Ординатура / Офтальмология / Английские материалы / Biomaterials and regenerative medicine in ophthalmology_Chirila_2010

8

Reconstruction of the ocular surface using biomaterials

T. V. Chirila, L. W. Hirst, Z. Barnard and Zainuddin, Queensland Eye Institute, Australia;

D.G. Harkin, Queensland University of Technology, Australia; I. R. Schwab, University of California,

Davis, USA

Abstract: This chapter discusses the effect on our vision of a large group of disorders, known as ocular surface disorders (OSDs), and presents the therapeutic strategies to reconstruct the afflicted ocular surface. If left untreated, OSDs lead to partial or total loss of eyesight. An overview

of various treatment strategies is presented, with the emphasis on the development of the ex vivo expansion of corneal limbal epithelial cells (presumed to be stem or progenitor cells) and the creation of transplantable epithelial constructs. The use of naturally derived biomaterials (collagen, fibrin, etc.) or synthetic polymers (polylactides, thermoresponsive polymers, etc.) as substrata in these constructs is critically analyzed. Emphasis is placed on the substrata from silk fibroin, currently being developed by the authors.

Key words: ocular surface disorders, corneal limbal epithelial cells, epithelial constructs, naturally derived biomaterials as substrata, synthetic biomaterials as substrata.

8.1Introduction

The quality of our vision is determined to a significant degree by the quality of the surface of our eyes, i.e. the ocular surface. A healthy, smooth and continuous ocular surface is essential for clear vision. The ocular surface is a complex entity that conceptually results from the functional integration of its anatomical components (conjunctival epithelium, corneoscleral limbus, corneal epithelium, tear film) with the adjacent structures (eyelid, eyelashes, lacrimal glands). Ultimately, the role of the ocular surface and adnexal tissues includes maintenance of corneal transparency, protection of the eye against external injury and infection, and comfort. The ocular surface is specialized to perform these functions. However, many acute, chronic or cicatrizing pathological conditions may lead to massive tissue destruction or trigger aggressive inflammatory responses from the ocular surface leading to irreversible scarring of the conjunctiva and opacification of the cornea. The spectrum of what is commonly covered by the term ‘ocular surface disorders’

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(or ‘diseases’), henceforth abbreviated as OSDs, is extensive, ranging from minor dry eye syndrome and blepharitis to potentially blinding conditions such as chemical and thermal injuries, or multiple surgeries. In an effort to classify the OSDs (Kruse, 2002), ten categories have been proposed, and more than 60 pathological conditions have been identified as OSDs, in addition to chemical, thermal, irradiation and mechanical injuries.

Of particular severity among OSDs are the limbal stem cell deficiency disorders. Epithelial corneal stem cells reside in the corneoscleral limbal region (Davanger and Evensen, 1971; Schermer et al., 1986; Cotsarelis et al., 1989; Dua and Azuara-Blanco, 2000; Kruse, 2002; Ang and Tan, 2004; Ahmad et al., 2006), and it is well established that their depletion is associated with events that lead to visual impairment or total visual loss (Dua and Azuara-Blanco, 2000; Sangwan, 2002; Ang and Tan, 2004). Experiments in animals show that the more limbal epithelium that is damaged, the more the capacity of the ocular surface for healing is reduced. When more than half of the limbal tissue was removed, the re-epithelialization was slow and resulted in a dysfunctional (usually conjunctivalized) corneal epithelium (Chen and Tseng, 1991; Huang and Tseng, 1991). This is frequently accompanied by opacity of the stroma. Conjunctivalization is described as the movement of conjunctiva-like tissue across the normal barrier of the limbus and on to the corneal surface causing pain and loss of vision. Less severe injury may result in partial limbal stem cell deficiency, when conjunctivalization of the corneal surface may not be evident or there may only be partial replacement of the corneal epithelium by conjunctival epithelium. Severe limbal stem cell deficiency involves the entire corneal surface and is associated with congenital diseases (e.g. aniridia, ectodermal displasia) (Sugar, 2002), or can be caused by chemical or thermal burns (Kim and Khosla-Gupta, 2002), iatrogenic factors such as chronic use of certain topical medication or repeated surgeries of limbal region or conjunctiva (Schwartz and Holland, 2002), inherited or bacterial keratitis, and immunological disorders (e.g. Stevens–Johnson syndrome, cicatricial pemphigoid) (Tauber, 2002). Contact lens wear, especially associated with the use of cleaning solutions and preservatives, can also cause significant stem cell loss (Sendele et al., 1983; Stenson, 1983; Bloomfield et al., 1984; Jenkins et al., 1993).

8.2Treatment of ocular surface disorders

The management of limbal stem cell deficiency is complicated, and surgery has always been, by necessity, the treatment of choice. While minor to moderate limbal stem cell deficiency may be treated medically (i.e. through observation and medication) or by surgical procedures such as debridement or removal of conjunctival tissue, surgical replacement of diseased tissue and restoration of epithelial progenitor/stem cells are essential in the treatment

of severe limbal stem cell deficiency. This requires transplantation of either autologous or allogeneic donor tissue. In the past, removal of abnormal epithelium (keratectomy) or penetrating or lamellar keratoplasty were the preferred surgical procedures, but it was soon realized that both have little chance of clinical success in the face of total limbal stem deficiency.

Keratectomy was followed inevitably by re-conjunctivalization, and keratoplasty provided a stable ocular surface lasting only for as long as the donor epithelium was present, and inevitably the surface was later covered by conjunctival epithelium (Holland and Schwartz, 2002). Subsequently, the progress in ophthalmic microsurgery and in elucidating the biology of stem cells led to the current treatments of OSDs, but such evolution has occurred in stages. Thoft reported the first conjunctival transplantation to treat chemical burns (Thoft, 1977). He used autografts from the normal eye of the same patient. Although it is unlikely that conjunctival transplantation is followed by transdifferentiation into corneal epithelium, the conjunctival autografts are still used in the management of certain OSDs that are not necessarily associated with limbal stem cell loss, such as pterygium (Hirst,

2003). Thoft was also the first surgeon to perform transplantation of donor peripheral corneal limbal epithelium with a stromal carrier from cadaveric eyes to treat bilateral chemical burns and severe atopic keratoconjunctivitis (Thoft, 1984). Presumably, some stem/progenitor cells were harvested with the transplants in certain patients, which may explain the visual improvement in a small series of patients (Holland and Schwartz, 2002).

The advances in stem cell biology had a crucial impact on the treatment of severe OSDs (Holland and Schwartz, 2002; Kim et al., 2003; Limb et al., 2006; Boulton et al., 2007; Revoltella et al., 2007), especially after the anatomical localization of corneal stem cells was established. This achievement opened the era of ‘cellular surgery’, a term coined by some investigators (Kinoshita and Nakamura, 2005) based on the fact that in such surgical procedures the ocular surface’s epithelial cells are harvested and expanded in an external environment prior to surgery. In the first clinical trial that applied this knowledge, conjunctival autografts were harvested deliberately to include cells from the corneal limbal region, and implanted in a series of 21 patients affected by some of the OSDs with the most devastating prognosis, such as chemical and thermal burns, keratopathy induced by contact lens wear and iatrogenic stem cell deficiency (Kenyon and

Tseng, 1989). The outcome was successful: healing and surface stabilization occurred in almost all cases, and visual acuity was improved in 17 cases. The limbal autograft transplantation is restricted by the amount of tissue that can be removed from the patients’ healthy contralateral eyes, as their healing can be seriously affected even when relatively small amounts of limbal epithelium are excised, and obviously it is not possible in cases of bilateral damage. Therefore, allograft transplantation techniques were also

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developed (Holland and Schwartz, 2002), where the limbal donor tissue was harvested from a living relative of the patient, or excised from cadaveric eyes. The transplantation of allogeneic tissue is restricted by the availability of suitable donor tissue and by immunological and biosafety concerns, and is generally associated with reduced clinical success because of the high rate of rejection. If this method of reconstruction of the ocular surface is to have any chance of success, it requires the administration of potent anti-rejection regimens, which can be associated with significant risks to the general health of the patient, especially considering that these treatments may be required for the rest of the patient’s life.

Transplantation of human amniotic membrane (amnion) is another strategy for the management of OSDs, an important development to which some landmark reviews were dedicated (Tseng, 2002; John, 2003; Bouchard and John, 2004; Dua et al., 2004). The amniotic membrane (AM) is the innermost layer of the placenta and one of the three foetal membranes. It consists of an epithelialized basement membrane resting on a relatively thick basement membrane and stroma. It has been used in the surgical reconstruction of a variety of tissues and organs since the beginning of the twentieth century. The first report on its use in the reconstruction of the ocular surface (conjunctiva) (de Rötth, 1940), was soon followed by reports on its transplantation in large series of patients with alkali burns (Sorsby and Symons, 1946; Sorsby et al., 1947). After a long hiatus, the modern era of AM transplantation began in the early 1990s, when Battle and Perdomo in the Dominican Republic communicated its use for the treatment of conjunctival disorders including chemical burns (Battle and Perdomo, 1993). Soon after, Tseng and his collaborators published the use of AM for ocular surface reconstruction in an animal model (Kim and Tseng, 1995). Tseng’s subsequent work in developing an adequate methodology for harvesting and preserving the membranes, as well as his further laboratory investigations and human clinical trials, provided a solid scientific and clinical foundation for the therapeutic use of AM in the management of some OSDs. It was also realized, however, that AM transplantation alone in patients with total limbal stem cell deficiency is unlikely to succeed. Consequently, limbal allografts and AM were transplanted in combination to enhance the clinical success in these cases (Tsubota et al., 1996; Tseng et al., 1998).

Some investigators believe that the success of AM transplantation relies on a series of processes potentially triggered by the presence of AM itself, including promotion of epithelialization, inhibition of conjunctival fibrosis, suppression of inflammatory cytokines and inhibition of protease activity

(Kinoshita and Nakamura, 2005). According to a recent report (Connon et al., 2006), transplanted AM can remain within the corneal tissue for long periods without being degraded and/or assimilated, but its persistence does not lead to inflammation, rejection or loss of transparency. Although the

use of AM gained enormous popularity, as illustrated in over 500 reports published by the year 2006 (Maharajan et al., 2007), there are some associated drawbacks that should not be ignored. In addition to its high cost, AM – as any human-derived tissue – is a potential vector for infectious diseases (Schwab et al., 2006). Variation in donors and harvesting or processing methods makes a qualitative standardization of the AMs available to the surgeon almost impossible, and significant variability in the mechanical properties of the commercially available AM preparations has been reported (Chuck et al., 2004). In conjunctival regeneration, the clinical success of AM transplantation is limited to several conjunctival defects (Hatton and Rubin, 2005). In many OSDs, the AM transplantation alone is not effective unless combined with transplantation of limbal epithelial stem cells or/and intraoperative topical use of mitomycin C (an anti-tumour antibiotic thought to reduce scar formation, but also with the potential for significant side effects). In addition, transplantation must be performed within days in the case of acute OSDs (e.g. burns) (Tseng, 2007). Finally, recent case studies (Maharajan et al., 2007; Saw et al., 2007) showed that in reality the AM transplantation can be associated with significant lack of clinical success.

8.3Ex vivo expansion of ocular surface epithelial cells

Localization of corneal and conjunctival stem/progenitor cells on the ocular surface was a crucial step in the development of modern strategies to treat OSDs associated with limbal stem cell deficiency. Advances in our understanding of the role and composition of the extracellular matrix led to the next crucial event: the development of an in vitro procedure to grow and propagate these cells. A method was developed (Lindberg et al., 1993) where the dissociated cells harvested from human corneal or conjunctival biopsies were serially co-cultured with γ-irradiated murine 3T3 fibroblasts (as feeder layers) in the presence of serum. This procedure made possible the creation of epithelial equivalent constructs, an alternative that is currently being investigated in many laboratories. Considering the problems encountered with the surgical approaches mentioned above, such a strategy has great promise (Nishida, 2003; Selvam et al., 2006; Shortt et al., 2007). An epithelial construct can be generated through the ex vivo expansion of human corneal limbal epithelial or conjunctival epithelial stem/progenitor cells. According to this strategy, a small tissue biopsy specimen is collected from the patient’s contralateral normal eye, if healthy, and is then either cultured as an explant or dissociated into isolated cells, which are grown in vitro while placed on (or within) a substratum (carrier). The resulting tissue construct, either as an independent sheet or attached to the substratum (which ideally should be biodegradable), is then transplanted to the site where new tissue formation is required. If

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the contralateral eye is not healthy enough for harvest, allogeneic tissue acquired from donor eyes may be expanded ex vivo and then attached to a substratum in the same fashion.

It was a few years later that De Luca and colleagues in Italy published their landmark report (Pellegrini et al., 1997) on the reconstruction of the damaged ocular surface in two patients using corneal limbal epithelial constructs expanded in vitro. Both patients (males) had severe alkali burns in one eye only, and the biopsies (1 mm2) were taken from the healthy contralateral eyes. The dissociated cells were co-cultured with murine 3T3 feeder cells in a complex growth medium containing foetal bovine serum (FBS). In preliminary experiments included in this study, cells were harvested from three different regions of the ocular surface (cadaver or consenting donor), namely bulbar conjunctiva, central cornea and limbus, and grown as described above. It was found that only the limbal cells were able to generate a stratified construct. For the autologous transplantation, grafts were prepared from confluent cultures of about 2 million limbal cells each, which were released from the culture dish and mounted on petrolatum gauze or on a soft contact lens. In one patient, the gauze was removed immediately after grafting and then the cell layer was covered with a contact lens. In the other patient, the contact lens with the cell layer on the concave side was placed directly on the eye. A stable ocular surface was achieved in both patients and maintained 2 years after grafting. In one patient, a penetrating keratoplasty was performed later and visual acuity was improved. The other patient was satisfied with the significant improvement in comfort and refused keratoplasty, an understandable attitude considering his experience with three previous failed attempts. We should point out that in this instance the epithelial cell sheets were not attached to a substratum at the time of grafting on to the damaged ocular surface. While the cells were grown on commercial tissue culture plastic, the gauze and contact lens on to which they were mounted after culturing were purely designed to make manipulation of the cell sheets easier. Although substratum-free epithelial cell constructs were successful in these two patients, and the approach was used at least in one other instance by others (Daya et al., 2005) (when the confluent cell sheet was mounted on nylon dressing prior to surgery and AM was used as a post-transplantation bandage), it is expected that the presence of a substratum (carrier) on which the limbal epithelial cells are not only grown and attached, but which is also transplanted together with the cell layer, would constitute a considerable surgical advantage. Consequently, the search for an adequate substratum in the creation of tissue-engineered constructs for the restoration of the ocular surface became part of the developing therapeutic strategies against OSDs and remains an ongoing interdisciplinary activity.

Although the conjunctival epithelial constructs have not been specifically mentioned in our exposition so far, there have been a significant number