Prelude to corneal tissue engineering – Gaining control of collagen organization

Abstract

By most standard engineering practice principles, it is premature to credibly discuss the “engineering” of a human cornea. A professional design engineer would assert that we still do not know what a cornea is (and correctly so), therefore we cannot possibly build one. The proof resides in the fact that there are no clinically viable corneas based on classical tissue engineering methods available. This is possibly because tissue engineering in the classical sense (seeding a degradable scaffolding with a population synthetically active cells) does not produce conditions which support the generation of organized tissue. Alternative approaches to the problem are in their infancy and include the methods which attempt to recapitulate development or to produce corneal stromal analogs de novo which require minimal remodeling. Nonetheless, tissue engineering efforts, which have been focused on producing the fundamental functional component of a cornea (organized alternating arrays of collagen or “lamellae”), may have already provided valuable new insights and tools relevant to development, growth, remodeling and pathologies associated with connective tissue in general. This is because engineers ask a fundamentally different question (How can that be done?) than do biological scientists (How is that done?). The difference in inquiry has prompted us to closely examine (and to mimic) development as well as investigate collagen physicochemical behavior so that we may exert control over organization both in cell culture (in vitro) and on the benchtop (de novo). Our initial results indicate that reproducing corneal stroma-like local and long-range organization of collagen may be simpler than we anticipated while controlling spacing and fibril morphology remains difficult, but perhaps not impossible in the (reasonably) near term.

Introduction

The term “tissue engineering” is thought to have been officially coined and defined in its modern sense by Robert Nerem in1988 at the Lake Granlibakken NSF workshop on the topic of engineering tissue (Viola et al., 2003). However, the concept was brought to widespread attention and formalized in a review paper in Science in 1993 (Langer and Vacanti, 1993) which paraphrased the definition: Tissue engineering is an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain or improve tissue function.

In the subsequent years since the field of tissue engineering was formalized, there has been an enormous scientific effort to produce tissue constructs for clinical use (Vacanti, 2006). The success of the approach has thus far been modest in relation to the expenditure of time and resources (Nerem, 2006) with few tissue engineered constructs approved for clinical use in the US (the most successful of these being artificial skin). The myriad limitations of current tissue engineering methods are enumerated by Dr. Vacanti in a recent article in the journal Tissue Engineering (Vacanti, 2006). Nonetheless, tissue engineering in general does hold great promise and the potential for producing tissue engineered replacements for diseased or injured corneas in the reasonably near term does exist. This is because corneal tissue is reasonably “simple”, thin and avascular (Ko et al., 2007). However, all tissues are extremely complex at some level (in the cornea it is at the level of matrix molecule organization). Thus, it is critical that our approach to this endeavour be circumspect and tempered with the appropriate scientific skepticism.

The term “engineering” as it relates to tissue engineering. An engineer is defined as “One who contrives, designs, or invents” (first definition, OED second edition 1989). In the sense of this definition, the term “tissue engineering” seems a perfectly reasonable description for what has been attempted in the field since its inception. However, in practice, the gerund “engineering” carries the implication that a particular task can be accomplished given the currently available knowledge base, adequate funding and adequate manpower. Indeed professional engineers usually have a large toolbox of established principles (i.e. boiler codes, steam tables, mechanical property indices, etc. for mechanical engineers) from which to draw upon to produce rationally designed solutions to practical problems. Unlike practitioners of most engineering disciplines (mechanical, chemical, civil and electrical), tissue engineers have a relatively empty toolbox at the moment. It is thus no surprise that things are progressing slowly and have been largely empirical in nature (because we are busy filling the toolbox!). Unfortunately, some declared attempts to produce “engineered” constructs as solutions to complex and poorly understood physiological/pathological problems are very premature. The problem resides with the semantic connotation of the term “tissue engineering”, which carries the implication of “doability” and may create expectations that cannot be met. The consequences of this semantics “gap” between what most tissue engineers actually do and what individuals outside the field expect of them have yet to be fully realized (and may be dire with regard to future funding of the field). Certainly, what our laboratories have been doing in the past few years is not engineering corneas or even corneal stromas. We have been relegated to “filling the toolbox” for future corneal tissue engineers.

Corneal disease and/or injury is the second leading cause of vision loss and affects more than 10 million people worldwide (Whitcher et al., 2001). Trachoma is the leading cause of corneal blindness in the third world (4.5 million people) followed by corneal injury (1.5 million people). The vast majority of these cases might benefit from a suitable corneal replacement (provided there was adequate follow up care – which is no small concern). In the developed world, conditions which indicate the use of donor corneal grafts include Fuch's dystrophy, keratoconus, pseudo and aphakic bullous keratopathy, corneal stromal dystrophies, corneal scarring and herpes simplex virus. There were approximately 33,000 corneal transplants per year performed in the United States as of the year 2000 (Aiken-O'Neill and Mannis, 2002). Though the donor cornea potential far outstrips the actual need of grafts, there are legitimate cultural, logistical and technical difficulties associated with the procurement of quality donor graft material. Under the best conditions, donor grafts are typically variable in quality and usually fail due to immunological rejection or endothelial decompensation resulting in an 18% failure rate for initial grafts (Thompson et al., 2003). In addition, LASIK surgery constitutes a rising threat to the number of viable donor corneas (LASIK procedures disqualify donor tissue for transplantation).

Therefore, aside from the academic challenge, there is appreciable clinical interest in the development of a suitable replacement for donor graft material. The cornea is a fairly simple, avascular, sparsely populated and multilaminar structure, which would seem to be an attractive target for tissue engineers. The fact that the cornea is thin and avascular is encouraging as it does not appear that significant diffusion limitations (a particularly vexing problem for tissue engineers in general (Vacanti, 2006)) will hamper the effort. However, the cornea is more complex than is apparent at first glance. There are three generally different cell types (epithelial, fibroblasts/keratocytes and endothelial) which require successful and compatible culturing techniques if a cornea is to be constructed by tissue engineering methods. In addition, at the heart of the cornea lies a complex, highly organized stromal extracellular matrix which provides the principal functions of the corneal tissue. Before the design of any engineered replacement for a tissue (or for any device) can begin, it is important to fully understand the function (and structure) of what it is that is to be replaced.

As part of the design process for the engineering of a tissue replacement, it is first necessary to attain a detailed understanding of the function that is performed by the native tissue in the context of its physiological environment. Our focus for this article is the corneal stroma. Nonetheless, it is instructive to review the functional role that the stroma plays within the whole cornea and the functional role of the cornea within the visual system. We will start with a brief review of corneal structure and function. There are three principal design requirements that the natural cornea must satisfy: (1) protection of the fragile intraocular contents, (2) transparency to visible light and (3) formation of a nearly perfect optical interface to refract light onto the retina. Over the millennia, Nature has evolved an elegant structure which is capable of simultaneously meeting all three of these critical design criteria.

The cornea comprises a highly structured, membrane bound but relatively acellular, transparent collagenous tissue that joins the more disorganized and opaque sclera at the limbus. The diameter of the human cornea is about 12 mm and the average radius of curvature of the central anterior surface is 7.8 mm. The cornea is roughly 520 μm thick at the center and 650 μm thick in the periphery. It is bounded anteriorly by a stratified squamous epithelium and posteriorly by an actively pumping monolayer of non-proliferative cells which are referred to as the corneal endothelium. At the heart of the cornea is the stromal tissue which comprises 90% of the total thickness where the three principal design requirements of the cornea (protection, transmission and refraction) are simultaneously satisfied by the long- and short-range extracellular matrix organization.

The three major functional layers of the cornea are the epithelium, stroma and endothelium. Fig. 1 shows the orderly structure of the corneal tissue in cross-section.

Epithelium. The corneal epithelium is a 50 μm thick, “tight”, stratified, squamous, multilaminar epithelium comprising three distinct cellular strata. The stratum germinatum is the posterior most layer and the only epithelial cell layer capable of undergoing mitosis (Hanna and O'Brien, 1960). The middle layer comprises daughter or wing cells which are pushed anteriorly during epithelial desquamation. The surface layer of squamous cells forms the complete tight junctions which generate the primary chemical and antigen protective barrier in the cornea. This continual desquamation of the corneal epithelium depends on a stable supply of stem cells which reside in limbal niches at the junction between the sclera and the cornea (Schermer et al., 1986). A healthy epithelium has 5–7 layers of cells and rests on a basement membrane comprising laminin, type IV collagen, identifiable hemidesmosomes and anchoring fibrils.

That the cornea requires a functional epithelium is demonstrated by pathological conditions which chronically disrupt the ocular surface mucosa (ocular cicatricial pemphigoid, Stevens–Johnson syndrome, etc.), disrupt tear production (Sjogren's) or by injuries which destroy the stem cell niches in the limbus (severe chemical or alkali burns). Losing the epithelial barrier typically results in corneal opacity and the loss of the surface air/tear interface for refraction. Natural or tissue engineered grafts cannot succeed without a functional epithelium.

Nonetheless, we assert that the epithelium is there to protect the stroma from invasion (and to maintain the tear/air interface). It is the stromal architecture that produces the necessary aberration-free curvature to generate the refractive power. In addition, given the significant advances in corneal epithelial culturing onto various substrates (Zieske et al., 1994), and the fact that donor corneas (which are de-epithelialized) gain epithelial coverage from the host, the epithelium is not currently a critical concern for corneal tissue engineers. It is important to mention the fact that there is a considerable effort underway to innervate engineered corneal constructs (Li et al., 2003, Li et al., 2005). The rationale for this approach is derived from data which suggest that denervation of the corneal stroma leads to poorly formed epithelium without proper cellular stratification (Alper, 1975).

Endothelium. The corneal endothelium is a transporting monolayer of about 400,000 hexagonal cells, 20 μm across and 4–6 μm in height. The endothelium maintains corneal transparency by keeping the corneal stroma in a state of relative deturgescence via a complex pump–leak mechanism ((Maurice, 1972) for pump discovery and see (Bonanno, 2003) for extensive review of endothelial ion transport). Without the endothelium the cornea has a natural tendency to imbibe fluid which can cause swelling, opacity and blindness. This dehydrating mechanism is part of a sophisticated corneal transport system which includes both limiting layers and is described in detail in Ruberti and Klyce (Ruberti and Klyce, 2002). As with the epithelium, an engineered construct that is perfectly mimetic of the natural stroma could not function without a patent, active endothelial layer. However, it may be quite possible to design a collagen-based cornea that does not swell by incorporating an analog to the sutural fibers in the dogfish (Smelser, 1962). Nonetheless, an endothelium is a far greater concern for tissue engineers than the epithelium, as it has been refractory to expansion in culture and the host endothelium will not effectively repopulate and deturgesce a bare donor stromal graft. Recently, researchers have been able to cultivate untransformed endothelial cells and expand them multiple times in a specialized culture medium (Engelmann et al., 1999, Engelmann et al., 2004, Joyce and Zhu, 2004, McAlister et al., 2005). Given these relatively successful parallel efforts associated with culturing and expanding endothelial cells in vitro, the endothelium does not appear to present a “significant” hurdle to corneal tissue engineering effort at this time. Ultimately, continued efforts to expand primary endothelial cells in culture will be critical to the success of engineered corneas.

Stroma. The adult human stroma is approximately 500 μm thick, relatively acellular (3–10% quiescent corneal keratocytes by volume), and comprises aligned arrays of hydrated type I/V heterotypic collagen fibrils (15% wet weight) of uniform diameter (32 ± 0.7 nm) (Meek and Leonard, 1993); glycosaminoglycans (GAGs) keratan sulfate and dermatan sulfate (1% wet weight (Anseth, 1961)); various proteoglycan (PG) core proteins (Axelsson and Heinegard, 1975) and other protein constituents including fibronectin, laminin and type VI collagen (among other collagens). The collagen fibrils are packed in 300–500 parallel arrays (lamellae) which are generally parallel to the corneal surface (Hamada et al., 1972) and are principally responsible for the observed tensile mechanical properties of the cornea (reviewed in Ethier et al. (2004)). The PGs and their associated GAGs contribute to the cornea's compressive and swelling material properties (Hedbys, 1961) and to the uniform spacing of the collagen fibrils (Scott, 1991).

We believe that the corneal stroma should be the current focus of researchers attempting to produce a corneal tissue analog and we have concentrated on this part of the cornea for a number of reasons: First, there have been very few concerted efforts aimed at reproducing the architecture of a natural corneal stroma (Orwin et al., 2003, Crabb et al., 2006a, Crabb et al., 2006b, Guo et al., 2007), while there have been multiple and relatively successful attempts to culture the limiting cell layers of the cornea (on a stromal scaffolding) (Minami et al., 1993, Zieske et al., 1994, Germain et al., 1999, Griffith et al., 1999, Griffith et al., 2002, Li et al., 2003, Germain et al., 2004, Li et al., 2005) fully reviewed in Ruberti et al. (2007). Second, the corneal stroma provides the majority of the principal functions of the corneal tissue. No corneal analog will work without a mechanically strong and clear, properly shaped stroma. Third, the stroma is extremely organized on the nanoscale making it a very difficult and interesting basic materials engineering problem. Finally, because of the highly organized nature of the stromal matrix, successful reproduction of the architecture requires detailed examination of in vivo/in vitro matrix assembly (from development to scar resolution) and the development of novel engineering methods to control collagen fibrillogenesis. Such methods have broader implications for connective tissue remodeling, homeostasis and pathology.

Note on the tear film: The tear film is a complex, multicomponent structured ocular surface coating which provides both a smooth optical interface and protection from pathogens. It is the secretary product of three-separate systems (conjunctival goblet cells, meibomian glands and lacrimal glands) none of which are part of the cornea proper. Therefore we will not consider the tear film in this chapter; although we note that it is a critical component of the ocular surface, without which neither donor corneal grafts nor tissue engineered replacements would survive.

The native cornea provides three fundamental functional attributes (which we consider essential design requirements for an artificial corneal construct) to the ocular optical system: protection, transmission and refraction of the incident light to the retina. The mechanisms by which the tissue simultaneously meets all three of these requirements are discussed below.

The cornea provides both transport protection (in the form of a barrier) and mechanical protection.

Transport protection. The transport of deleterious chemicals and pathogens is impeded by the tight junctions of superficial squamous cells of the corneal epithelium (Sugrue and Zieske, 1997). Thus, any natural, stromal analog should be inductive for the migration of epithelial cells from the host peripheral corneal tissue and support the formation of a multilaminar, adherent epithelium with complete tight junctions (as donor corneas do (Boot et al., 1991)).

Mechanical protection. Protection of the fragile intraocular contents is provided by the tensile properties of the stromal extracellular matrix. The tensile mechanical strength of the tough ocular tunic (of which the cornea is a continuous part) must be high enough to withstand chronic tensile stress induced by the intraocular pressure (IOP) and permit survival of significant traumatic impacts without rupture. The overall biomechanical properties of the cornea proper are complex because the stromal tissue is highly anisotropic, heterogeneous and viscoelastic. A full review of corneal mechanics is provided in Ethier et al. (2004). Biomechanical properties are typically derivative of the orientation of collagen fibrils in load-bearing connective tissue. This is also true for the cornea when one is considering the tensile modulus in the direction of tangential loads. As might be expected, in the human corneal stroma, the preferred collagen fibril orientation as determined by X-ray (Aghamohammadzadeh et al., 2004, Meek and Boote, 2004) and SEM (Radner et al., 1998, Radner and Mallinger, 2002) (Fig. 2A) is reflected in the measured tensile strength of excised test specimens (Fig. 2B). It is also reflected clinically by the tendency of astigmatic axes to be aligned with either tangential or sagittal meridians. It is important to note that the fibril organization model of Meek and Boote (Aghamohammadzadeh et al., 2004) is very recent (2004) and is the result of full-thickness integrations or averages of X-ray interactions with the collagen fibrils. We thus are only beginning to understand the complex arrangement of fibrils in the corneal stroma, which begs the question: How can we recreate what we do not truly know?

The compressive behavior of the cornea is dominated not by collagen, but by its associated proteoglycans. More specifically, compressive corneal mechanics are dependent upon hydration and are primarily the result of the 40 mEq of fixed negative charges (at normal hydration) bound to the stromal proteoglycans (Hedbys, 1961, Kostyuk et al., 2002). Thus the cornea typically exhibits a net negative swelling pressure of approximately 60 mmHg (Hedbys et al., 1963), which is the result of the combined action of the epithelial barrier and the endothelial active pump (reviewed in Ruberti and Klyce (2002)). It has been known for over 100 years that stromas will swell and become opaque if excised and placed in hypotonic fluid (Leber, 1873). We assert that the compressive properties of the cornea are not critical to protect the globe, but are a consequence of the presence of the proteoglycans which are thought to maintain the collagen fibrillar spacing (Scott, 1991). Nonetheless, any accurate reproduction of a natural collagen/GAG based stroma will also require the same sort of hydration control system (due to the fixed charges on the GAGs) to maintain transparency. It is important to note that if a corneal analog can be produced which does not swell and remains clear, a transport system which deturgesces the cornea may not be required. This is the ostensibly valid argument of researchers pursuing synthetic hydrogel-based corneas which promote epithelialization (Sweeney et al., 2003). In the case of these synthetic porous implants, the epithelium would still be required to provide a barrier function and to maintain the tear/air interface. Research has demonstrated however that endothelial cells may still be necessary to aid the formation of healthy epithelium (Zieske et al., 1994, Orwin and Hubel, 2000).

The native cornea is an extremely efficient transmitter of incident visible light (Cox et al., 1970), with such efficiency dependent on the relative content and distribution of water in the stroma (Goldman et al., 1968). The transmission of light is critical to the function of the tissue. Thus the transparency of the stromal matrix, which constitutes the majority of the tissue thickness (and the light interaction) is also critical to corneal function. To understand the optics of corneal matrix transparency, it is necessary to examine the stromal collagen architecture at the nanoscale and to examine the role of the keratocytes.

Keratocytes and corneal crystallins. Within the last decade, it has been shown that keratocytes contribute actively to corneal transparency (Jester et al., 1999a, Jester et al., 1999b). Following corneal wounding, such as PRK surgery, the dedifferentiation of keratocytes to fibroblast and/or myofibroblasts can lead to a clinically relevant corneal haze (Jester et al., 1999a, Jester et al., 1999b). The haze has been attributed to poor optical properties of the cells which have undergone dedifferentiation. Their ability to “index match” to allow transmission of light appears to be a function of the content of soluble enzyme crystallins expressed in the cytoplasm of cells with a keratocyte phenotype (Karring et al., 2004). Thus, the cell population in corneal constructs should be of the appropriate phenotype to reduce cell-induced optical haze. For a review of factors controlling corneal stromal cell phenotype see West-Mays and Dwivedi (2006).

Stromal fibril organization and transparency. In Maurice's (1957) landmark paper on corneal transparency, he concluded that a regular crystalline arrangement of the monodisperse diameter collagen fibrils in the cornea was required to maintain transparency to incident light (Maurice, 1957). Later Hart and Farrell and then Benedek showed theoretically that corneas could be transparent even if there was only limited correlation in the spacing of the collagen (Hart and Farrell, 1969, Benedek, 1971). Benedek demonstrated theoretically that for transparency, it was important that the collagen fibrils should not pack together and that areas of collagen depletion (lakes) larger than the wavelength of light must not exist (Benedek, 1971). The fundamental argument against the requirement of extremely ordered fibrils was derived from examination of Bowman's membrane in the shark which is both transparent and disordered (Fig. 3) (Goldman and Benedek, 1967). Thus, while it is not necessary that the collagen fibrils in an engineered stroma be uniformly spaced, they should be much smaller than the wavelength of light, have a reasonably monodisperse diameter distribution and exhibit uniform center-to-center spacing. Further, there should not be large regions (on the order of the wavelength of light) devoid of fibrils.

Refraction of light. The combination of the nearly perfectly spherical corneal anterior surface and the index of refraction change at the air/tear film interface generate approximately 80% (42 diopters) of the total refractive power of the human ocular system. Precisely how the cornea forms such an effective refractive surface remains a matter of speculation, but is likely to reside in the details of the stromal collagen nanoscale arrangement coupled with the distribution of the mechanical tensile load (which is a consequence of IOP via Pascal's Principle and Laplace's Law). For instance, the fact that there are (putatively) no covalent crosslinks binding collagen fibrils to one another, fibrils may move relatively freely to distribute the load induced by the IOP. The precise pattern of in vivo corneal collagen load-bearing remains difficult to determine. After a detailed mechanical analysis of corneal transport, Friedman concluded that the anterior most lamellae carry the load from the IOP (Friedman, 1972). In his paper, he cited the clinical manifestations of sub-epithelial edema as evidence supporting this proposed load distribution. More recently however, evidence has been provided that indicates that in the rabbit (McPhee et al., 1985, Hennighausen et al., 1998) and in the human (Hjortdal, 1996) that the IOP load is distributed evenly through the thickness of the corneal stroma. In areas where the stromal lamellae are parallel to the corneal surface and given the monodispersity in the fibril diameters, this finding implies that the collagen fibrils should “feel” approximately the same strain (or stretch). How this load sharing, which requires precise control of fibril length and prestrain, is accomplished is an excellent research question. Some mechanisms must ensure during both development and growth, that the corneal curvature remains reasonably spherical. This requires that the load be distributed with precision among the collagen fibrils. The solution to this question may involve a matrix remodeling control system such as that putatively responsible for ocular globe length control (Troilo and Wallman, 1991, Troilo, 1992).

The stroma comprises 90% of the total corneal thickness, thus it is not surprising that it plays a major role in providing the principal functions of the cornea. We suggest that the stroma owes its success in simultaneously meeting the three corneal design requirements (protection, transmission and refraction) to its exquisite nanoscale organization. Fig. 4 shows the organization of the stromal collagen fibrils, which have a virtually monodisperse diameter distribution (transparency), reasonably uniform local interfibrillar spacing (transparency), no interfibrillar covalent crosslinks (refraction?) and are arranged in parallel arrays which are generally tangential to the corneal surface (mechanical strength).

We assert that this remarkable and persistent nanoscale arrangement of fibrils which persists throughout the cornea (with the exception of Bowman's membrane) is directly responsible for corneal function and thus links corneal form and function at the level of the nanoscale. The stromal organization appears to be the principal reason why attempts to engineer a viable cornea have been unsuccessful to date. Any corneal analog comprising a natural collagenous extracellular matrix (ECM) must closely mimic this arrangement or risk being too weak, too opaque or too irregular to form an appropriate refractive surface. It is for this reason that we believe that efforts to produce a natural cornea via tissue engineering should focus on reproducing the stroma and in particular on reproducing the nanoscale stromal architecture.

There has already been extensive and reasonably successful effort expended on reproducing the epithelium and endothelium in vitro from primary human cells. This suggests that if a viable stroma is produced, methods are already available to populate it with limiting functional cell layers.

The epithelium is responsible for the protection of the stroma and the maintenance of the tear film. Though it is not critical to produce corneal constructs with adherent epithelia to replace failed corneas (donor corneas are debrided of their epithelium prior to implantation), it is important that constructs induce the attachment, spreading and ultimately growth to confluence of epithelial cells to form a patent anterior corneal barrier. A healthy corneal epithelium should form a confluent multilaminar (5–7 cell layers) stratified structure with complete tight junctions surrounding the surface cells. Desmosomes should be present between the cells and hemidesmosomes with anchoring fibrils projecting into an adherent basement membrane. Culture of epithelial cells onto various substrates has been achieved by numerous investigators: rabbit epithelial cells on plastic (Sundar-Raj et al., 1980); on denuded rabbit corneal stroma (Friend et al., 1982) and on collagen gels (Geggel et al., 1985). It was realized early in this effort that culture technique and cell response to substrates were critical to epithelial morphology and protein expression, leading Trinkaus-Randall to demonstrate that epithelial cells responded differently to different substrates (Trinkaus-Randall et al., 1988, Trinkaus-Randall and Gipson, 1985) and Minami et al. to show that airlifted cultures produced appropriately stratified epithelial layers (Minami et al., 1993). In 1994, Zieske et al. demonstrated that rabbit epithelium basement membrane and differentiation was profoundly influenced by the presence of endothelial cells in an airlifted culture system (Zieske et al., 1994). Fig. 5 shows the morphology of the epithelium grown at the moist air interface and in the presence of the endothelial cells.

Culture of human corneal epithelial cells onto collagen-based substrates for the purposes of corneal tissue engineering has been performed with reasonable success by a number of investigators: primary cells on collagen gels (and on stromal blocks) (Ohji et al., 1994), immortalized cells on glutaraldehyde fixed collagen/chondroitin sulfate gels, (Griffith et al., 1999) primary limbal epithelial cells on collagen populated with keratocytes (Germain et al., 1999), primary epithelial cells on dehydrothermally cross-linked collagen sponges populated with keratocytes and in the presence of endothelial cells (Orwin and Hubel, 2000). One of the more important results of this body of work is the fact that careful attention should be paid to the location of cell harvesting because there are spatial differences in the proliferative capacity of human corneal epithelial cells, with limbally derived cells showing the greatest ability to undergo multiple passages in culture (Lindberg et al., 1993). In general, progress in this area has been exceptional and it appears certain that once a more appropriate stromal analog has been produced, attempts to epithelialize it will be well informed.

Corneal tissue has a tendency to imbibe fluid and become opaque as a consequence of the large imbibition pressure imparted by the stromal GAGs (Hedbys et al., 1963). In 1972, David Maurice (Maurice, 1972) determined that the mechanism responsible for countering the swelling pressure and keeping the cornea in a state of relative deturgescence (and thus transparent) is an active transporter located in the corneal endothelium. For tissue engineered corneas with a natural stromal analog, a functioning confluent endothelial layer would provide a critical component of the corneal transport system. In addition, it has also been shown that endothelial cell co-culture plays a role in generation of appropriate structure and differentiation behavior of corneal epithelium (Zieske et al., 1994, Orwin and Hubel, 2000). Thus, even if the stromal analog comprises a material which does not require “pumping down” to become transparent, unknown signaling molecules derived from a functioning endothelium may still be required to guarantee the patency of the corneal epithelium. Unfortunately, unlike those of other mammals such as rabbit and pig, human corneal endothelial cells do not proliferate in vivo, instead, they undergo polymegathism and pleomorphism to cover exposed Descemet's membrane following the loss of neighboring cells (Yee et al., 1985). Human corneal endothelial cells (HCECs), which are contact inhibited and arrested in the G1 phase of the cell cycle (Joyce et al., 1996a, Joyce et al., 1996b), are perniciously non-proliferative and unique culture methods and cell selection protocols have been evolved specifically to encourage proliferation (Engelmann et al., 1999, Chen et al., 2001). The recent progress in endothelial culture techniques has made the difficult task of supplying enough vital, differentiated and functional untransformed human corneal endothelial cells to populate corneal constructs a distinct possibility (Bohnke et al., 1999, Engelmann et al., 1999, Chen et al., 2001, Joyce and Zhu, 2004). The laboratory of Dr. Joyce has been particularly successful in inducing the proliferation of HCECs in culture (Chen et al., 2001, Joyce and Zhu, 2004, Zhu and Joyce, 2004, Konomi et al., 2005). In 2001, they successfully cultured HCECs from donors 50 to 80 years old onto denuded Descemet's membrane of recipient corneas and achieved remarkably high cell densities. The methods are not trivial and the Joyce laboratory employs donor selection criteria to increase the probability of obtaining highly dense cell cultures. These extremely encouraging results have, for the first time, engendered the possibility of transplanting untransformed HCECs onto a patient's denuded Descemet's membrane or of seeding HCECs in high density on artificial corneal constructs. The ability to culture untransformed HCECs is a major advance toward the goal of producing a tissue engineered cornea.