanted to restore the normal corneal surface on limbal deficient corneas. The feasibility of this new approach based on an autologous source has been demonstrated in a short-term rabbit study³⁸ and in long-term human patients.^36,68,69This new approach paves the way to use AM as a tissue engineering sub- strate and may open up new therapeutics by incorporating gene therapies in the future.

There are certain limitations to AMT because this is a substrate transplantation and thus can- not be used to treat ocular surface disorders that are characterized with a total loss of limbal epithelial stem cells or conjunctival epithelial stem cells. Because AMT still relies on the host tissue to supply epithelial and mesenchymal cells, it cannot be used to reconstruct the ocular surface that has severe aqueous tear deficiency, diffuse keratinization,⁵⁵ absence of blinking in severe neurotrophic state, and stromal ischemia.

If not overcome, these conditions present as contraindications for AMT.

Limbal Autografts

In cases of unilateral limbal stem cell deficiency, a limbal conjunctival autograft (CLAU) can be harvested from the healthy eye. The transplanta- tion limbal tissue from the fellow eye, using adjacent conjunctiva as the carrier tissue, was first reported by Kenyon and Tseng²⁰ in 1989.

CLAU has become the popular choice in the treatment of unilateral limbal deficiency.

Reports continue to support CLAU as the treat- ment of choice for unilateral disease such as chemical and thermal burns^70,71 [Figures 20.3 (see color section)].

The principles of the standard procedure is to transplant two segments of conjunctival limbal tissue at the 12 and 6 o’clock positions, mainly because these areas are protected by the lids, and are often the sights of conjunctival invasion.

The procedure is performed under general or bilateral retrobulbar and/or topical anesthesia.

In the injured eye, a 360˚ conjunctival peritomy 2 mm posterior to the limbus is performed. Bare

scleral dissection to the limbus is performed, and the ring of tissue is removed. This is fol- lowed by removal of abnormal corneal epithe- lium and vascular tissue (pannus). The donor limbal epithelium is harvested from the nonin- jured fellow eye. Two grafts, each with about a 4–clock hour circumferential length, are taken.

The two incisions, conjunctival and corneal, are then joined by a radial incision at each end. The graft extends 0.5 mm onto clear cornea and 2 mm onto the bulbar conjunctiva. It therefore includes limbal stem cells. The donor site is left open and heals rapidly. The donor tissue is transplanted to the injured eye. It is sutured to the cornea. The donor tissue size of two 4–clock hour circumfer- ence provides a sufficient number of stem cells to the injured eye and avoids limbal deficiency in the donor eye. Postoperative care consists of top- ical antibiotics, steroids, cycloplegics, and non- preserved artificial tears [Figure 20.3 (see color section)]. The presence of limbal tissue may act as a physical barrier against invading tissue. Dua et al.³⁸recommended that any invading conjunc- tiva after CLAU be removed, so that corneal epithelium of donor origin may migrate to reep- ithelialize the entire cornea.

The use of CLAU for recurrent pterygia⁷² is also effective in preventing recurrence. However, whether stem cells are in fact required in this case is still debatable. Healthy limbal tissue in pterygia patients is usually sufficient in provid- ing transient amplifying cells to cover for resected pterygium tissue.

Although CLAU cannot be used in bilateral disease, conjunctival limbal allograft tissue from living relatives (lr-CLAL ) is an alternative method, especially when the same human leuko- cyte antigen haplotypes are available (i.e., sib- lings).⁷³ Although there are no incidents of limbal dysfunction after procurement of donor tissue from a healthy eye, caution is required in cases with unilateral chemical burns, because claims that only one eye was inflicted may not be entirely true. Removal of limbal tissue from a partially stem cell-deficient eye may cause irre- versible damage.

The most significant advantage of CLAU is the abolishment of any risks of immunologic rejection. However, persistent inflammation of the ocular surface cause by the original disease or surgical trauma can also cause loss of donor limbal tissue, and care must be taken to control the original disease and inflammation in these patients.

Limbal Allografts

In cases of bilateral disease, a living relative may provide healthy stem cells (lr-CLAL).^73,74 However, reports of studies in the rabbit eye have shown that the removal of two thirds of the limbal zone can result in delayed epithelial heal- ing, vascularization, and conjunctival epithelial ingrowth.⁷⁶Because of this potential complica- tion and because a healthy contralateral con- junctiva is not always available, allograft limbal transplantation with cadaver eyes may be con- sidered.⁷⁷ In such cases, either a conjunctival limbal allograft (c-CLAL) or a keratolimbal allo- graft (KLAL) is performed.⁷⁸

Conjunctival limbal allograft⁷⁹ is technically similar to conjunctival limbal autograft, except for the need for a living donor⁸⁰or a cadaver.⁸¹

Keratoepithelioplasty was first described by Thoft.⁸² In this technique, four lenticules of peripheral corneal epithelium with superficial corneal stroma are harvested from a fresh donor eye. Originally, limbal tissue was not harvested with the lenticules. Only later was the technique modified to include limbal tissue.^83,84The lentic- ules are secured to the corneoscleral limbus on the recipient eye 90˚ apart. Limbal transplant from cadaveric donors (KLAL) is the treatment of choice in bilateral disease, and can restore a cornea epithelial phenotype in approximately 50% to 70% of cases.⁸⁵In KLAL, cadaveric lim- bal tissue is transplanted by using the peripheral cornea as carrier tissue, and therefore the proce- dure has two specific advantages over other lim- bal stem cell techniques: Stored donor tissue is readily available, and because three separate 180˚ segments of limbal tissue are used, KLAL affords the largest number of transplanted lim- bal stem cells compared with any other limbal stem cell transplantation technique. Although encouraging results have been reported,⁸⁶ dry eye and preoperative conjunctival keratiniza- tion were initially associated with poor results after keratolimbal allografts.⁸⁶

With allografts, either from a living relative or from cadaver eyes, the possibility of rejection and/or infection must be considered. The com- bination of allograft limbal transplantation, AMT, treating severe conjunctival scarring and limbal stem cell deficiency in cases of cicatricial pemphigoid and Stevens-Johnson syndrome was reported.⁸⁷ However, longer follow-up in these patients showed that many of the severe

patients with Stevens-Johnson syndrome and ocular cicatricial pemphigoid, are at risk of ves- sel invasion and conjunctivalization after months of a seemingly smooth course. Chronic deterioration of donor stem cells can be caused by a number of factors, many of which still need to be addressed. Discontinuation of immuno- suppression may cause rejection of grafts (see below, immunotherapy). Lid deformities and dry eye may inflict chronic mechanical damage leading to persistent epithelial defects and stem cell depletion. Recurrence of original disease may cause nonspecific inflammation, triggering any number of events leading to graft failure.

The limbus acting as a physical barrier may also be important, because a single-piece lamellar keratoplasty (LKP) and KLAL graft (large graft) had less success than a two-piece or a two-stage procedure (KLAL and LKP).^88,89There is a con- troversy regarding the staging of the proce- dure. Ilary and Daya⁹⁰ reported no difference in KLAL survival whether it was performed simultaneously with a keratoplasty or as a later procedure. However, KLAL combined with ker- atoplasty seemed to have a shorter survival time than KLAL followed by keratoplasty.

Holland and Schwartz⁹¹suggested waiting at least 3 months after an epithelial transplantation before considering a corneal graft to allow stabi- lization of the transplanted epithelial tissue.

Conversely, Rao et al.⁷³ advocated a combined approach with the rationale of avoiding the need for a second procedure and preserving donor transient amplifying cells. Because successful epithelial transplantation often obviates the need for keratoplasty, Daya and Ilari⁹² recommended waiting at least 1 year before performing a kerato- plasty, and, in cases in which there is a normal endothelium, a deep lamellar keratoplasty is preferable in their opinion.

Holland et al.⁹³reported the results of KLAL in aniridia (Figures 20.4 and 20.5); the outcome seemed improved if surgery was performed ear- lier in the disease process – specifically, before vision was impaired from irreversible stromal opacification. Because aniridic keratopathy involves only the corneal epithelium in the early stages, KLAL alone may be sufficient for visual rehabilitation when performed on younger aniridic patients. However, if an aniridic patient is merely observed through young adulthood, the epithelial keratopathy will typically lead to stro- mal scarring, and the patient will likely need PK in addition to KLAL to restore baseline visual acuity.

The identification of allograft rejection is of high priority because failure to diagnose rejec- tion will compromise graft survival. Daya et al.⁹⁴ showed pathologic findings of clinically diag- nosed KLAL rejection. Donor limbal segments removed during a second KLAL showed infiltra-

tion of T cells, and loss of corneal epithelium- like cells that express cornea-specific keratin.

Clinical signs associated with these pathologic findings were intense sectoral injection, perilim- bal conjunctival injection, limbal edema, and cellular infiltration of KLAL grafts. These signs are followed by persistent epithelial defects, ves- sel invasion, and keratinization. It is not known whether rejection in KLAL occurs via a similar mechanism as penetrating keratoplasty. The inci- dence of rejection does not need to involve both tissues at the same time, and has been observed to occur in only one of the grafts. In fact, immuno- logic rejection to the central graft seems to be higher when accompanied by KLAL.⁹⁵

The usefulness of Cyclosporine A as an immunosuppressant agent has shown conflict- ing results. The rationale for the use of immuno- suppressants is to increase graft survival rate by decreasing progressive destruction of limbal stem cells from acute or chronic allograft rejec- tion. Acute allograft rejection rate as reported by others varies from none⁸² to 30%,⁹⁶ and in this study it was 39.4%. There is no consensus regarding specific immunosuppressive regi- mens after KLAL. Systemic Cyclosporine A was used in more severe cases and in higher doses where there was recurrent inflammation. In the study published by Ilari and Daya,⁹⁰ no differ- ence in KLAL survival was found between patients treated or not treated with long-term Cyclosporine A, primary failure excluded.

However, there was a higher rate of acute allo- graft rejections in patients treated with oral Cyclosporine A as compared with the patients not receiving Cyclosporine A, and this probably reflected patient selection for using oral Cyclosporine A. In their study, although there were fewer episodes of acute rejection in the group not receiving Cyclosporine A, KLAL sur- vival was shorter (13.5 months compared with 22 months). This possibly reflects a process of chronic low-grade rejection as suggested by Daya et al.⁹⁴and Holland and Schwartz,⁹¹which may be prevented or delayed by the use of Cyclosporine A.

In addition, management of all other aspects of the patient’s ocular health is essential to ensure the best opportunity for allograft survival (Table 20.2). The presence and severity of glaucoma need to be elucidated, because it is important that the management of intraocular pressure be stable before limbal allograft is performed. Holland and Schwartz⁹⁷ recommended aggressive and early

7.5 mm 1 mm

Scleral rim

a

b

c 1/3

2/3

Figure 20.4. Diagram of key steps in preparation of tissue for keratolimbal allograft and a recipient eye with three keratolimbal crescents sutured in place. b The central corneal button is removed with a trephine. c The remaining limbal ring is divided into two 180˚ cres- cents. d The crescents are thinned by removing the posterior two-thirds of the corneoscleral tissue via sharp dissection. [Reprinted from Holland EJ, Djalilian AR, Schwartz GS. Management of aniridic keratopathy with keratolimbal allograft: a limbal stem cell transplantation technique.

Ophthalmology 2003;110:126–127, Figures 2 and 3. With permission from the American Academy of Opthalmology.]

a

Patient's limbus

Donor limbus

d

placement of a tube shunt in patients receiving more than one topical glaucoma medication. The rationale for this aggressive approach is that an increase in intraocular pressure after limbal allo- graft is quite common. In addition, multiple top- ical medications and their preservatives can be toxic to the transplanted epithelial surface. Next, the status of the eyelids and lashes are evaluated.

Surgical correction of existing exposure, lagoph- thalmos, and ectropion as well as aggressive management of trichiasis and distichiasis must be performed before limbal allograft. Failure of the ocular surface secondary to nonimmune inflammation can occur from exposure and trauma secondary to misdirected eyelashes.

Aggressive management of preoperative inflam- mation is the next factor that must be considered and aggressively managed before limbal allograft.

Limbal allografts that are transplanted into an inflamed ocular surface have a significantly poorer prognosis than those in which the inflam- mation has been minimized. Therefore, topical

and systemic immunosuppression are initiated weeks to months before limbal allograft to achieve the greatest chance for success. Once the glaucoma is stabilized, the lid anatomy is restored, and the ocular inflammation is reason- ably controlled, a limbal transplantation tech- nique will be performed. The selection of which ocular surface procedure to be used is based on several factors. If the patient has unilateral dis- ease, Holland and Schwartz recommended CLAU as the procedure of choice because this procedure does not run the risk of failure secondary to immune rejection. For patients with bilateral dis- ease, the choice is between KLAL and lr-CLAL.

For the majority of patients with limbal defi- ciency without extensive conjunctival disease, the authors advocated KLAL, because of the avail- ability of cadaver donor tissue as well as the increased quantity of stem cells available for transplantation (18 clock hours of limbus).

However, if the patient has extensive conjunctival disease, they recommended lr-CLAL procedure,

Figure 20.5. Slit lamp photograph of severe aniridic keratopathy, prior and 1 year after keratolimbal allograft. [Reprinted from Holland EJ, Djalilian AR, Schwartz GS. Management of aniridic keratopathy with keratolimbal allograft: a limbal stem cell transplantation technique. Ophthalmology 2003;110:126–127, Figures 1 and 4.]

because it provides much needed healthy con- junctival cells in addition to limbal tissue. More recently, we have combined KLAL and lr-CLAL in patients with the most severe ocular surface dis- ease to maximize the advantages inherent in each procedure. In those patients in whom a stable ocular surface has been obtained, consideration of a subsequent keratoplasty can be entertained. If the patient has significant stromal scarring with good endothelial function, a lamellar keratoplasty should be considered. In patients with stromal and endothelial disease, penetrating keratoplasty is often required for visual rehabilitation.

Stem Cell Therapy

As an alternative to limbal grafting, corneal stem cell therapy may be considered for some patients. The aims of stem cell therapy are to promote reepithelialization of the cornea,

provide stable corneal epithelium, prevent regression of new vessels, and restore epithelial clarity. A pioneering approach published by Rheinwald and Green⁹⁸that optimized the pro- duction of cultured cutaneous epithelium suit- able for grafting burn patients has been successfully adopted for the culture of multilay- ered corneal epithelium.⁹⁹ Pellegrini et al.¹⁰⁰ have shown that this can be produced for graft- ing of corneal patients with unilateral limbal stem cell deficiency. The discovery that cultured limbal cells include stem cells, detectable as holoclones (clones that are derived from human epithelial stem cells and that have high prolifer- ative potential), permitted the development of limbal cultures for the treatment of patients with a partial deficiency of limbal stem cells. Limbal epithelial cells were obtained from a small biopsy specimen from a healthy area of the patient’s cornea; after culturing, these cells developed into corneal epithelium, which was successfully transplanted back into the patient.

Further results have shown clinical improve- ment of the corneal surface after application of cultured autologous corneal epithelial cells.⁶⁸ Recent work has also shown the potential for using limbal tissue stored in eye banks as a source of cells for producing cultured corneal epithelial allografts.¹⁰¹ Providing the cultured epithelium with a basement membrane is likely to improve graft “take” and may even promote survival of any cultured stem cells, allowing them to establish themselves in the host stem cell niche. Tseng et al.¹⁰²have developed a tech- nique in which epithelial cells from a limbal biopsy are explanted directly onto AM in cul- ture. After 2 to 3 weeks, the composite graft is then ready for the patient. Significant improve- ments in corneal clarity and surface stability have been achieved using this technique. This technique of ex vivo expanded limbal transplan- tation provides a novel method for transplanta- tion of either an autograft (EVELAU), using the patient’s own limbal stem cells, or from a living- related donor (lr-EVELAU).

Tissue Engineering

The next stage in OSR is the identification of corneal epithelial stem cells and the transplanta- tion of bioengineered tissue, including isolated stem cells. Because the potential markers for

Table 20.2 Algorithm for an approach to treat patients with severe ocular surface disease

1. Management of glaucoma

a. Tube shunt for patients on more than 1 topical medication 2. Correction of eyelid and eyelash abnormalities

a. Exposure: lagophthalmos, ectropion

b. Misdirected lashes: entropion, trichiasis, distichiasis 3. Suppression of inflammation

a. Topical corticosteroids and cyclosporin A b. Systemic immunosuppression

i. Oral corticosteroids ii. Tacrolimus or cyclosporin A iii. Mycophenolate or azathioprine 4. Ocular surface transplantation

a. Conjunctival limbal autograft (CLAU) for unilateral disease b. Keratolimbal allograft (KLAL) for bilateral limbal deficiency

with minimal to moderate conjunctival disease c. Living-related conjunctival limbal allograft (lr-CLAL) for

bilateral limbal deficiency with moderate to severe conjunc- tival disease

d. Combined conjunctival-keratolimbal allograft (C-KLAL) for bilateral limbal deficiency with severe conjunctival disease 5. Keratoplasty

a. Lamellar (LK) for patients with stromal opacification with normal endothelium

b. Penetrating (PK) for patients with stromal opacification with loss of endothelial function

Source: Holland EJ, Schwartz GS. The Paton lecture: ocular surface transplantation: 10 years’ experience. Cornea 2004;23:425-431.

stem cell identification have been reported,^103,104 the next challenge will be to isolate these cells, and to maintain cell cultures with the potential to provide an unlimited stock of undifferenti- ated cells. Culture conditions can be modified to induce these cells to differentiate as required.

Stem cells may be further engineered for low immunogenicity by the induction of genes, and perhaps express genes that will help these cells proliferate.

The AM has a thick basement membrane that is able to support the growth of corneal epithe- lial cells.³⁶This will allow AM to be used as a car- rier for transplanting cultured cells, and in fact, animal studies³⁵and clinical reports^38,68,105show that cultured epithelial transplants using AM substrate are effective in selected cases. However, the long-term results of this procedure are unknown, and yet we still await the demonstra- tion of the stem cell itself. There is no guarantee that these transplantable sheets of epithelium contain stem cells, even though the cells may be of limbal origin. An entirely different strategy will be required for developing epithelial trans- plants to be used as a temporary graft, and those that are intended to seed stem cells.

A possible alternative is under development of natural and/or synthetic biopolymers that will support cellular components of the cornea. An in vitro model of such design is already a real- ity,¹⁰⁶and further studies in polymer design and cell cultures may someday produce a trans- plantable artificial cornea.

Summary

Visual function requires an intact ocular sur- face. The integrity of this surface is maintained in humans by two highly specialized epithelia – the conjunctival epithelium and the limbal corneal epithelium. Although anatomically con- tinuous with each other at the corneoscleral lim- bus, the two cell phenotypes represent quite distinct subpopulations. A population of ker- atinocyte stem cells in defined locations governs the renewal of these stratified epithelia. Stem cells for the cornea reside at the corneoscleral limbus. Corneal stem cells are segregated in the basal layer of the limbus, the transitional zone between the cornea (the transparent part of the ocular surface)and the bulbar conjunctiva (which covers the white part of the ocular

surface). The microenvironment of the limbus is considered to be important in maintaining the stemness of stem cells. These stem cells generate transient amplifying cells that terminally differ- entiate after a discrete number of cell divisions.

Limbal stem cells also act as a “barrier” to con- junctival epithelial cells and normally prevent them from migrating onto the corneal surface.

Under certain conditions, however, the limbal stem cells may be partially or totally depleted, resulting in varying degrees of stem cell defi- ciency 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 remain- ing intact limbal epithelium. In total stem cell deficiency, several surgical techniques have been developed. Conjunctival transplantation procedures can be either autografts or allografts, depending on the source of donor tissue. A con- junctival autograft (CAU) uses tissue from the fellow or same eye. A conjunctival allograft can use donor tissue from a cadaver or living relative and be designated as a cadaveric conjunctival allograft (c-CAL) or living-related conjunctival allograft (lr-CAL). Limbal transplantation pro- cedures can be subdivided based on the donor and the carrier tissue. A conjunctival limbal autograft (CLAU) uses tissue from the fellow eye, and conjunctiva is the carrier. A cadaveric conjunctival limbal allograft (c-CLAL) uses a cadaveric donor for conjunctiva and limbus.

A living-related conjunctival limbal allograft (lr-CLAL) is a procedure in which a living rela- tive donates conjunctiva and limbal tissue. A ker- atolimbal allograft (KLAL) utilizes a cadaveric donor, and peripheral cornea is used to transfer the limbal stem cells. With the latter option, sys- temic immunosuppression is required: The initial systemic immunosuppression protocol consisted of oral prednisone, cyclosporine A, and azathio- prine. Later, the protocol was changed to oral prednisone, tacrolimus, and mycophenolate.

Ex vivo expanded limbal transplantation is the newest technique to provide a source of donor limbal tissue. With this technology, lim- bal tissue from a donor is expanded in culture before transplantation. In ex vivo expanded