Control of Scar Tissue Formation in the Cornea: Strategies in Clinical and Corneal Tissue Engineering

Corneal structure is highly organized and unified in architecture with structural and functional integration which mediates transparency and vision. Disease and injury are the second most common cause of blindness affecting over 10 million people worldwide. Ninety percent of blindness is permanent due to scarring and vascularization. Scarring caused via fibrotic cellular responses, heals the tissue, but fails to restore transparency. Controlling keratocyte activation and differentiation are key for the inhibition and prevention of fibrosis. Ophthalmic surgery techniques are continually developing to preserve and restore vision but corneal regression and scarring are often detrimental side effects and long term continuous follow up studies are lacking or discouraging. Appropriate corneal models may lead to a reduced need for corneal transplantation as presently there are insufficient numbers or suitable tissue to meet demand. Synthetic optical materials are under development for keratoprothesis although clinical use is limited due to implantation complications and high rejection rates. Tissue engineered corneas offer an alternative which more closely mimic the morphological, physiological and biomechanical properties of native corneas. However, replication of the native collagen fiber organization and retaining the phenotype of stromal cells which prevent scar-like tissue formation remains a challenge. Careful manipulation of culture environments are under investigation to determine a suitable environment that simulates native ECM organization and stimulates keratocyte migration and generation.

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title: Application of structural analysis to the mechanical behaviour of the cornea
abstract: Structural engineering analysis tools have been used to improve the understanding of the biomechanical behaviour of the cornea. The research is a multi-disciplinary collaboration between structural engineers, mathematical and numerical analysts, ophthalmologists and clinicians. Mathematical shell analysis and nonlinear finite-element modelling have been used in conjunction with laboratory experiments to study the behaviour of the cornea under different loading states and to provide improved predictions of the mechanical response to disease and injury. The initial study involved laboratory tests and mathematical back analysis to determine the corneal material properties and topography. These data were then used to facilitate the construction of accurate finite-element models that are able to reliably trace the performance of cornea upon exposure to disease, injury or elevated intra-ocular pressure. The models are being adapted to study the response to keratoconus (a disease causing loss of corneal tissue) and to tonometry procedures, which are used to measure the intra-ocular pressure. This paper introduces these efforts as examples of the application of structural engineering analysis tools and shows their potential in the field of corneal biomechanics.

title: Collagen fibril orientation and corneal curvature.
abstract: This issue of Structure presents a report (Aghamohammadzadeh et al., 2004) that maps collagen fibril orientation throughout the cornea and explains how this information can be used to understand why the cornea is curved.

title: Functional human corneal equivalents constructed from cell lines.
abstract: Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

title: Bioengineered corneas for transplantation and in vitro toxicology.
abstract: Bioengineered corneas have been designed to replace partial or the full-thickness of defective corneas, as an alternative to using donor tissues. They range from prosthetic devices that solely address replacement of the cornea's function, to tissue engineered hydrogels that permit regeneration of host tissues. In cases where corneal stem cells have been depleted by injury or disease, most frequently involving the superficial epithelium, tissue engineered lamellar implants reconstructed with stem cells have been transplanted. In situ methods using ultraviolet A (UVA) crosslinking have also been developed to strengthen weakened corneas. In addition to the clinical need, bioengineered corneas are also rapidly gaining importance in the area of in vitro toxicology, as alternatives to animal testing. More complex, fully innervated, physiologically active, three-dimensional organotypic models are also being tested.

title: A viscoelastic biomechanical model of the cornea describing the effect of viscosity and elasticity on hysteresis.
abstract: To develop a method for evaluating viscosity and elasticity of the cornea and to examine the effect that both properties have on hysteresis.



A three-component spring and dashpot model was created in Simulink in Matlab to represent the purely elastic and viscoelastic behavior of the cornea during a measurement using device called an ocular response analyzer (ORA). Values for elasticity and viscosity were varied while sinusoidal stress was applied to the model. The simulated stresses were used to determine how hysteresis is affected by the individual components of elasticity, viscosity, and maximum stress. To validate the model, high-speed photography was used to measure induced strain in a corneal phantom during ORA measurement. This measured strain was compared with the strains simulated by the model.



When the spring in the viscoelastic portion of the model was stiffened, hysteresis decreased. When the spring in the purely elastic element was stiffened, hysteresis increased. If both springs were stiffened together, hysteresis peaked strongly as a function of the viscosity of the viscoelastic element. Below the peak value, lower elasticity was associated with higher hysteresis. Above the peak value, higher elasticity was associated with higher hysteresis. In addition, hysteresis increased as the air maximum pressure was increased. Measurements from phantom corresponded to predictions from the model.



A viscoelastic model is presented to illustrate how changing viscosity and elasticity may affect hysteresis. Low hysteresis can be associated with either high elasticity or low elasticity, depending on the viscosity, a finding consistent with clinical reports.

title: 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.

title: The keratocyte: corneal stromal cell with variable repair phenotypes.
abstract: Keratocytes, also known as fibroblasts, are mesencyhmal-derived cells of the corneal stroma. These cells are normally quiescent, but they can readily respond and transition into repair phenotypes following injury. Cytokines and other growth factors that provide autocrine signals for stimulating wound responses in resident cells are typically presented by platelets at the site of an injury. However, due to the avascular nature of the cornea many of the environmental cues are derived from the overlying epithelium. Corneal epithelial-keratocyte cell interactions have thus been extensively studied in numerous in vivo corneal wound healing settings, as well as in in vitro culture models. Exposure to the different epithelial-derived factors, as well as the integrity of the epithelial substratum, are factors known to impact the keratocyte response and determine whether corneal repair will be regenerative or fibrotic in nature. Finally, the recent identification of bone-marrow derived stem cells in the corneal stroma suggests a further complexity in the regulation of the keratocyte phenotype following injury.

title: Can we produce a human corneal equivalent by tissue engineering?
abstract: Tissue engineering is progressing rapidly. Bioengineered substitutes are already available for experimental applications and some clinical purposes such as skin replacement. This review focuses on the development of reconstructed human cornea in vitro by tissue engineering. Key elements to consider in the corneal reconstruction, such as the source for epithelial cells and keratocytes, are discussed and the various steps of production are presented. Since one application of this human model is to obtain a better understanding of corneal wound healing, the mechanisms of this phenomenon as well as the function played both by membrane-bound integrins and components from the extracellular matrix have also been addressed. The analysis of integrins by immunohistofluorescence labelling of our reconstructed human cornea revealed that beta(1), alpha(3), alpha(5), and alpha(6) integrin subunits were expressed but alpha(4) was not. Laminin, type VII collagen and fibronectin were also detected. Finally, the future challenges of corneal reconstruction by tissue engineering are discussed and the tremendous applications of such tissue produced in vitro for experimental as well as clinical purposes are considered.

title: STUDIES ON THE CORNEA. II. THE FINE STRUCTURE OF DESCEMET'S MEMBRANE
abstract: Descemet's membrane, previously thought to be "structureless," has been found to be characterized by a fine structure of great regularity. Sections perpendicular to the plane of the membrane surface appeared to be cross-striated, with narrow dark bands separated by wide light bands traversed by fine filaments. Tangential sections showed a two-dimensional array of dark nodes and thin internodal filaments which connected each node with the six others around it to form a hexagonal figure. The average distance between nodes, and between the dark bands in transverse sections, was about 1070 A; the width of the nodes was about 270 A; and the width of the connecting filaments was less than 100 A. This pattern has been found in all species of Descemet's membrane so far examined, although it appeared to be better developed in some forms than in others. So far as is known, it has not been observed in any other type of tissue. The differentiation of Descemet's membrane has been followed in the chick embryo. It appeared late and developed slowly but showed the characteristic fine structure from the time it could be clearly identified. The evidence for placing Descemet's membrane in the collagen class of proteins is strongly supplemented by the results obtained from x-ray diffraction examination by Rougvie and Bear.

title: Hematopoietic colony-forming cells derived from human embryonic stem cells
abstract: Human embryonic stem (ES) cells are undifferentiated, pluripotent cells that can be maintained indefinitely in culture. Here we demonstrate that human ES cells differentiate to hematopoietic precursor cells when cocultured with the murine bone marrow cell line S17 or the yolk sac endothelial cell line C166. This hematopoietic differentiation requires fetal bovine serum, but no other exogenous cytokines. ES cell-derived hematopoietic precursor cells express the cell surface antigen CD34 and the hematopoietic transcription factors TAL-1, LMO-2, and GATA-2. When cultured on semisolid media with hematopoietic growth factors, these hematopoietic precursor cells form characteristic myeloid, erythroid, and megakaryocyte colonies. Selection for CD34+ cells derived from human ES cells enriches for hematopoietic colony-forming cells, similar to CD34 selection of primary hematopoietic tissue (bone marrow, umbilical cord blood). More terminally differentiated hematopoietic cells derived from human ES cells under these conditions also express normal surface antigens: glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. The in vitro differentiation of human ES cells provides an opportunity to better understand human hematopoiesis and could lead to a novel source of cells for transfusion and transplantation therapies.

title: The use of X-ray scattering techniques to quantify the orientation and distribution of collagen in the corneal stroma
abstract: The bulk of the corneal stroma is comprised of a layered network of fibrillar collagen. Determining the architecture of this unique structure may help us to better understand the cornea's biomechanical and optical function. The analysis of diffraction patterns obtained when X-rays are passed through the regularly arranged collagen molecules and fibrils of the stromal matrix yields quantitative data on fibrillar organisation, including the orientation and distribution of collagen lamellae within the corneal plane. In recent years, by exploiting the radiation from powerful synchrotron sources, techniques have been developed to enable the mapping of collagen fibril, and therefore lamellar, directions across whole corneas. This article aims to summarise the use of X-ray diffraction to map the orientation and distribution of collagen in the corneal stroma. The implications of the knowledge gained so far are discussed in relation to the optical and biomechanical properties of the cornea, and their alteration due to disease and surgical intervention.

title: Corneal stromal cells use both high- and low-contractility migration mechanisms in 3-D collagen matrices.
abstract: Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency.

title: Recreating the microenvironment of the native cornea for tissue engineering applications.
abstract: A viable tissue-engineered corneal replacement needs to be transparent and mechanically resilient. One necessary element for achieving this level of functionality is a scaffolding material that minimizes backscattered light, supports cellular growth, and maintains the transparent cellular phenotype. We hypothesize that the best scaffolding material will mimic the microenvironment of the natural corneal extracellular matrix (ECM). This work describes a method for electrospinning collagen type I fibers that replicates the unique morphology and arrangement of collagen type I fibers in the native cornea. In the cornea the collagen type I fibers are approximately 30 nm in diameter and aligned within stacked lamellae. After comparing several methods, the optimal method for creating uniformly aligned fibers was achieved by electrospinning onto a dual plate device with a quartz glass substrate. The fibers were crosslinked in glutaraldehyde vapor for 3 days and then further crosslinked and sterilized with liquid glutaraldehyde. Rabbit corneal fibroblasts were cultured on the fiber constructs for 7 days. Qualitative analysis of the cell morphology and intracellular protein expression suggests that the electrospun fibers provide a viable scaffold material for engineering a corneal tissue replacement.

title: The architecture of the corneal stroma.
abstract: In recent years the evolution of modern refractive surgery has focused attention on the architecture and biological properties of the cornea. In this issue of the BJO (p 437) Muller et al address the differential behaviour of the anterior and posterior stroma during corneal swelling and draw interesting conclusions about the factors maintaining corneal shape.

Transparency of the corneal stroma depends particularly on the degree of spatial order of its collagen fibrils which are narrow in diameter and closely packed in a regular array.1-8 The collagen fibrils themselves are weak scatterers, since their fibril diameter is less than the wavelength of light, and fibril refractive index is close to that of the ground substance. There is little variation in fibril diameter and separation between the anterior and posterior cornea.

The stromal fibrils are further organised into bundles, or lamellae, of which there are approximately 300 in the central cornea and 500 close to the limbus.9 The posterior lamellae course directly across the full width of the cornea without a break, having their origins in fibres which wind around the limbus at the corneoscleral junction10-12 or, according to Radner,9 have a pseudocircular organisation at the limbus, forming the ligamentum circulare corneae. On the basis of x ray diffraction studies, about 49% of the stromal lamellae are preferentially aligned orthogonally, along the vertical and horizontal meridians, while about 66% lie within a 45° sector.1112 Fibrils within a lamella are in parallel array, except where branching of lamellae occurs. Branching in the horizontal plane occurs throughout the stroma, whereas anteroposterior branching is found only in the anterior third.13

The anterior and posterior stroma differ in specific ways. In general the posterior stroma is more ordered,14 more hydrated,15 more easily swollen, and has a lower …

title: Lamellar orientation in human cornea in relation to mechanical properties.
abstract: We have applied wide-angle X-ray scattering to the human cornea in order to quantify the relative number of stromal collagen fibrils directed along the two preferred corneal lamellar directions: superior-inferior and nasal-temporal. The data suggest that, on average, the two directions are populated in equal proportion at the corneal centre. Here, approximately one-third of the fibrils throughout the stromal depth tend to lie within a 45 degrees sector of the superior-inferior meridian, and similarly for the nasal-temporal direction. However, in some eyes we have measured significant differences between the two preferential fibril populations, with some corneas exhibiting as much as 25% more collagen in one direction than the other. Based on these findings, a mechanical model of the normal cornea may be envisaged, whereby the fibril tension in the underlying "background" of isotropically arranged collagen helps to balance the intraocular pressure; while the extra preferentially aligned fibrils take up the additional tensile stress along the superior-inferior and nasal-temporal meridians exerted by the rectus muscles and the orbicularis. It is possible that, through a direct impact on the elastic modulus of the tissue, an imbalance of superior-inferior and nasal-temporal fibrils in some eyes might affect corneal shape.

title: Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.
abstract: The need for successful ex-vivo expansion and directed differentiation of haematopoietic stem cells (HSCs) for therapeutic applications has increased over the past decade. Haematopoietic cell cultures are complex and full characterisation of the process environment has yet to be achieved. The complexity and transient nature of HSC cultures make the identification, maintenance and control of optimal operating conditions challenging. Application of real-time, on-line monitoring techniques and process control strategies enhances the ability to operate bioprocesses of desired reproducibility and high product quality. In this review, we discussed the methods by which in vitro culture information necessary for bioprocess control may be obtained, including process considerations, monitoring and analytical tools, and design of experiments (DOE). The successful application of these tools may result in time- and cost-effective cultures for directed differentiation and expansion of haematopoietic components intended for clinical use.

title: Biomechanics and wound healing in the cornea.
abstract: The biomechanical and wound healing properties of the cornea undermine the predictability and stability of refractive surgery and contribute to discrepancies between attempted and achieved visual outcomes after LASIK, surface ablation and other keratorefractive procedures. Furthermore, patients predisposed to biomechanical failure or abnormal wound healing can experience serious complications such as keratectasia or clinically significant corneal haze, and more effective means for the identification of such patients prior to surgery are needed. In this review, we describe the cornea as a complex structural composite material with pronounced anisotropy and heterogeneity, summarize current understanding of major biomechanical and reparative pathways that contribute to the corneal response to laser vision correction, and review the role of these processes in ectasia, intraocular pressure measurement artifact, diffuse lamellar keratitis (DLK) and corneal haze. The current understanding of differences in the corneal response after photorefractive keratectomy (PRK), LASIK and femtosecond-assisted LASIK are reviewed. Surgical and disease models that integrate corneal geometric data, substructural anatomy, elastic and viscoelastic material properties and wound healing behavior have the potential to improve clinical outcomes and minimize complications but depend on the identification of preoperative predictors of biomechanical and wound healing responses in individual patients.

title: The organization of collagen in the corneal stroma.
abstract: The cornea has evolved to fulfil the dual functions of enclosing and protecting the inner contents of the eye, and focussing light onto the retina with minimum scatter and optical degradation. It does this by means of the arrangement of the constituent collagen fibrils, an arrangement that is unique in connective tissues. This article reviews our current knowledge about the detailed organization of collagen in the corneal stroma, and presents new data suggesting that a significant proportion of collagen fibrils running across the cornea, change direction near the limbus and fuse with the circumferential limbal collagen.

title: Orthogonal scaffold of magnetically aligned collagen lamellae for corneal stroma reconstruction.
abstract: The creation of 3D scaffolds that mimic the structure of physiological tissue required for normal cell function is a major bioengineering challenge. For corneal stroma reconstruction this necessitates the creation of a stroma-like scaffold consisting of a stack of orthogonally disposed sheets of aligned collagen fibrils. This study demonstrates that such a scaffold can be built up using magnetic alignment. By allowing neutralized acid-soluble type I collagen to gel in a horizontal magnetic field (7 T) and by combining a series of gelation-rotation-gelation cycles, a scaffold of orthogonal lamellae composed of aligned collagen fibrils has been formed. Although initially dilute, the gels can be concentrated without noticeable loss in orientation. The gels are translucent but their transparency can be greatly improved by the addition of proteoglycans to the gel-forming solution. Keratocytes align by contact guidance along the direction of collagen fibrils and respect the orthogonal design of the collagen template as they penetrate into the bulk of the 3D matrix. The scaffold is a significant step towards the creation of a corneal substitute with properties resembling those of native corneal stroma.

title: An active artificial cornea with the function of inducing new corneal tissue generation in vivo-a new approach to corneal tissue engineering.
abstract: An active artificial cornea which can perform the function of inducing new cornea generation in vivo but does not need culture cells in vitro and which has similar optical and mechanical properties to those of the human cornea was constructed. An animal keratoplasty experiment using the artificial cornea as the implant showed that the animals' corneas could keep smooth surface and clear stroma postoperatively, and that the repopulation of the host's keratocytes, the degradation of the implant and new corneal tissue generation were completed at 5-6 months after surgery. Such an artificial cornea has several advantages over other corneal equivalents constructed in the typical way of tissue engineering: in having similar mechanical and optical properties to those of the human cornea and with no exogenetic cells, it can be used universally in different implantation surgeries without immunoreaction; it is easy to prepare and process into different shapes and sizes on a large scale, and suitable for long-distance transportation and long-term storage. All these characteristics make it a new approach to cornea tissue engineering having potential in many clinical applications.

title: Bioengineered corneas: how close are we?
abstract: Bioengineered corneas are substitutes for human donor tissue that are designed to replace part or the full thickness of damaged or diseased corneas. They range from prosthetic devices that solely address replacement of the cornea's function to tissue-engineered hydrogels that allow some regeneration of the host tissue. In addition, there are also bioengineered lenticules that may be implanted into the cornea to improve vision by altering the refractive properties of the eye, an alternative procedure to refractive surgery. In recent years, there have been significant developments in many areas of bioengineered corneas, such as the clinical trials of an artificial cornea designed as a prosthesis, the development of completely natural corneal replacements, and the development of biosynthetic matrices that permit host tissue regeneration. For correction of refractive errors, a synthetic corneal onlay that allows stable overgrowth of epithelium appears to be promising.

title: Corneal blindness: a global perspective.
abstract: Diseases affecting the cornea are a major cause of blindness worldwide, second only to cataract in overall importance. The epidemiology of corneal blindness is complicated and encompasses a wide variety of infectious and inflammatory eye diseses that cause corneal scarring, which ultimately leads to functional blindness. In addition, the prevalence of corneal disease varies from country to country and even from one population to another. While cataract is responsible for nearly 20 million of the 45 million blind people in the world, the next major cause is trachoma which blinds 4.9 million individuals, mainly as a result of corneal scarring and vascularization. Ocular trauma and corneal ulceration are significant causes of corneal blindness that are often underreported but may be responsible for 1.5-2.0 million new cases of monocular blindness every year. Causes of childhood blindness (about 1.5 million worldwide with 5 million visually disabled) include xerophthalmia (350,000 cases annually), ophthalmia neonatorum, and less frequently seen ocular diseases such as herpes simplex virus infections and vernal keratoconjunctivitis. Even though the control of onchocerciasis and leprosy are public health success stories, these diseases are still significant causes of blindness--affecting a quarter of a million individuals each. Traditional eye medicines have also been implicated as a major risk factor in the current epidemic of corneal ulceration in developing countries. Because of the difficulty of treating corneal blindness once it has occurred, public health prevention programmes are the most cost-effective means of decreasing the global burden of corneal blindness.

title: Serial analysis of gene expression in the corneal endothelium of Fuchs' dystrophy.
abstract: To compare the gene expression profiles of normal human corneal endothelium with Fuchs' corneal endothelium, by using serial analysis of gene expression (SAGE).



Three pairs of normal human corneas were obtained from eye banks. Thirteen bisected Fuchs' corneal buttons were processed at the time of corneal transplantation. The endothelia of normal and Fuchs'-affected corneas were stripped, and total RNA was isolated. Serial analysis of gene expression (SAGE) was performed to identify and quantify gene transcripts. Genes over- and underexpressed by Fuchs' endothelium were limited to P < 0.01 by the method of Audic and Claverie.



A total of 19,136 tags were identified with 9,530 from normal and 9,606 from Fuchs' endothelium. The expression of 18 transcripts was upregulated, and 36 transcripts were downregulated in Fuchs' endothelium compared with normal tissue. Upregulated transcripts included serum amyloid A1 and A2, metallothionein, and apolipoprotein D. Of the downregulated transcripts, 26 matched known genes, 3 matched expressed sequence tags (ESTs), and 7 were unknown to current databases. One downregulated transcript involved a newly reported bicarbonate transporter. Decreased transcripts related to antioxidants and proteins conferring protection against toxic stress were noted in Fuchs' versus normal endothelium including nuclear ferritin, glutathione S-transferase-pi, and heat shock 70-kDa protein. Nine different SAGE tags matching mitochondrial sequences accounted for 25% of the ESTs that were decreased in Fuchs' endothelium.



SAGE analysis comparing normal to Fuchs' endothelium demonstrates diminished expression of mitochondrial, pump function, and antiapoptotic cell defense genes.

title: The effect of growth factor signaling on keratocytes in vitro and its relationship to the phases of stromal wound repair.
abstract: To determine the relationship between signaling by different growth factors and the phases of corneal stromal wound repair. The authors hypothesize that the process involves sequential signaling, resulting first in proliferation and then in extracellular matrix (ECM) synthesis.



The effects of IGF-I, TGF-beta1, FGF-2, and PDGF on proliferation and ECM production by primary cultured bovine keratocytes were evaluated. DNA synthesis was determined by (3)H-thymidine incorporation, and maximal cell density was determined by measurement of DNA content. Relative levels of ECM components synthesized by keratocytes and secreted into the media were evaluated by (3)H-glycine incorporation into total ECM protein and collagen, by (3)H-glucosamine incorporation into chondroitin sulfate, keratan sulfate, and hyaluronan, and by Western blotting with antibodies specific to procollagen types Iota and IotaIotaIota.



FGF-2 stimulated the highest level of proliferation and the lowest level of glycosaminoglycan synthesis and inhibited the synthesis of collagen types Iota and IotaIotaIota. IGF-I, in contrast, stimulated the lowest level of proliferation and the highest levels of collagen synthesis. PDGF and TGF-beta1 had intermediate effects on proliferation and collagen synthesis. Although FGF-2 inhibited collagen production, it could be restored by subsequent treatment with IGF-I, TGF-beta1, and PDGF.



The results of this study showed that the level of proliferation induced by the growth factors was inversely related to the levels of collagen production. The authors suggest that FGF-2 initiates the hypercellular phase of corneal wound healing and that IGF-I and PDGF are involved in the restoration of a normal ECM.

title: Corneal epithelial wound healing.
abstract: One of the important functions of the cornea is to maintain normal vision by refracting light onto the lens and retina. This property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Epithelial renewal is essential because it enables this tissue to act as a barrier that protects the corneal interior from becoming infected by noxious environmental agents. The renewal process also maintains the smooth optical surface of the cornea. This rate of renewal is closely maintained by an integrated balance between the processes of corneal epithelial proliferation, differentiation, and cell death. Attempts to understand this complex cascade make it evident that the appropriate integration and coordination of corneal epithelial renewal depends on the actions of a myriad of cytokines. We have attempted in this review to collate the receptor and cell signaling events and cytokine studies that are responsible for mediating corneal wound healing.

title: Activation of epidermal growth factor receptor during corneal epithelial migration.
abstract: Epidermal growth factor (EGF) and related growth factors: transforming growth factor (TGF)-alpha, heprin-ding (HB)-EGF, and amphiregulin (AR), have been shown to stimulate events associated with epithelial wound repair. These growth factors function by binding to a common EGF receptor (EGFR), tyrosine kinase. We have used in vivo and organ culture wound-healing models to examine the kinetics and extent of EGFR activation during corneal epithelial wound repair and whether the epithelium itself produces EGFR ligands capable of stimulating the healing process.



In the in vivo model, 3-mm debridement wounds were made in rat corneas and allowed to heal in situ. Activation of EGFR was analyzed by 1) indirect immunofluorescence microscopy, 2) immunoprecipitation using anti-EGFR and anti-phosphotyrosine (anti-PT), and 3) binding-site localization using EGF-fluorescein isothiocyanate (FITC). Relative levels of mRNA for EGF, TGF-alpha, HB-EGF, and AR were determined using reverse transcription-polymerase chain reaction. To determine whether inhibiting EGFR activation slows epithelial migration, wounded corneas were allowed to heal in organ culture in the presence of tyrphostin AG1478 (0-50 microM), a specific inhibitor of EGFR kinase activity.



In unwounded corneas, EGFR was localized in basal cells and appeared to be membranous. Within 1 hour after wounding, EGFR was no longer immunolocalized in the membranes of cells migrating into the wound area. EGF-FITC-binding assays indicated that EGFR ligands could penetrate all the way to the limbus. Immunoprecipitation showed that EGFR was phosphorylated on tyrosine residues within 30 minutes after wounding and that phosphorylation levels increased after wounding. Levels of mRNA for TGF-alpha, HBEGF, and AR all appeared to increase after wounding. In organ culture experiments, tyrphostin AG1478 inhibited migration rates in a dose-dependent manner.



These data indicate that EGFR was activated during corneal epithelial wound healing in vivo. Furthermore, this activation appears to be a necessary component of the process, because inhibition of the EGFR signaling cascade significantly slowed migration rates.

title: Keratocyte and fibroblast phenotypes in the repairing cornea
abstract: In mammals, tissue damage is usually repaired by activation of a fibrotic response which saves the life of the organism, but which can never restore function to the damaged organ. In addition, fibrotic responses form the basis for diverse pathologies, including many that occur in the eye. It is intriguing, therefore, to observe the occasional circumstances in which repair in mammals appears to take on a regenerative character, such as during fetal wound healing or in certain types of corneal wounds. The thesis of this chapter is that the choice between regeneration or fibrosis lies in the control of fibroblast phenotype. The cornea of the eye has several features which make it a particularly useful model for the study of fibroblast phenotype. Studies discussed herein, identify failure to activate the transcription factor NF-kappaB as a control mechanism for inhibiting fibroblast activation in the cornea. Evidence is further presented for the view that transition in fibroblast phenotype in repair tissue is not simply a matter of differential gene expression, but is a developmental event which reflects changes in the hard wiring of signalling pathways by which the cell responds to environmental input.

title: Matrix metalloproteinase distribution during early corneal wound healing
abstract: AimTo compare matrix metalloproteinase (MMP) localisation in anterior keratectomy (AK) and lamellar keratectomy (LK) wounds.MethodsWounds were produced in one eye of 24 rabbits. The AK wounds were made to approximately 120 μm in depth and then allowed to re-epithelialise. The LK wounds were of similar depth, but the anterior stroma and epithelium were replaced after a second deeper keratectomy had been performed. Immunohistochemistry was used to localise the MMP-1, -2, -3, and -9 at intervals from 4 h to 14 days following surgery. The contralateral eyes acted as controls.ResultsAfter an AK wound MMP-1 was present at the leading edge of migrating epithelium after 18 h, while MMP-2 and -9 were localised behind the advancing epithelial edge. The presence of these enzymes rapidly fell to low levels after epithelial closure. There was only faint MMP-3 localisation between days 3 and 7. After an LK wound, MMP-1, -3, and -9 were not detected in the stromal interface, but MMP-2 was present at all time points.ConclusionsThis study suggests that after an AK wound, MMP-1 is a key mediator of epithelial migration, while MMP-2 and -9, and to a lesser extent MMP-3, may participate in the remodelling of corneal stroma and the reformation of epithelial basement membrane. In contrast, an LK wound results in a much lower stimulus for MMP activation. The action of MMP-2 in stromal repair is thus partly independent of epithelial injury.

title: Concentration-dependent effects of transforming growth factor β1 on corneal wound healing
abstract: There is an unmet challenge to promote wound healing in non-healing wounds such as in the post-LASIK (laser-assisted in situ keratomileusis) cornea. Using human corneal fibroblasts (HCFs) in cell culture, we investigated the concentration dependence of the growth factor transforming growth factor β1 (TGFβ1) on wound closure. Although high concentrations of TGFβ1 leads to scarring, we asked whether low concentrations of TGFβ1 could promote wound healing without generating a large fibrotic response.  HCFs were cultured in supplemented serum-free media (SSFM). Cell migration was assessed by scratch-wounding. SMAD 2/3 and p38 mitogen-activated protein kinase (p38MAPK) localization and α-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. Active TGFβ was quantified using a luciferase bio-assay.  We found that neutralizing antibody to TGFβ1 reduced cell migration by 73%, compared to immunoglobulin G (IgG) control, establishing that endogenous TGFβ1 (determined to be 0.01 ng/ml) is necessary to promote cell migration. To evaluate the concentration-dependent effects of TGFβ1 on wound closure, HCF migration was quantified to determine the impact of increasing concentrations of TGFβ1 (0.01–1.0 ng/ml). Compared to control (cells in SSFM), the higher concentrations (0.1 and 1.0 ng/ml TGFβ1) significantly decreased cell migration (63%–86%), induced myofibroblast differentiation (83%–88%), increased SMAD 2/3 localization into the nucleus (72%–79%) and inhibited the activation of p38MAPK (51%–63%). In contrast, addition of the lower concentration of TGFβ1 (0.01 ng/ml TGFβ1) promoted a cell migration rate that was similar to endogenous TGFβ, reduced SMAD 2/3 nuclear localization, and stimulated p38MAPK activation. A TGFβ1 blocking antibody and the p38MAPK inhibitor, SB202192, was used to demonstrate that p38MAPK activation is necessary for TGFβ1-induced cell migration.  Together, our data demonstrate that low concentrations of TGFβ1 promote p38MAPK activation that is a key to HCF migration, suggesting that a low concentration of TGFβ may be useful in treating non-healing corneal wounds.

title: Direct epithelial–stromal interaction in corneal wound healing: Role of EMMPRIN/CD147 in MMPs induction and beyond
abstract: In the cornea, the epithelium and the underlying stroma are separated by the basement membrane and Bowman's layer. The disruption of these anatomical barriers during wound healing represents a key step which initiates tissue remodeling through the modification of the epithelial-stromal interactions (ESI). Diffusible cytokines are generally viewed as central modulators in the bidirectional communication between these epithelial and stromal compartments and their implication in all stages of the wound healing process has been an active area of research for many years. Our studies which aimed to explore mechanisms of matrix degradation in pathological corneal wound healing have shown that EMMPRIN, a glycoprotein expressed on corneal epithelial cell surface, can induce matrix metalloproteinase (MMP) production and myofibroblasts differentiation after direct interaction with corneal fibroblasts. EMMPRIN appears therefore as a potential mediator of ESI by direct cell-cell contact which represents a new mechanism for dysregulated MMPs' induction observed in corneal ulcerations. These direct epithelial-stromal interactions (direct-ESI) can occur when delayed epithelial healing prevents regeneration of the basement membrane and allows the two cell types to come into close proximity. We propose that prevention of these interactions through inhibition of EMMPRIN may represent a promising therapeutic strategy in the inhibition of MMP induction in ulceration.

title: Photorefractive keratectomy: implications of corneal wound healing.
abstract: Photorefractive keratectomy (PRK) with an argon fluoride laser (193 nm) was developed to alter the refractive power of the eye predictably and permanently. At PRK the central corneal epithelium is debrided mechanically and a disc 4 to 6 mm in diameter is ablated into the anterior stroma using computer controlled algorithms to control the depth of the wound. Theoretically for corrections of up to -7 dioptres less than 10% of the axial corneal thickness is removed.' As there is little loss of corneal strength and miniimal tissue distortion during surgery, reproducibility is enhanced, producing a potentially superior refractive technique to radial keratotomy. Initial enthusiasm for PRK was based on the remarkable precision and smoothness of the ablated surface"A which it was hoped would be translated to an accurate change in refraction. An ideal result would be achieved if the corneal epithelium then assumed the contour of the ablated surface and there was no stromal regeneration. However, it is now apparent that an initial period of overcorrection is followed by regression to a relatively stable refraction and that in a proportion of cases stromal scarring can produce glare and a loss of best corrected visual acuity.--' Despite the precision of the PRK ablation the accuracy of the final refraction is at present only comparable to radial keratotomy. Accordingly 5 months after PRK approximately 55% of eyes have an unaided vision of 6/6, while 95% of eyes with -2-00 dioptres of error are within 1 dioptre of their intended refraction,7 but with considerable variation between individuals.578 This error in final refraction is due to the normal response of the cornea to an anterior keratectomy, which is to regenerate tissue to restore the integrity of the eye. Anterior stromal haze after PRK is thought to result from light scattered by scar tissue and has been shown to be more severe with increased ablation depth.5 7'" Gartry et al detected a reduction in corneal transparency in 92% ofpatients, ofwhom 18% had lost one to two lines of best corrected Snellen acuity 1 year after surgery.7 Additionally, if a small ablation zone (4 mm) is used positive spherical aberration (creating a halo effect) occurs in conditions of reduced illumination as the pupil dilates past the edge of the ablation.57 Although these halos can be eliminated by using a larger ablation zone,7 this entails an increased depth of ablation with its associated increased risk of  A potential loss of transparency in the optical axis of an otherwise healthy eye must give cause for concern. Unfortunately, topical corticosteroid therapy following PRK has not been demonstrated to reduce subepithelial haze or regression  Trials are therefore in progress to evaluate different postoperative regimens to minimise scar formation. As modification of the wound healing response may become an integral part of the management of patients following PRK it is opportune to review our current concepts of corneal scar formation.

title: Effects of fibroblast origin and phenotype on the proliferative potential of limbal epithelial progenitor cells.
abstract: The current study investigates potential differences in fibroblast phenotype across the anterior segment of the human eye with the aim to understanding factors that support the regenerative function of human limbal epithelial progenitor cells (LEPs) during wound healing. Separate cultures of fibroblasts were established from the cornea, limbus and sclera by growth in serum-supplemented medium. The resulting cultures were examined for potential differences in morphology and growth rate, as well as expression of CD34, CD45, CD90, CD141, CD271, vimentin and α-smooth muscle actin (α-sma). Finally, cultures were examined for their ability to support the growth of LEPs. While all cultures grew at a similar rate, scleral cultures often contained larger and more irregularly shaped cells which stained positive for α-sma. Western blotting confirmed a gradient of α-sma expression with lowest levels in corneal cultures. All three cultures stained positively for CD90 and vimentin, and were negative for CD34, CD45, CD141 and CD271. Only limbal or corneal irradiated fibroblasts supported the establishment of LEP cultures. While LEP colony forming efficiency and prominent expression of ABCG2, C/EBPδ and p63 was similar with either limbal or corneal fibroblasts, limbal fibroblasts supported significantly better growth. These results indicate that scleral fibroblasts have an increased capacity for myofibroblast formation which appears to negatively impact on their ability to support LEP growth. Superior growth of LEPs in the presence of limbal fibroblasts indicates a role for limbal fibroblasts in promoting the proliferation of limbal epithelium during wound healing.

title: Urokinase receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation.
abstract: Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.

title: Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea
abstract: Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease.

title: Development of the corneal stroma, and the collagen–proteoglycan associations that help define its structure and function
abstract: The cornea of the eye is a unique, transparent connective tissue. It is comprised predominantly of collagen fibrils, remarkably uniform in diameter and regularly spaced, organized into an intricate lamellar array. Its establishment involves a precisely controlled sequence of developmental events in which the embryonic cornea undergoes major structural transformations that ultimately determine tissue form and function. In this article, we will review corneal developmental dynamics from a structural perspective, consider the roles and interrelationships of collagens and proteoglycans, and comment on contemporary concepts and current challenges pertinent to developmental processes that result in an optically clear, mature cornea. Developmental Dynamics 237:2607–2621, 2008.

title: Growth factors in corneal wound healing following refractive surgery: A review.
abstract: The first part of this review article aims to provide important basic definitions of growth factor terminology, and to put forward a model for understanding the role of growth factors in a wound healing context. In the second part of the paper, we review the literature on growth factors in the cornea, including that on changes associated with wound healing following refractive surgery in the epithelial, stromal, and endothelial layers. The role of growth factors in stromal removal, corneal neovascularization, corneal innervation and wound healing is considered. The importance of the epithelial-stromal interaction is discussed, including the role of growth factors in keratocyte apoptosis. In the final section, we review the current literature on endogenous and exogenous modulation of growth factors in corneal wound healing. This includes important in vitro work but aims to emphasize clinically relevant results. Photorefractive keratectomy (PRK) may have short-term complications such as pain and haze, whereas laser in situ keratomilieusis (LASIK) may have longer-term adverse effects on corneal biomechanics. The emerging technique of laser epithelial keratomilieusis (LASEK) provides an interesting alternative wound which may be less susceptible to the inherent complications of PRK and LASIK. At present, the phenomenon of iatrogenic keratectasia following LASIK is not fully understood, but these features of wound healing following PRK may be amenable to growth factor modulation.

title: Growth factors and ocular wound healing
abstract: Protein growth factors regulate many of the processes in vitro that are essential for the process of normal ocular wound healing, including migration, mitosis and differentiation of cells. This has led to the hypothesis that peptide growth factors play key roles in regulating normal ocular wound healing in vivo. A corollary to this concept is that insufficient action of growth factors causes impaired healing, and prolonged action of growth factors produces excessive scarring. If both of these concepts are correct, then the addition of exogenous protein growth factors should enhance healing of chronic ocular wounds and reducing prolonged actions of growth factors should limit excessive scarring. Although much remains to be understood about the role of growth factors in ocular development and wound healing, results of a substantial number of laboratory and clinical experiments indicate that these hypotheses are generally correct. This article reviews the results of pre-clinical experiments and clinical trials investigating the roles of protein growth factors in ocular development and wound healing.

title: Collagen ultrastructural changes during stromal wound healing in organ cultured bovine corneas.
abstract: Corneal collagen ultrastructural changes occur during the healing process. The present study was designed to compare collagen ultrastructural changes after trephine wounding or flap creation. Bovine corneas were injured and maintained in organ culture for up to 4 weeks. Samples were removed from culture at 0, 1, 2, 3 and 4 weeks and snap frozen in liquid N(2). X-ray scattering was used to measure changes in collagen interfibrillar spacing, intermolecular spacing and fibrillar diameter. Some samples were fixed in 10% Neutral Buffered Formalin solution and wax embedded for immunohistochemistry to monitor myofibroblast differentiation in corneal flaps. Swelling effects (i.e. changes in interfibrillar spacing) were more severe in trephined corneas than in those with stromal flaps. Collagen fibrillar diameter remained normal in the periphery of injured corneas, but increased significantly in areas within and around the wound in trephined samples and in the epithelial incision site in corneal flaps. Intermolecular spacing was unchanged in all samples. In the flaps, alphaSMA expression was only detected in an area adjacent to the epithelial plug, and cell numbers gradually increased during the culture. We conclude that stromal swelling is more rapid for trephine-wounded corneas than in stromal flaps, indicating that the intensity of the corneal healing response depends on the type of injury.

title: Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction.
abstract: The purpose of this study was to evaluate specific keratocyte phenotypes (keratocyte, fibroblast, myofibroblast) for cell contractility and ability to contract extracellular matrix. Rabbit keratocyte phenotype was modulated by exposure to optimal proliferative doses of IGF-I, IL-1alpha, FGF2, PDGF-AB, and TGFbeta(1). Cells were then evaluated by immunocytochemistry, western blot, collagen gel contraction and LPA stimulation to measure: (1) focal adhesion (FA), fibronectin (FN) and f-actin assembly; (2) expression of alpha-smooth muscle actin (alpha-SMA); (3) ability to contract extracellular matrix and (4) determine contractile ability, respectively. Untreated keratocytes showed no ability to contract collagen matrix. IGF-I and IL-1alpha increased cell proliferation (70.2 and 74.3%, respectively) but did not alter keratocyte phenotype or ability to contract matrix. FGF2 and PDGF induced fibroblast differentiation with FA and FN assembly and significant (p<0.05) extracellular matrix contraction. TGFbeta(1) induced myofibroblast differentiation with prominent FA and FN assembly, expression of alpha-SMA and significantly greater (p<0.05) matrix contraction. Addition of LPA induced actin filament assembly in growth factor starved fibroblasts and myofibroblasts but had no effect on the cultured keratocyte phenotype. We report for the first time that the keratocyte phenotype is non-contractile and that cell quiescence is not a defining characteristic. We further establish that changes in environmental conditions modulate the keratocyte phenotype resulting in physiologically functional differences regarding cell contractility and capacity to contract extracellular matrix.

title: Development of an optimised culture medium for keratocytes in monolayer.
abstract: Our objective was to formulate a medium for monolayer culture optimising both keratocyte growth and preservation of the keratocyte phenotype.



An experimental matrix selected 14 media to test, using 7 components. Selection criteria were growth rates over 5 passages and expression of the CD34 marker.



Acetylcholine, insulin and vitamin C had no effect on growth and differentiation. The DMEM + Ham F12 1 : 1 based medium was selected for its initial effect on growth. At concentrations of 5 ng/ml, b-FGF improved the percentage of CD34+ cells without reducing growth rates. New-born calf serum (NCS) had a greater effect on growth than foetal calf serum (FCS). We showed three major interactions: between b-FGF and IGF-1, FCS and IGF-1 and NCS and b-FGF.



We selected the following medium, which provides optimal growth and preservation of the CD34+ phenotype: DMEM/HAM-F12 + 10% NCS + 5 ng/ml b-FGF + antibiotics.

title: Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum.
abstract: To determine whether keratocytes made fibroblastic in vitro by addition of fetal bovine serum to the medium regain the keratocyte phenotype after culture in serum-free medium.



Collagenase-isolated keratocytes from bovine corneas were plated in DMEM/F-12 containing 1% horse plasma, to allow cell attachment, and then cultured until day 4 in either DMEM/F-12 alone, to retain the keratocyte phenotype, or in DMEM containing 10% fetal bovine serum, to cause the keratocytes to become fibroblastic. Medium for the fibroblastic cells was replaced on day 4 with serum-free medium, and cells were cultured until day 12. Cell phenotypes were determined on days 4 to 5 and 11 to 12 of culture as follows: (1) by the morphologic appearance on phase-contrast microscopy; (2) by the levels of aldehyde dehydrogenase in the cells, determined by SDS-PAGE and Coomassie blue staining; (3) by the relative synthesis of collagen types I and V, determined by (14)C-proline radiolabeling; (4) by pepsin digestion and analysis of collagen types by SDS-PAGE autoradiography; (5) by relative synthesis of cornea-specific proteoglycan core proteins determined by analysis of chondroitinase- or endo-beta-galactosidase-generated radiolabeled core proteins by SDS-PAGE autoradiography; and (6) by the relative synthesis of keratan sulfate and chondroitin sulfate determined by (35)SO(4) radiolabeling and measuring the sensitivity to endo-beta-galactosidase and chondroitinase ABC.



Keratocytes cultured in serum-free medium appeared dendritic and became fibroblastic in appearance when exposed to medium containing serum. Keratocytes and fibroblasts synthesized a similar proportion of collagen types I and V. However, compared with the keratocytes, the fibroblasts possessed no aldehyde dehydrogenase and synthesized significantly higher levels of decorin and significantly lower levels of prostaglandin D synthase (PGDS) and keratan sulfate. Subsequent culture of the fibroblasts in serum-free medium did not restore aldehyde dehydrogenase to keratocyte levels but did restore the cell morphology to a more dendritic appearance and returned the synthesis of decorin, PGDS, and keratan sulfate to keratocyte levels.



The results of these studies indicate that primary cultures of keratocytes made fibroblastic by exposure to serum can return to their keratocyte phenotype in synthesizing extracellular matrix. These results also indicate that the differences in the organization of the collagenous matrix produced by keratocytes and fibroblasts may be related more to the different proteoglycan types than to the collagen types produced.

title: Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes.
abstract: After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.

title: Expression of alpha-smooth muscle (alpha-SM) actin during corneal stromal wound healing.
abstract: The purpose of this study was to correlate the temporal expression of alpha-smooth muscle specific actin (alpha-SM actin), a molecular marker for myofibroblast transformation, with corneal wound contraction.



After full-thickness, central corneal injury in rabbit eyes, the anterior width of the wound (wound gape) was measured in the same animals using in vivo confocal microscopy. In addition, animals were sacrificed at various times after injury for the determination of alpha-SM actin expression by immunofluorescent microscopy using a mouse monoclonal antibody specific for human alpha-actin. Antibody specificity was confirmed by Western blot analysis of normal and wound fibroblasts. Expression of alpha-SM actin also was related spatially to f-actin and the wound margin by co-localization with phalloidin and DTAF (5([4,6-dichlorotriazin-2yl]amino)fluorescein), a fluorescent marker bound to the wound margin.



Wound contraction was most evident from days 7 to 42, when wound gape progressively decreased from 574 +/- 120 microns to 250 +/- 61 microns. Thereafter, the wound remained stable to day 84 (304 +/- 58 microns). Expression of alpha-SM actin directly correlated with wound contraction--appearing across the wound at day 7, the full thickness of the wound at day 14, and the posterior wound at day 28. alpha-SM actin was localized exclusively to phalloidin-stained, f-actin microfilament bundles or stress fibers within wound healing fibroblasts, and the disappearance of alpha-SM actin correlated with the concomitant disappearance of stress fibers at days 28 to 42. Staining of the wound margin with DTAF confirmed that the expression of alpha-SM actin was limited to fibroblasts within the wound.



The expression of alpha-SM actin was directly correlated to corneal wound contraction, appearing at the initiation of and disappearing at the completion of the contraction process. Furthermore, the exclusive expression of alpha-SM actin by fibroblasts present only within the wound suggests that local environmental factors unique to the wound may play an important role in myofibroblast transformation.

title: Application of the reconstructed rabbit corneal epithelium model to assess the in-vitro eye irritant test of chemicals.
abstract: The rabbit corneal epithelium model (RCE model) was developed as a three-dimensional in vitro model to replace animal testing for the assessment of eye irritation. In the model, a stratified culture of rabbit corneal epithelial cells is grown at the air-liquid interface on collagen gel that acts as a parabasal membrane. Histological cross-sections show that the structure of the RCE model closely parallels that of the rabbit corneal epithelium. The eye irritation potency of test samples is estimated from the measurement of viability using the MTT assay in conjunction with the RCE model. A set of 30 chemicals belonging to different families with known in vivo Draize score was investigated with the in vitro eye irritation test using the RCE model in order to internally validate the protocol. Use of the RCE model at concentrations of 0.05%, 0.50%, and 1.00% and the calculation of the IC(50) and percentage of viability allowed the irritants to be divided into four classes. The performance of the in vitro eye irritation test at a concentration of 0.50% using the RCE model was characterized by good sensitivity (92.3%), good specificity (100%), and good accuracy (93.3%) compared with the irritation classification predicted by in vivo Draize score at concentrations of 10% and 100%. These results indicate that the RCE model may provide a useful and sensitive in vitro eye irritation test as an alternative method to the Draize test.

title: Radial keratotomy. II. Role of the myofibroblast in corneal wound contraction.
abstract: The cellular mechanism of corneal wound contraction after radial keratotomy (RK) was studied in a feline eye model. A total of 10 cat eyes were evaluated at various times from 0-30 days after surgery. Changes in the distribution of intracellular filamentous actin, nonmuscle myosin, alpha-actinin, surface membrane alpha 5 beta 1 integrin, and extracellular fibronectin were studied using immunofluorescence and laser confocal and electron microscopy. From day 3-7, staining for fibronectin increased along the wound margin. By day 7, keratocytes adjacent to the wound margin showed increased f-actin staining with intense staining for fibronectin compared with normal keratocytes. Myosin and alpha 5 beta 1 integrin expression was very weak at this time; alpha-actinin was not found. By day 14, fibroblasts within the wound formed f-actin microfilament bundles (stress fibers) which colocalized with fibronectin. Wound-healing fibroblasts also stained positively for alpha 5 beta 1 integrin, myosin, and alpha-actinin (the latter two were colocalized). The presence of myosin and alpha-actinin in the wound fibroblasts and the re-organization of f-actin into stress fibers by day 14 correlated with the development of wound contraction. A comparison of the cellular distribution of actin, myosin, and alpha-actinin with alpha 5 beta 1 integrin 14 days after injury suggested that integrin was localized along stress fiber bundles during wound contraction. The data from this study suggest that modulation of wound gape during healing of RK wounds may involve transformation of the corneal keratocyte to a myofibroblast-like cell and the subsequent formation of intracellular stress fibers composed of f-actin, nonmuscle myosin, and alpha-actinin. Based on the colocalization of fibronectin filaments and f-actin filaments and the unique distribution of alpha 5 beta 1 integrin, these findings support the hypothesis that the tension within the wound is generated by the formation of intracellular stress fibers and the interactions between stress fibers and the extracellular matrix, mediated by specific membrane receptor molecules.

title: Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture.
abstract: To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes.



Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes.



Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum.



A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.

title: Keratocyte Phenotype Mediates Proteoglycan Structure
abstract: In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-β-induced myofibroblasts, which expressed high levels of α-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.

title: Dense fibrillar collagen matrices: a model to study myofibroblast behaviour during wound healing.
abstract: Fibroblastic cells play an important part in wound healing. Human dermal fibroblasts seeded onto three-dimensional fibrillar collagen matrices migrate into the collagen network and differentiate into myofibroblasts. In order to evaluate the use of collagen matrices as model systems for studying myofibroblast phenotype during wound healing, myofibroblast behaviour migrating into dense or loose matrices was compared. The effect of collagen concentration on cell morphology, remodelling, proliferation and apoptosis of human myofibroblasts was evaluated. Myofibroblasts within dense collagen matrices (40 mg/ml) were spindle shaped, similar to cells observed during tissue repair. In contrast, cells within loose matrices (5mg/ml) were more rounded. Matrix hydrolysis activities (MT1-MMP and MMP2) did not differ between the two collagen concentrations. The myofibroblast proliferation rate was measured after 24h bromodeoxyuridine incorporation (BrdU). Cells in dense collagen matrices proliferated at a higher rate than cells in loose matrices at each culture time point tested. For example, 40% of cells in dense matrices were replicating compared to 10% of cells in loose matrices after 28 days in culture. Apoptotic cells were only detected in dense matrices from day 21 onwards when cells had already migrated into the collagen network. Taken together, these results show that a high collagen concentration has a stimulatory effect on myofibroblast proliferation and apoptosis, two important events in wound healing. Thus, dense matrices can be used to create controlled conditions to study myofibroblast phenotype.

title: Injured corneal epithelial cells promote myodifferentiation of corneal fibroblasts.
abstract: To determine whether injured corneal epithelial cells stimulate myodifferentiation in corneal fibroblasts and whether transforming growth factor (TGF)-beta is involved.



Rabbit corneal fibroblasts were cultured on collagen gel, with or without cocultured corneal epithelial cells or with partially scraped epithelial cells, on a companion plate separated by a permeable membrane. To evaluate fibroblast-induced gel contraction, gel thickness was measured daily relative to the original thickness. Total fibroblasts on the gel were counted. Myofibroblasts were counted by using immunocytochemical identification with anti-alpha-smooth muscle actin (alpha-SMA). TGF-beta was assayed in the media on days 3 and 6. These procedures also were performed in the presence of anti-TGF-beta antibody.



Gel contraction, alpha-SMA-positive cells, and total cell number were significantly greater on gels with injured epithelial cells than on gels without epithelial cells or with uninjured epithelial cells, as was TGF-beta concentration in the media. Anti-TGF-beta antibody eliminated these differences.



Injured epithelial cells stimulate myodifferentiation in fibroblasts through one or more soluble factors, including TGF-beta.

title: Stromal haze, myofibroblasts, and surface irregularity after PRK.
abstract: The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze. Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups: [see table in text]. Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin alpha4 to delineate the epithelial basement membrane. Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2+/-0.2 cells/400x field) and group V (1.8+/-0.4), with significantly more in groups II (3.5+/-1.8), III (6.8+/-1.6), VII (7.9+/-3.8), IV (12.4+/-4.2) and VI (14.6+/-5.1). The screened groups were significantly different from each other (p < 0.05), with myofibroblast generation increasing with higher surface irregularity in the -4.5 diopter PRK groups. The -9.0 diopter PRK group VI had significantly more myofibroblast generation than the -9.0 diopter PRK with PTK-smoothing group VII (p < 0.01). Areas of basement membrane disruption were demonstrated by staining corneas for integrin alpha4 and were prominent in corneas with grade I or higher haze. SMA-positive myofibroblasts tended to be present sub-adjacent to basement membrane defects. Late apoptosis was detected at 1 month after surgery within clusters of myofibroblasts in the sub-epithelial stroma. In conclusion, these results demonstrated a relationship between the level of corneal haze formation after PRK and the level of stromal surface irregularity. PTK-smoothing with methylcellulose was an effective method to reduce stromal surface irregularity and decreased both haze and associated myofibroblast density. We hypothesize that stromal surface irregularity after PRK for high myopia results in defective basement membrane regeneration and increased epithelium-derived TGFbeta signalling to the stroma that increases myofibroblast generation. Late apoptosis appears to have a role in the disappearance of myofibroblasts and haze over time.

title: Chemical and Topographical Effects on Cell Differentiation and Matrix Elasticity in a Corneal Stromal Layer Model
abstract: Control and maintenance of the keratocyte phenotype is vital to developing in vitro tissue engineered strategies for corneal repair. In this study the influence of topographical and chemical cues on the mechanical, phenotypical and genotypical behaviour of adult human derived corneal stromal (AHDCS) cells in three dimensional (3D) multi-layered organised constructs is examined. Topographical cues are provided via multiple aligned electrospun nanofiber meshes, which are arranged orthogonally throughout the constructs and are capable of aligning individual cells and permitting cell migration between the layers. The influence of chemical cues is examined using different supplements in culture media. A non-destructive indentation technique and optical coherence tomography are used to determine the matrix elasiticity (elastic modulus) and dimensional changes, respectively. These measurements were indicative of changes in cell phenotype from contractile fibroblasts to quiescent keratocytes over the duration of the experiment and corroborated by qPCR. Constructs containing nanofibers have a higher initial modulus, reduced contraction and organised cell orientation compared to those without nanofibers. Cell-seeded constructs cultured in serum-containing media increased in modulus throughout the culture period and underwent significantly more contraction than constructs cultured in serum-free and insulin-containing media. This implies that the growth factors present in serum promote a fibroblast-like phenotype; qPCR data further validates these observations. These results indicate that the synergistic effect of nanofibers and serum-free media plus insulin supplementation provide the most suitable topographical and chemical environment for reverting corneal fibroblasts to a keratocyte phenotype in a 3D construct.

title: Growth factor regulation of corneal keratocyte mechanical phenotypes in 3-D collagen matrices.
abstract: To assess how wound healing cytokines and the extracellular matrix (ECM) environment regulate the keratocyte mechanical phenotype.



Rabbit corneal keratocytes were plated within standard bovine or rat tail type I collagen matrices (2.5 mg/mL), compressed collagen matrices (approximately 100 mg/mL), or on collagen-coated dishes and cultured for up to 7 days in serum-free media, platelet derived growth factor BB (PDGF BB), insulin-like growth factor (IGF), TGFβ1, TGFβ2, or FGF2. F-actin, α-smooth muscle actin (α-SMA) and collagen fibrils were imaged using confocal microscopy. Cell morphology, local matrix reorganization, and global matrix contraction were quantified digitally.



IGF and PDGF BB stimulated elongation of keratocytes and extension of dendritic processes within 3-D matrices, without inducing stress fiber formation or collagen reorganization. In contrast, treatment with TGFβ1 and TGFβ2 increased keratocyte contractility, as indicated by stress fiber formation and matrix compaction and alignment. This transformation was enhanced at higher cell densities within standard 3-D matrices, in which α-SMA was incorporated into stress fibers. In contrast, α-SMA was expressed within compressed 3-D matrices even at low cell density. FGF2 did not produce significant cytoskeletal or matrix reorganization in standard 3-D matrices; however, stress fibers were consistently expressed within compressed collagen matrices and on rigid two-dimensional substrates. Inhibiting Rho kinase blocked both TGFβ- and FGF2-induced stress fiber formation.



Keratocytes cultured in IGF or PDGF BB maintain a quiescent mechanical phenotype over a range of matrix environments. In contrast, the mechanical phenotypes induced by FGF and TGFβ vary in response to the structural and/or mechanical properties of the ECM.

title: Growth factors: importance in wound healing and maintenance of transparency of the cornea
abstract: The mechanism of corneal wound healing has not been clarified yet. However, evidence has accumulated that various kinds of growth factor such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) play a key role in corneal wound healing. For example, these growth factors are expressed in the corneal epithelial cells, keratocytes and endothelial cells, and their receptors are expressed in the corneal cells. Furthermore, these growth factors promote the proliferation of corneal cells and induce the migration of corneal cells. In addition to the growth factors, inflammatory cytokines such as interleukin (IL)-1, IL-6 and TNF-alpha are involved in corneal wound healing. These cytokines are expressed in the normal and inflammatory cornea after infections, alkaliburn, etc. where they control the growth of corneal cells and induce the migration of corneal cells. Thus, a number of growth factors and cytokines function in the regulation of corneal cell proliferation and in the maintenance of corneal transparency.

title: Growth factors in the anterior segment: role in tissue maintenance, wound healing and ocular pathology.
abstract: A number of growth factors and their associated receptors, including epidermal growth factor, transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, fibroblast growth factor and platelet-derived growth factor have been detected in the anterior segment of the eye. On binding to cellular receptors, these factors activate signalling cascades, which regulate functions including mitosis, differentiation, motility and apoptosis. Production of growth factors by corneal cells and their presence in the tear fluid and aqueous humour is essential for maintenance and renewal of normal tissue in the anterior eye and the prevention of undesirable immune or angiogenic reactions. Growth factors also play a vital role in corneal wound healing, mediating the proliferation of epithelial and stromal tissue and affecting the remodelling of the extracellular matrix (ECM). These functions depend on a complex interplay between growth factors of different types, the ECM, and regulatory mechanisms of the affected cells. Imbalances may lead to deficient wound healing and various ocular pathologies, including edema, neovascularization and glaucoma. Growth factors may be targeted in therapeutic ophthalmic applications, through exogenous application or selective inhibition, and may be used to elicit specific cellular responses to ophthalmic materials. A thorough understanding of the mechanism and function of growth factors and their actions in the complex environment of the anterior eye is required for these purposes. Growth factors, their function and mechanisms of action as well as the interplay between different growth factors based on recent in vitro and in vivo studies are presented.

title: Transforming growth factor‐β3 regulates assembly of a non‐fibrotic matrix in a 3D corneal model
abstract: Corneal tissue engineering has attracted the attention of many researchers over the years, in part due to the cornea's avascularity and relatively straightforward structure. However, the highly organized and structured nature of this optically clear tissue has presented a great challenge. We have previously developed a model in which human corneal fibroblasts (HCFs) are stimulated by a stable vitamin C (VitC) derivative to self‐assemble an extracellular matrix (ECM). Addition of TGFβ1 enhanced the assembly of ECM; however, it was accompanied by the upregulation of specific fibrotic markers. In this study, we tested the effects of all three TGFβ isoforms (‐β1, ‐β2 and ‐β3) on ECM production, as well as expression of fibrotic markers. HCFs were grown in four media conditions for 4 weeks: control, VitC only; T1, VitC + TGFβ1; T2, VitC + TGFβ2; and T3, VitC + TGFβ3. The cultures were analysed with western blots, TEM and indirect immunofluorescence (IF). Compared to controls, all TGFβ isoforms stimulated matrix production by about three‐fold. IF showed the presence of type III collagen and smooth muscle actin (SMA) in T1 and T2; however, T3 showed little to no expression. In western blots, T3 stimulated a lower type III:type I collagen ratio when compared to the other conditions. In addition, TEM indicated that T3 stimulated a higher level of matrix alignment and organization. HCFs stimulated by VitC and TGFβ3 appear to generate a matrix that mimics the normal adult or developing human cornea, whereas TGF‐β1 and ‐β2 drive the constructs towards a more fibrotic path.

title: Differential regulation of key stages in early corneal wound healing by TGF-beta isoforms and their inhibitors.
abstract: Inhibition of TGF-beta reduces myofibroblast differentiation and fibrosis in the cornea. Determining the actions of distinct TGF-beta isoforms and their inhibitors during early corneal wound healing is an essential step in guiding therapeutic intervention.



Bovine serum-free corneal cell and wounded organ cultures were challenged with a range of concentrations of TGF-beta1, -beta2, and -beta3; IL-10; and neutralizing human monoclonal antibodies (mAbs) against TGF-beta1 (CAT-192) or -beta2, (CAT-152). Cultures were assessed for re-epithelialization, proliferation (cell counts and cresyl violet assay), morphology (histologic examination), repopulation of the area under the wound, and myofibroblast transformation (alpha-smooth muscle actin) between 0 and 5 days.



TGF-beta1 delayed re-epithelialization, increased repopulation of the stroma, increased keratocyte proliferation and was the only isoform to promote myofibroblast differentiation. The anti-TGF-beta1 mAb, CAT-192 promoted re-epithelialization and reduced repopulation of the stroma. Exogenous TGF-beta3 had little effect on re-epithelialization but reduced repopulation of the stroma. IL-10 promoted corneal re-epithelialization at low doses but inhibited this response at high doses. Stromal repopulation was prevented by all doses of IL-10. TGF-beta2 or the anti-TGF-beta2 mAb, CAT-152 had little effect on any repair parameter.



The results confirm TGF-beta1 as the principal isoform in corneal wound healing and suggest that inhibition of the action of TGF-beta1 can promote corneal wound healing. Treatment with the anti-TGF-beta1 mAb CAT-192 accelerates corneal re-epithelialization but reduces cell repopulation of the stroma. The cytokines TGF-beta3 and IL-10 have opposing actions to that of TGF-beta1.

title: Proteoglycan Expression during Transforming Growth Factor β-induced Keratocyte-Myofibroblast Transdifferentiation*
abstract: Keratocytes of the corneal stroma secrete a unique population of proteoglycan molecules considered essential for corneal transparency. In healing corneal wounds, keratocytes exhibit a myofibroblastic phenotype in response to transforming growth factor β (TGF-β), characterized by expression of α-smooth muscle actin. This study examined proteoglycan and collagen expression by keratocytesin vitro during the TGF-β-induced keratocyte-myofibroblast transition. TGF-β-treated primary bovine keratocytes developed myofibroblastic features, including actin stress fibers anchored to paxillin-containing focal adhesions, cell-associated fibronectin, α5 integrin, and α-smooth muscle actin. Collagen I and III protein and mRNA increased in response to TGF-β. Secretion of [35S]sulfate-labeled keratan sulfate proteoglycans decreased markedly in response to TGF-β. Dermatan sulfate proteoglycans, however, increased in size and abundance. Protein and mRNA transcripts for normal stromal proteoglycans (lumican, keratocan, mimecan, and decorin) all decreased in response to TGF-β, but protein expression and mRNA for biglycan, a proteoglycan present in fibrotic tissue, was markedly up-regulated. These results show that TGF-β in vitroinduces a proteoglycan expression pattern similar to that of corneal scars in vivo. This altered proteoglycan expression occurred coordinately with transdifferentiation of keratocytes to the myofibroblastic phenotype, implicating these cells as the source of fibrotic tissue in nontransparent corneal scars.

title: Human corneal fibrosis: an in vitro model.
abstract: Corneal injury may ultimately lead to a scar by way of corneal fibrosis, which is characterized by the presence of myofibroblasts and improper deposition of extracellular matrix (ECM) components. TGF-beta1 is known to stimulate overproduction and deposition of ECM components. Previously, an in vitro three-dimensional (3-D) model of a corneal stroma was developed by using primary human corneal fibroblasts (HCFs) stimulated with stable vitamin C (VitC). This model mimics corneal development. The authors postulate that with the addition of TGF-beta1, a 3-D corneal scar model can be generated.



HCFs were grown in four media conditions for 4 or 8 weeks: VitC only; VitC+TGF-beta1 for the entire time; VitC+TGF-beta1 for 1 week, then VitC only for 3 or 7 weeks; and VitC for 4 weeks, then VitC+TGF-beta1 for 4 weeks. Cultures were analyzed with TEM and indirect immunofluorescence.



Compared with the control, addition of TGF-beta1 increased construct thickness significantly, with maximum increase in constructs with TGF-beta1 present for the entire time-2.1- to 3.2-fold at 4 and 8 weeks, respectively. In all TGF-beta-treated cultures, cells became long and flat, numerous filamentous cells were seen, collagen levels increased, and long collagen fibrils were visible. Smooth muscle actin, cellular fibronectin, and type III collagen expression all appeared to increase. Cultures between weeks 4 and 8 showed minimal differences.



Human corneal fibroblasts stimulated by VitC and TGF-beta1 appear to generate a model that resembles processes observed in human corneal fibrosis. This model should be useful in examining matrix deposition and assembly in a wound-healing situation.

title: Fibroblast mechanics in three-dimensional collagen matrices.
abstract: Fascia provides mechanical support and frameworks for the other tissues of the body. Type 1 collagen is the major protein component of fascia, and fibroblasts are the cell type primarily responsible for its biosynthesis and remodeling. Research on fibroblasts interacting with collagen matrices provides new insights regarding how cell-matrix tension state and growth factor specificity regulate cell migration and matrix remodeling.

title: Stromal-epithelial interactions in the cornea
abstract: Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.

title: Altered KSPG expression by keratocytes following corneal injury.
abstract: Keratocytes synthesize keratan-sulfate proteoglycans (KSPG), lumican and keratocan, to develop and maintain proper collagen interfibrillar spacing and fibril diameter characteristics of the transparent cornea. The purposes of this study are to compare the expression patterns of KSPGs and keratin 12 (K12) respectively by corneal keratocytes and epithelial cells after three different types of injuries; partial and total epithelial debridement and alkali burn.



Corneas of 8-12 week old C57Bl/6J or FVBN mice were wounded by partial epithelial (2 mm in diameter) and total epithelial debridement, and alkali burn (0.1 M NaOH, 30 s) and were allowed to heal for various periods of time, from 1 to 84 days. The corneas were then subjected to light microscopy, in situ and Northern hybridization and RT-PCR for examining the expression of K12 and KSPG in the corneal epithelium and stroma, respectively. Immunohistochemistry with anti-alpha-smooth muscle actin (alpha-SMA) was used to identify myofibroblasts in the stroma of injured cornea.



In 2-3 days, partial epithelial denuded corneas were resurfaced by corneal epithelium positive for K12, and stromal edema caused by debridement disappeared. Total epithelial debridement wounded corneas were resurfaced by conjunctival epithelial cells in 2 weeks. Stromal edema in the total epithelial debridement corneas began to subside after 6 weeks. Corneal epithelial cells resurfaced alkali burned corneas within 3-5 days. In situ and Northern hybridization showed a decrease in keratocan and lumican expression at 6 weeks and increased at 12 weeks post-injury in all wound types. Alpha-SMA positive myofibroblasts in the cornea were detected via immunostaining at the time point when KSPG expression was lowest, 6 weeks post-injury.



The results suggest keratocan and lumican are down-regulated during wound healing at 6 weeks and returned to higher levels at 12 weeks post-injury; implicating that the cells repopulating the injured corneal stroma regained the characteristic function of keratocytes independent of the wound types. However, complete epithelial removal results in irreversible loss of K12 expression.

title: Lumican is required for neutrophil extravasation following corneal injury and wound healing
abstract: An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum−/− mice and a novel transgenic mouse, Lum−/−,Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum−/− mice and this defect was rescued by the expression of lumican in the corneas of Lum−/−,Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

title: Decorin biology, expression, function and therapy in the cornea.
abstract: Decorin is a small leucine-rich proteoglycan (SLRP) that plays a vital role in many important cellular processes in several tissues including the cornea. A normal constituent of the corneal stroma, decorin is also found in the majority of connective tissues and is related structurally to other small proteoglycans. It interacts with various growth factors such as epidermal growth factor (EGF) and transforming growth factor beta (TGFβ) to regulate processes like collagen fibrillogenesis, extracellular matrix (ECM) compilation, and cell-cycle progression. Studies have linked decorin dysregulation to delayed tissue healing in patients with various diseases including cancer. In the cornea, decorin is involved in the regulation of transparency, a key function for normal vision. It has been reported that mutations in the decorin gene are associated with congenital stromal dystrophy, a disease that leads to corneal opacity and visual abnormalities. Decorin also antagonizes TGFβ in the cornea, a central regulatory cytokine in corneal wound healing. Following corneal injury, increased TGFβ levels induce keratocyte transdifferentiation to myofibroblasts and, subsequently, fibrosis (scarring) in the cornea. We recently reported that decorin overexpression in corneal fibroblasts blocks TGFβ-driven myofibroblast transformation and fibrosis development in the cornea in vitro suggesting that decorin gene therapy can be used for the treatment of corneal scarring in vivo.

title: Decorin and biglycan of normal and pathologic human corneas.
abstract: Corneas with scars and certain chronic pathologic conditions contain highly sulfated dermatan sulfate, but little is known of the core proteins that carry these atypical glycosaminoglycans. In this study the proteoglycan proteins attached to dermatan sulfate in normal and pathologic human corneas were examined to identify primary genes involved in the pathobiology of corneal scarring.



Proteoglycans from human corneas with chronic edema, bullous keratopathy, and keratoconus and from normal corneas were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), quantitative immunoblotting, and immunohistology with peptide antibodies to decorin and biglycan.



Proteoglycans from pathologic corneas exhibit increased size heterogeneity and binding of the cationic dye alcian blue compared with those in normal corneas. Decorin and biglycan extracted from normal and diseased corneas exhibited similar molecular size distribution patterns. In approximately half of the pathologic corneas, the level of biglycan was elevated an average of seven times above normal, and decorin was elevated approximately three times above normal. The increases were associated with highly charged molecular forms of decorin and biglycan, indicating modification of the proteins with dermatan sulfate chains of increased sulfation. Immunostaining of corneal sections showed an abnormal stromal localization of biglycan in pathologic corneas.



The increased dermatan sulfate associated with chronic corneal pathologic conditions results from stromal accumulation of decorin and particularly of biglycan in the affected corneas. These proteins bear dermatan sulfate chains with increased sulfation compared with normal stromal proteoglycans.

title: Decorin transfection suppresses profibrogenic genes and myofibroblast formation in human corneal fibroblasts.
abstract: Decorin, a small leucine-rich proteoglycan, is a natural inhibitor of transforming growth factor beta (TGFbeta). Myofibroblast and haze formation in the cornea have been attributed to TGFbeta hyperactivity released from corneal epithelium following injury to eye. This study tested the hypothesis that decorin-gene transfer inhibits TGFbeta-driven myofibroblast and haze formation in the cornea. Human corneal fibroblast (HSF) cultures generated from donor human corneas were used. Decorin cDNA was cloned into mammalian expression vector. Restriction enzyme analysis and DNA sequencing confirmed the nucleotide sequence of generated vector construct. The decorin gene cloned into mammalian expression vector was introduced into HSF with lipofectamine transfection kit. Expression of decorin in selected clones was characterized with RT-PCR, immunocytochemistry and western blotting. Phage contrast microscopy and trypan blue exclusion assay evaluated the effects of decorin-gene transfer on HSF phenotype and viability, respectively. Real-time PCR, western blot and immunocytochemistry were used to analyze inhibitory effects of decorin-gene transfer on TGFbeta-induced myofibroblast formation by measuring differential expression of alpha smooth muscle actin (SMA), a myofibroblast marker, mRNA and protein expression. Analysis of variance (ANOVA) and the Bonferroni-Dunn adjustment for repeated measures were used for statistical analysis. Our data indicate that decorin-gene transfer into HSF do not alter cellular phenotype or viability. Decorin over-expressing HSF clones grown in the presence of TGFbeta1 under serum-free conditions showed a statistically significant 80-83% decrease in SMA expression (p value < 0.01) compared to naked-vector transfected clones or un-transfected HSF controls. Decorin-transfected, naked-vector transfected and un-transfected HSF grown in the absence of TGFbeta1 showed no or extremely low expression of SMA. Furthermore, decorin over-expression did not affect HSF phenotype and decreased TGFbeta-induced RNA levels of profibrogenic genes such as fibronectin, collagen type I, III, and IV that play important role in stromal matrix modulation and corneal wound healing. The results of study suggest that decorin-gene transfer effectively prevents TGFbeta-driven transformation of keratocyte and corneal fibroblast to myofibroblasts. We postulate that decorin-gene therapy can be used to treat corneal haze in vivo.

title: Heterogeneity of collagens in rabbit cornea: type III collagen.
abstract: Whole neonate rabbit corneas and adult corneas containing 2-week-old scars were incubated in the presence of [14C] glycine. Radiolabeled collagen extracted from the corneas and scar tissue were analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography to determine the types and relative quantity of collagen polypeptides present and synthesized by these tissues. In addition to other collagen types, type III was found in both neonate cornea and scar tissue from adult cornea, albeit in relatively small quantities. Type III collagen in normal cornea was associated with the residue after pepsin digestion and formic acid extraction of the tissue, and the same type of collagen was extracted from scar tissue after similar treatment. Type III collagen-specific monoclonal antibody bound to developing normal corneas and healing adult tissue sections, as determined by immunofluorescence. Antibody binding was localized to the endothelium and growing Descemet's membrane in fetal and neonate corneas, and restricted to the most posterior region of the corneal scar tissue. Although monoclonal antibody to keratan sulfate, used as a marker for stromal fibroblasts, bound to most of the scar tissue, the antibody failed to bind to the posterior scar tissue positive for type III collagen. We conclude that endothelial cells from fetal and neonate rabbit cornea and endothelium-derived fibroblasts from healing wounds of adult cornea synthesize and deposit type III collagen. Moreover, this collagen appears to be incorporated into the growing Descemet's membrane of normal corneas and narrow posterior portion of the scar tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Synthesis of type III collagen by fibroblasts from the embryonic chick cornea
abstract: Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cells at Hamburger-Hamilton stages 20--30 but not in the stroma at any age. Intact corneas from embryos older than stage 30 contain and synthesize type I collagen but no detectable type III collagen. However, whole stromata subjected to collagenase treatment and scraping (to remove epithelium and endothelium) and stromal fibroblasts from such corneas inoculated in vitro begin synthesis of type III collagen within a few hours while continuing to synthesize type I collagen. As demonstrated by double-antibody staining, most corneal fibroblasts contain collagen types I and III simultaneously. Collagen type III was identified biochemically in cell layers and media after chromatography on carboxymethylcellulose be detection of disulfide-linked alpha l (III)3 by SDS gel electrophoresis. The conditions under which the corneal fibroblasts gain the ability to synthesize type III collagen are the same as those under which they lose the ability to synthesize the specific proteoglycan of the cornea: the presence of corneal-type keratan sulfate.

title: Online monitoring of the mechanical behavior of collagen hydrogels: influence of corneal fibroblasts on elastic modulus.
abstract: Collagen hydrogels have been widely used to model biological systems and examine cell behavior in vitro. Of increasing interest is how cells affect the mechanical characteristics of their surrounding matrix and vice versa over long culture periods. In this study, the change in mechanical properties of collagen hydrogels embedded with human corneal fibroblasts was examined over a 6-week culture period using a novel online spherical indentation system. The elastic modulus of the hydrogels was found to increase during the first 2 weeks of culture and decrease after 4 weeks in culture. The effects of actin polymerization and matrix metalloproteinase inhibitors were also examined, which verified some mechanisms involved in the alteration of the mechanical properties such as cell tensile forces and other potential factors. This online monitoring technique demonstrates the ability to examine the mechanical properties of cell-seeded constructs in response to the culture environments--in particular, the response to the addition of drugs or chemical reagents--which will provide a useful tool in studying the mechano-feedback loop between cells and their surrounding matrix.

title: Mechanical characterization of biomimetic membranes by micro-shaft poking
abstract: The popularity of biomimetic membranes has recently increased due to their biomedical applications such as tissue engineering/regenerative medicine and biosensors. Characterization of the viscoelastic properties of these membranes is important in developing functional membranes. A new micro-shaft poking technique has been developed, which is free from the complication of substrate backing, and which is normally an intractable problem in conventional indentation testing of membrane materials. A tailored indentation apparatus with a spherical indenter and a force resolution and displacement of 1 μN and 1 μm was constructed. Alginate and agarose were used to fabricate biomimetic membranes. Chicken epidermis was examined to represent a real biological tissue. The results show that the elastic modulus increased with concentration in hydrogels. Epidermis moduli appeared to increase with increased strain. Stress relaxation tests have also been conducted to examine the time-dependent behaviours of various hydrogels and a viscoelastic model has been correspondingly developed and applied to describe the experimental results. Potential applications of this new instrument to other membranes, both artificial and biological, have also been addressed.

title: An Indentation Technique to Characterize the Mechanical and Viscoelastic Properties of Human and Porcine Corneas
abstract: Cornea is a load-bearing tissue whose mechanical and viscoelastic characteristics are not well understood, due to the challenge associated with most of the measurements. A novel indentation technique has been developed for mechanical characterization of human and porcine corneal tissue, using a tailored depth-sensing microindentation instrument. During indentation, the corneas were suspended by clamping the edges of the cornea, thus allowing depth-sensing measurement free from the complication of the backing substrate. The deformation displacement and the amount of force applied by the indenter were used to obtain hysteresis and stress relaxation data for both human and porcine corneas. Optical coherence tomography was used to measure the thickness of the cornea. Simple theoretical analyses have been undertaken to explain the loading–unloading and the stress relaxation data. The effect of swelling on the mechanical properties of the cornea was also examined. Porcine corneas appeared to be less stiff and to demonstrate more linear response than human corneas under loading. More importantly, it is shown that swelling reduced the strength of the corneas. Our results demonstrate that this new indentation system can be used to characterize the mechanical and viscoelastic properties of corneas.

title: Effect of human corneal keratocytes and retinal pigment epithelial cells on the mechanical properties of micropatterned collagen films.
abstract: Collagen-based micropatterned films were seeded with human corneal keratocyte and epithelial cells to study their mechanical properties as tissue engineering substrates. The patterns were in the form of parallel channels with slanted walls. Influence of cell presence, type and growth on the mechanical properties of the films was investigated. Unseeded films showed gradual strength reduction from an initial value of 0.046 N/mm, possibly due to degradation, down to 0.032+/-0.012 N/mm in 2 weeks. Keratocyte growth was found to significantly improve the mechanical behavior of the films upon 1 week of incubation (0.067+/-0.017 N/mm) and the improvement continued gradually over the next 2 weeks. Films seeded with D407 retinal pigment epithelial cells, on the other hand, experienced a decrease (0.023+/-0.011 N/mm), followed by a slight increase in mechanical properties in the 21-day period. A steady increase in the number of keratocytes along the channels, cytoskeleton alignment and extracellular matrix (ECM) secretion restricted to the channels was observed. Increase in strength observed with keratocytes and, to a lesser extent, with the epithelial cells can be attributed to directional ECM synthesis and the orientation of the cells and their cytoskeleton which contribute to the strength in the direction of the channels. This study showed that cell, especially keratocyte, presence compensates for the degradation of collagen films and improve the overall mechanical properties of the engineered tissue.

title: Laser epithelial keratomileusis in 2010 – a review
abstract: Photorefractive keratectomy, laser epithelial keratomileusis (LASEK) and Epi-LASIK are all variants of a similar type refractive surgery involving laser on the surface of the cornea and differ mainly in management of the epithelium. Although laser in situ keratomileusis (LASIK) is currently the most popular form of refractive surgery, LASEK is the procedure of choice in some patients. We highlight potential complications of LASEK and how these may be managed. Following laser refractive surgery, corneal thickness is reduced, which has implications for intraocular pressure measurement and glaucoma screening and management. This is particularly important following surface laser procedures where no evidence of previous surgery may be visible. In the event that cataract surgery is required at a later date, correct calculation of the appropriate intraocular lens power can be difficult and it thus important that patients are given their preoperative keratometry readings and refraction. Compared with LASIK patients, those who undergo LASEK are considered to be at lower risk of corneal ectasia. Improved understanding of wound healing post LASEK and better postoperative pain management are ongoing challenges.

title: A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes.
abstract: The aim of this study was to construct a rabbit anterior cornea replacement with an acellular porcine cornea matrix (APCM) as a scaffold. The scaffold was prepared from fresh porcine corneas which were treated with 0.5% (wt./vol.) sodium dodecyl sulfate (SDS) solution and stirred for 24 h in a 4 degrees C refrigeration chamber. The complete removal of corneal cells was confirmed by H&E and DAPI staining. The stroma structure and mechanical properties were well preserved. The extracts had no cytotoxicity to rabbit corneal keratocytes, epithelial and endothelial cells as determined by MTT assay. Moreover, there was no sign that an immune reaction occurred in or around the transplanted disks within 6 months of animal implantation. To construct a rabbit anterior cornea replacement, keratocytes were injected into APCM and cultured for 7 days in a dynamic culturing system, followed by culturing corneal epithelial cells on the stroma construct surface for another 7 days. The phenotype of the construct was similar to normal rabbit corneas, with high expression of cytokeratin 3 in the epithelial cell layer and expression of vimentin in the stromal cells. These results suggested that the APCM developed by using SDS might be a suitable scaffold for cornea tissue engineering.

title: The immune privilege of corneal grafts
abstract: Keratoplasty is the oldest and one of the most successful forms of solid tissue transplantation. In the United States, over 33,000 corneal transplants are performed each year. Unlike other forms of tissue transplantation, keratoplasties are routinely performed without the aid of tissue typing or systemic immunosuppressive drugs. In spite of this, 90% of the first‐time corneal transplants will succeed–a condition that demonstrates the immune privilege of keratoplasties. The avascular nature of the corneal allograft bed led many to suspect that corneal grafts were sequestered from the immune apparatus. Although pleasing in its simplicity, this explanation has given way to a more comprehensive hypothesis that embodies multiple, interdependent mechanisms, which promote the long‐term survival of corneal allografts. These mechanisms conspire to interrupt the transmission of immunogenic stimuli to peripheral lymphoid tissues; induce the generation of a deviated immune response; and neutralize immune effector elements at the host‐graft interface. This paradigm is analogous to a three‐legged stool. Disassembly of any one of the three components results in the collapse of immune privilege. Strategies to re‐establish corneal immune privilege may have clinical application for high‐risk hosts who have rejected previous corneal allografts.

title: Corneal Graft Rejection: Incidence and Risk Factors
abstract: To determine the incidence and risk factors of late corneal graft rejection after penetrating keratoplasty (PKP).  Records of all patients who had undergone PKP from 2002 to 2004 without immunosuppressive therapy other than systemic steroids and with at least one year of follow up were reviewed. The role of possible risk factors such as demographic factors, other host factors, donor factors, indications for PKP as well as type of rejection were evaluated.  During the study period, 295 PKPs were performed on 286 patients (176 male, 110 female). Mean age at the time of keratoplasty was 38±20 (range, 40 days to 90) years and mean follow up period was 20±10 (range 12-43) months. Graft rejection occurred in 94 eyes (31.8%) at an average of 7.3±6 months (range, 20 days to 39 months) after PKP. The most common type of rejection was endothelial (20.7%). Corneal vascularization, regrafting, anterior synechiae, irritating sutures, active inflammation, additional anterior segment procedures, history of trauma, uncontrolled glaucoma, prior graft rejection, recurrence of herpetic infection and eccentric grafting increased the rate of rejection. Patient age, donor size and bilateral transplantation had no significant influence on graft rejection.  Significant risk factors for corneal graft rejection include corneal vascularization, anterior synechiae, irritating sutures, active inflammation, regrafting, additional surgery, trauma, uncontrolled intraocular pressure, history of graft rejection, recurrent herpetic infection, eccentric grafting and corneal scarring. Recipient age and donor cornea size do not seem to be risk factors for corneal graft rejection.

title: Mechanisms of corneal allograft rejection and regional immunosuppression
abstract: Corneal transplantation has not matched the improvements in outcome seen with other clinical transplantation procedures. The therapeutic strategies, which have improved the outcomes of solid vascularised organs are not applicable to corneal transplantation. Corneal transplantation is different with respect to relevant transplantation biology and the clinical context in which it is practiced. New approaches need to be developed which provide regional rather than systemic immunosuppression. The accessibility of the cornea makes it particularly suitable for topical medication and for gene therapy approaches. Engineered antibodies, small enough to pass through the cornea, and directed at key molecules in the allograft response have been developed. Gene therapy had been developed using viral vectors to transfect the corneal endothelium with the genes for immunosuppressive lymphokines. Both approaches show promise.

title: Xenotransplantation and risks of zoonotic infections
abstract: The shortage of human organs and tissues for transplantation and the advances in immunology of rejection and in genetic engineering have renewed interest in xenotransplantation - the transplantation of animal organs, tissues or cells to humans. Clinical trials have involved the use of non-human primate, porcine, and bovine cells/tissues/organs. In recent years, research has focused mainly on pigs as donors (especially, pigs genetically engineered to carry some human genes). One of the major concerns in xenotransplantation is the risk of transmission of animal pathogens, particularly viruses, to recipients and the possible adaptation of such pathogens for human-to-human transmission. Porcine endogenous retroviruses (PERVs) have been of special concern because of their ability to infect human cells and because, at present, they cannot be removed from the source animal's genome. To date, retrospective studies of humans exposed to live porcine cells/tissues have not found evidence of infection with PERV but more extensive research is needed. This article reviews infectious disease risks associated with xenotransplantation, some measures for minimizing that risk, and microbiological diagnostic methods that may be used in the follow-up of xenotrasplant recipients.

title: WZS‐pig is a potential donor alternative in corneal xenotransplantation
abstract: To explore the characteristics of rejection of Wuzhishan (WZS) pig‐to‐rhesus monkey corneal transplants and to evaluate WZS pig corneas as potential donor material for a clinical setting.

title: The host response in experimental corneal xenotransplantation
abstract: In addressing the worldwide shortage of human donor cornea for transplantation, animal cornea may be a substitute if mechanisms of xenogeneic (cross-species) rejection can be identified and controlled. Xenotrans-plantation of solid organs is followed by hyperacute rejection within minutes due to humoral graft rejection. In an experimental model corneal xenografts in rats survived for 2-3 days, depending on the phylogenetic disparity of the donor animal. Endothelial injury was the specific cause of graft failure, probably mediated by humoral rejection mechanisms. A later cell-mediated rejection response was seen. The potent humoral response is the most important feature differentiating xenograft from allograft rejection.

title: New Dacron tissue colonisable keratoprosthesis: clinical experience.
abstract: Keratoprostheses (KPs) are made of an optical cylinder integrated with a supporting element which conditions their biocompatibility. A new KP with a Dacron tissue colonisable support has been designed in order to reduce significantly the complication rate. This new KP was implanted into 20 eyes of 20 patients with bilateral corneal blindness unsuited to a corneal implant. The follow up ranged from 24 to 96 months. All 20 patients had some improved visual acuity with 13 retaining this improvement for more than 2 years. These favourable results may be indicative of the reliability of the new KP.

title: In vitro culture characteristics of corneal epithelial, endothelial, and keratocyte cells in a native collagen matrix.
abstract: The objective of this investigation was to demonstrate the effectiveness of a tissue-engineered collagen sponge as a substrate for the culture of human corneal cells. To that end, human kerotocyte, epithelial, and endothelial cells were cultured separately on collagen sponges composed of native fibrillar collagen with a pore size of approximately 0.1 mm. Co-culture experiments were also performed (epithelial/endothelial and epithelial/keratocyte cultures). Proliferation of keratocytes and matrix production was assessed. The morphology of the epithelial and endothelial cell cultures was characterized by histology and scanning electron microscopy. Keratocytes cultured on collagen sponges exhibited increased matrix synthesis over time as well as proliferation and repopulation of the matrix. Epithelial and endothelial cells showed the ability to migrate over the collagen sponge. The thickness of the epithelial layer was influenced by soluble factors produced by endothelial cells. The morphology of the bottom layer of epithelial cells was influenced by the presence of keratocytes in the culture. These studies indicate that human corneal cells exhibit normal cell phenotype when cultured individually on an engineered collagen sponge matrix and co-culture of different cell types in the cornea can influence cell behavior.

title: Cataract surgery with the AlphaCor artificial cornea
abstract: To review the outcomes of AlphaCor artificial cornea (Argus Biomedical Pty Ltd.) implantation concurrent with or subsequent to cataract surgery.  Nonrandomized multicenter clinical trial.  AlphaCor devices were implanted under an approved protocol. Extracapsular cataract extraction with intraocular lens implantation was concurrently performed in 5 cases; an additional case had a displaced cataractous lens removed after severe blunt trauma subsequent to AlphaCor implantation.  Cataract surgery was difficult because the view was restricted and residual soft lens matter was retained. There was an increased incidence of postoperative complications (eg, melt, glaucoma) in cataract cases compared with noncataract AlphaCor cases. The mean best corrected visual acuity was worse in cases with cataract than in cases without.  The technique for cataract surgery in AlphaCor cases needs to be refined. Imaging studies performed postoperatively may help ascertain whether soft lens matter remains so additional antiinflammatory medication can be considered. Melting appears to be preventable by topical medroxyprogesterone 1% therapy.

title: A model for the preliminary biological screening of potential keratoprosthetic biomaterials.
abstract: A series of in vitro screening assays for the preliminary selection of biomaterials for use in the fabrication of artificial corneas (keratoprostheses) (KPros) have been investigated. These screening assays assessed the initial binding of inflammatory and cell adhesive proteins, activation of inflammatory proteins, adhesion of keratocytes, epithelial cells and macrophages and the production of inflammatory cytokines by keratocytes contacting biomaterials. Central optic biomaterials were selected on the basis of low-inflammatory and cell adhesion potential. Peripheral skirt materials were selected on the basis of low-inflammatory potential but good cell adhesion to anchor the implant within the host cornea. Green fluorescent protein (GFP) gene transfer was used in a novel context to investigate cell invasion in the absence of external staining techniques. Confocal laser scanning microscopy and scanning electron microscopy were used to investigate GFP positive keratocyte invasion of porous materials. The results of in vitro assays were compared to a corneal organ culture system in which the biomaterials were assessed within a stromal environment. A range of polyurethane-based interpenetrating polymers with a range of water contents were screened. All materials showed low-inflammatory potential. A reduction in biomaterial water content induced an increase in complement C3 and fibronectin binding and in cell adhesion to materials, whilst differences in co-monomer formulation had little impact. The screening methods used in the current study provide a suitable preliminary assessment regime for the in vitro evaluation of potential KPro materials.

title: An overview of the development of artificial corneas with porous skirts and the use of PHEMA for such an application.
abstract: An overview of the efforts to develop functional polymeric artificial corneas (keratoprostheses) by incorporating a porous skirt is presented. The development of such a device by the author's group using poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels, as a combination of their homogeneous and heterogeneous states, and the rationale of this choice are also discussed. The latest results of the clinical trials with the PHEMA keratoprosthesis in human patients indicate a lower risk of the complications traditionally associated with the implantation of artificial corneas.

title: Functional innervation in tissue engineered models for in vitro study and testing purposes.
abstract: The biotechnology industry is rapidly expanding and the emerging field of tissue engineering is projected to have a high impact in the near future. Recently the field of cellular, drug, and prosthetic delivery has melded with the field of tissue engineering to make simulated tissues. In addition to their roles as tissue substitutes for transplantation, these simulated tissues may provide more accurate models and environments for toxicology testing and the study of peripheral nerves. The current study demonstrates the importance of innervation, in general, for the function of engineered tissues. We observe that the presence of nerves in a tissue engineered (TE) human cornea model enhances the growth of the epithelium and the formation of its protective mucin layer. Innervation also confers protection to the epithelium from chemical insult, as determined by the level of post-treatment epithelial cell death. We demonstrate differential responses of the nerves to chemical stimuli by changes in intracellular sodium as measured by 2-photon microscopy. The 2-photon imaging techniques also allow for the visualization and study of the fine sensory axon fibers within the 3-dimensional tissue. This work demonstrates a role for innervation in the protective quality and function of the engineered tissue, and the potential to use the nerves themselves as indicators of the severity of an insult. These results are important to consider for the development of any optimized TE models for in vitro study and testing purposes.

title: Development of a rabbit corneal equivalent using an acellular corneal matrix of a porcine substrate
abstract: The tissue equivalent that mimics the structure and function of normal tissue is a major bioengineering challenge. Tissue engineered replacement of diseased or damaged tissue has become a reality for some types of tissue such as skin and cartilage. The tissue engineered corneal epithelium, stroma, and endothelium scaffold are promising concepts in overcoming the current limitations of a cornea replacement with an allograft.  The acellular corneal matrix from porcine (ACMP) was examined as a potential corneal cell sheet frame. The physical and mechanical properties of strength, expansion, transparency, and water content of the ACMP were measured. The major antigens of the cell components were completely removed with series of extraction methods, the major antigens of the cell components were identified by hematoxylin and eosin (HE), immunofluorescence staining, and scanning electron microscopy. The structural properties were investigated by HE stain and scanning electron microscopy. The three types of rabbit corneal cells were cultured in vitro, and characteristics were investigated by colony formation efficiency (CFE), BrdU staining, immunofluorescence staining, and western blot assay of keratin 3 (K3), vimentin, and aquaporin A. The biocompatibility of the ACMP was investigated for one month using rabbit corneal stroma and three types of cultured corneal cells both in vivo and in vitro. The three types of cultured rabbit corneal cells were seeded onto ACMP of each side at a cell density of 5.0×103 cells/mm2.  The optical and mechanical properties of the ACMP were similar to the normal porcine cornea. The collagen fiber interconnected to the network, formed regular collagen bundles of the ACMP, and was parallel to the corneal surface. The ACMP was transferred to the rabbit cornea stroma, which showed an intact epithelium and keratocytes in the implant region. There were no inflamed cells or new vessel invasion one month after transplantation. The three types of cultured rabbit corneal cells were positive for K3, vimentin, and aquaporin A. CFE and BrdU (5-bromo-2′-deoxyuridine) staining showed no statistical difference. The cultured rabbit corneal limbal epithelial cells, keratocyte cells, and endothelial cells formed a confluent cell sheet on the ACMP, which consisted of one to two cell layers. Immunofluorescence and scanning electron microscopy examination showed that the cells steadily adhered to the surface of the ACMP and maintained their conformation and special molecule expression such as K3, vimentin, and aquaporin A. Rabbit corneal epithelium-ACMP, keratocytes-ACMP, and endothelium-ACMP scaffold was built in vitro.  The rabbit corneal scaffold was made by the ACMP as a frame with three types of allogeneic rabbit corneal cells. This is a new concept in treating injured corneas.

title: Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds.
abstract: Tissue engineering is a new and exciting technique which has the potential to create tissues and organs de novo. It involves the in vitro seeding and attachment of human cells onto a scaffold. These cells then proliferate, migrate and differentiate into the specific tissue while secreting the extracellular matrix components required to create the tissue. It is evident, therefore, that the choice of scaffold is crucial to enable the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Current scaffolds, made by conventional scaffold fabrication techniques, are generally foams of synthetic polymers. The cells do not necessarily recognise such surfaces, and most importantly cells cannot migrate more than 500 microm from the surface. The lack of oxygen and nutrient supply governs this depth. Solid freeform fabrication (SFF) uses layer-manufacturing strategies to create physical objects directly from computer-generated models. It can improve current scaffold design by controlling scaffold parameters such as pore size, porosity and pore distribution, as well as incorporating an artificial vascular system, thereby increasing the mass transport of oxygen and nutrients into the interior of the scaffold and supporting cellular growth in that region. Several SFF systems have produced tissue engineering scaffolds with this concept in mind which will be the main focus of this review. We are developing scaffolds from collagen and with an internal vascular architecture using SFF. Collagen has major advantages as it provides a favourable surface for cellular attachment. The vascular system allows for the supply of nutrients and oxygen throughout the scaffold. The future of tissue engineering scaffolds is intertwined with SFF technologies.

title: Ex vivo construction of an artificial ocular surface by combination of corneal limbal epithelial cells and a compressed collagen scaffold containing keratocytes.
abstract: We have investigated the use of a laminin-coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane (AM). Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibers within the compressed gel were dense, homogeneous, and similar in structure to those within denuded AM. Limbal epithelial cells were successfully expanded upon the compressed collagen, resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology, and cell stratification. Similar protein expression profiles for cytokeratin 3 (CK3) and CK14 and no significant difference in CK12 mRNA expression levels by real-time polymerase chain reaction were also observed. This study provides the first line of evidence that a laminin-coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification, and differentiation to a degree that is comparable to the leading conventional scaffold, denuded AM.

title: New technologies in limbal epithelial stem cell transplantation.
abstract: Destruction of the limbal epithelial stem cell (LESC) population in the cornea can lead to disorders that result in chronic inflammation, pain and impaired vision. Amniotic membrane (AM) is commonly used as a substrate for LESC transplantation for ocular surface repair but it is not an ideal substrate and so attempts have been made to find a more suitable alternative. The possible substitutes reviewed here include modified AM, cell carriers such as contact lenses or gauze as well as natural substrates fibrin and silk fibroin and many collagen-based scaffolds. Although there are a number of interesting systems in development, the search for an appropriate alternative continues.

title: Development of a reconstructed cornea from collagen-chondroitin sulfate foams and human cell cultures.
abstract: To develop an artificial cornea, the ability to coculture the different cell types present in the cornea is essential. Here the goal was to develop a full-thickness artificial cornea using an optimized collagen-chondroitin sulfate foam, with a thickness close to that of human cornea, by coculturing human corneal epithelial and stromal cells and transfected human endothelial cells.



Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells. Scaffolds were characterized for bulk porosity and pore size distribution. The performance of the three-dimensional construct was studied by histology, immunofluorescence, and immunohistochemistry.



The scaffold had 85% porosity and an average pore size of 62.1 microm. Keratocytes populated the scaffold and produced a newly synthesized extracellular matrix as characterized by immunohistochemistry. Even though the keratocytes lost their CD34 phenotype marker, the absence of smooth muscle actin fibers showed that these cells had not differentiated into myofibroblasts. The epithelial cells formed a stratified epithelium and began basement membrane deposition. An endothelial cell monolayer beneath the foam was also apparent.



These results demonstrate that collagen-chondroitin sulfate scaffolds are good substrates for artificial cornea construction with good resilience, long-term culture capability, and handling properties.

title: Constructing an in vitro cornea from cultures of the three specific corneal cell types
abstract: SummaryThis paper presents a reliable method for establishing pure cultures of the three types of corneal cells. This is believed to be the first time, corneal cells have been cultured from fetal pig corneas. Cell growth studies were performed in different media. Subcultures of the three corneal cell types were passaged until the 30th generation without their showing signs of senescence. For engineering an in vitro cornea, corneal epithelial cells were cultured over corneal stromal cells in an artificial biomatrix of collagen with an underlying layer of corneal endothelial cells. The morphology, histology, and differentiation of the in vitro cornea were investigated to determine the degree of comparability to the cornea in vivo. The in vitro construct displayed signs of transition to an organotypic phenotype of which the most prominent was the formation of two basement membranes.

title: What biomechanical properties of the cornea are relevant for the clinician?
abstract: Although the effects of central corneal thickness and corneal curvature on intraocular pressure measurement are well known, it has only recently become possible to measure the biomechanical properties of the cornea in vivo. This article reviews the structural and material properties of the cornea and considers the effects of corneal parameters, including biomechanics, on IOP measurement. The role of corneal biomechanics as a potential indicator of the structural integrity of the globe will also be discussed. Current evidence suggests that the importance of corneal biomechanics to the glaucoma clinician rests primarily with its effects on IOP measurement. However, the possibility that corneal biomechanics may give an indication of the structural integrity of the optic nerve head cannot be completely excluded. Further population and longitudinal studies are needed to clarify whether current in vivo measures of corneal biomechanical properties, including corneal hysteresis, prove to be independent predictors of glaucoma susceptibility.

title: Ultrarapid Engineering of Biomimetic Materials and Tissues: Fabrication of Nano‐ and Microstructures by Plastic Compression
abstract: Currently, the concept of engineered tissues depends on the ability of cultured cells to fabricate new tissue around a scaffold. Ibis is inherently slow and expensive and has had limited success so far. We report here a new process for the cell-independent, controlled engineering of biomimetic scaffolds by rapid removal of fluid from hyperhydrated collagen gel (or other) constructs, using plastic compression (PC). PC fabrication produces dense, cellular, mechanically strong native collagen structures with controllable nano- and microscale biomimetic structures. The huge-scale shrinkage (> 100-fold) provides the ability to introduce controllable mechanical properties, microlayering, and embossed interface. topography without cell participation, but with high cell viability. Critically, this takes minutes rather than the conventional days and weeks. The rapidity and biomimetic potential of the PC fabrication process at the mesoscale opens a new route for the production of biomaterials and patient-customized tissues. It also represents a new concept in 'engineering' tissues.

title: Cellular disorders induced by high magnetic fields
abstract: To evaluate whether static high magnetic fields (HMFs), in the range of 10–17 T, affect the cytoskeleton and cell organization in different types of mammalian cells, including fibroblasts, epithelial cells, and differentiating neurons.

title: A review on electrospinning design and nanofibre assemblies.
abstract: Although there are many methods of fabricating nanofibres, electrospinning is perhaps the most versatile process. Materials such as polymer, composites, ceramic and metal nanofibres have been fabricated using electrospinning directly or through post-spinning processes. However, what makes electrospinning different from other nanofibre fabrication processes is its ability to form various fibre assemblies. This will certainly enhance the performance of products made from nanofibres and allow application specific modifications. It is therefore vital for us to understand the various parameters and processes that allow us to fabricate the desired fibre assemblies. Fibre assemblies that can be fabricated include nonwoven fibre mesh, aligned fibre mesh, patterned fibre mesh, random three-dimensional structures and sub-micron spring and convoluted fibres. Nevertheless, more studies are required to understand and precisely control the actual mechanics in the formation of various electrospun fibrous assemblies.

title: Effect of Microtopographical Cues on Human Keratocyte Orientation and Gene Expression
abstract: To determine if microtopographical cues can influence the orientation and extracellular matrix production of human keratocytes in vitro.  Human keratocytes were cultured on grooved and ungrooved polycaprolactone films for up to 3 weeks. The cell morphology was examined using ordinary light microscopy, reflective microscopy, and scanning electron microscopy. The cells gene expression was examined using a GEArray Q series gene expression kit.  Cells initially appeared to orientate in the direction of the grooves. Cells cultured on ungrooved films exhibited random orientations. For longer culture periods on grooved membranes, a second cell layer formed on top of the initial layer at an angle orientation to the initial layer. Analysis of mRNA showed that several genes involved in the production of integrins and matrix metalloproteinases were either up-regulated or down-regulated in the presence of the grooves.  The introduction of microtopographical cues has been shown to influence the orientation of keratocytes and alter their gene expression. This pilot study reveals some important findings that can be used in the development of bioengineered corneas.

title: The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells.
abstract: Corneal stroma is an avascular connective tissue characterized by layers of highly organized parallel collagen fibrils, mono-disperse in diameter with uniform local interfibrillar spacing. Reproducing this level of structure on a nano- and micro-scale may be essential to engineer corneal tissue with strength and transparency similar to that of native cornea. A substrate of aligned poly(ester urethane) urea (PEUU) fibers, 165 ± 55 nm in diameter, induced alignment of cultured human corneal stromal stem cells (hCSSCs) which elaborated a dense collagenous matrix, 8-10 μm in thickness, deposited on the PEUU substratum. This matrix contained collagen fibrils with uniform diameter and regular interfibrillar spacing, exhibiting global parallel alignment similar to that of native stroma. The cells expressed high levels of gene products unique to keratocytes. hCSSCs cultured on PEUU fibers of random orientation or on a cast film of PEUU also differentiated to keratocytes and produced abundant matrix, but lacked matrix organization. These results demonstrate the importance of topographic cues in instructing organization of the transparent connective tissue of the corneal stroma by differentiated keratocytes. This important information will help with design of biomaterials for a bottom-up strategy to bioengineer spatially complex, collagen-based nano-structured constructs for corneal repair and regeneration.

title: Effects of hepatocyte growth factor, transforming growth factor-beta1 and epidermal growth factor on bovine corneal epithelial cells under epithelial-keratocyte interaction in reconstruction culture.
abstract: In the cornea, corneal epithelial cells are in close contact with keratocytes: the epithelial cells organize thickened lamellar structure on a layer of keratocytes embedded in extracellular matrix (ECM). Thus, growth factors are expected to critically regulate corneal component cells under epithelial-keratocyte interaction. The purpose of this study is to clarify effects of hepatocyte growth factor (HGF), transforming growth factor-beta1 (TGF-beta1) or epidermal growth factor (EGF) on corneal epithelial cells under epithelial-keratocyte interaction. We examined proliferation and differentiation of the epithelial cells in a simple corneal reconstruction culture composed of an epithelial cell layer on the keratocyte-containing stromal layer, using three-dimensional collagen gel matrix culture. We observed the morphological change by phase contrast microscopy, and conducted histological and immunohistochemical examinations. The epithelial proliferation was examined by nuclear bromodeoxy-uridine (BrdU) uptake. In the reconstructed cornea under epithelial-keratocyte interaction, EGF-, TGF-beta1- and HGF-treated cells formed a thickened epithelial layer consisting of 5-6, 5-6 and 3-4 cells, respectively. In fact, both EGF and TGF-beta1 induced significantly higher intakes of nuclear BrdU of the epithelial cells than HGF. In lamellar differentiation of the epithelial cells, TGF-beta1- or HGF-treated cells formed a triple lamellar structure specific for the in vivo corneal epithelium: basal, middle and superficial layers are composed of cuboidal basal-like cells, spindle wing-like cells and flat superficial-like cells, respectively. TGF-beta1-treated cells formed a more markedly thickened epithelial layer than HGF-treated cells. In contrast, EGF formed a single lamellar structure consisting of cuboidal cells. These results suggest that those growth factors regulate proliferation and/or lamellar differentiation of corneal epithelial cells under epithelial-keratocyte interaction. The most interesting result was that TGF-beta1 promotes proliferation and lamellar differentiation of corneal epithelial cells through keratocyte-mediated stimulation.

title: Corneal wound healing: I. The players
abstract: With the advent of molecular biology and its precise sophisticated techniques, it has been possible to immunolabel and otherwise identify the presence and distribution of many of the compounds that participate in wound healing and to gain some understanding of their interaction. Many are currently being tested for their possible pharmacologic application. This article identifies the compounds and cellular activities that are part of wound healing in order to facilitate an understanding of this complex process in the cornea.

title: Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma.
abstract: To investigate the potential of human corneal stromal stem cells to assume a keratocyte phenotype and to organize extracellular matrix (ECM) in vitro similar to corneal stromal tissue.



Human corneal stromal stem cells (hCSSC) were isolated as side population cells by flow cytometry. Cloned hCSSC were cultured as free-floating pellets in serum-free media for 3 weeks. Gene expression was examined using gene array, quantitative RT-PCR, immunostaining, and immunoblotting. Transmission electron microscopy showed collagen fibril size and alignment.



Pellet cultures of hCSSC in serum-free media upregulated the expression of keratocyte-specific genes and secreted substantial ECM containing characteristic stromal components: keratocan, keratan sulfate, collagen I, collagen V, and collagen VI. Abundant connexin 43 and cadherin 11 in pellets demonstrated cell-cell junctions typical of keratocytes in vivo. Electron microscopy of the pellet cultures revealed abundant fibrillar collagen, some of which was aligned in parallel arrays similar to those of stromal lamellae. Gene array identified expression in pellets of several genes highly expressed by keratocytes. Transcripts for these keratocyte genes -- FLJ30046, KERA, ALDH3A1, CXADR, PTGDS, PDK4, MTAC2D1, F13A1 -- were increased by as much as 100-fold in pellets compared with hCSSC. Simultaneously, expression of stem cell genes BMI1, KIT, NOTCH1, SIX2, PAX6, ABCG2, SPAG10, and OSIL was reduced by a similar factor in pellets compared with hCSSC.



Scaffolding-free pellet culture of hCSSC induces keratocyte gene expression patterns in these cells and secretion of an organized stroma-like ECM. These cells offer a novel potential for corneal bioengineering.

title: Keratocan Expression of Murine Keratocytes Is Maintained on Amniotic Membrane by Down-regulating Transforming Growth Factor-β Signaling*
abstract: Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-β2 and TGF-β receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express α-smooth muscle actin, even when 10 ng/ml TGF-β1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-β signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-β. Collectively, these results indicate that down-regulation of Smad-mediated TGF-β signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-β signaling, achieved by AM stromal matrix in part via suppression of TGF-β gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

title: Epithelial-stromal interactions: specific stimulation of corneal epithelial cell growth in vitro by a factor(s) from cultured stromal fibroblasts.
abstract: By using a recently modified method of isolating and culturing rabbit corneal cells, this study investigated the presence of a diffusible substance(s) in stromal fibroblast conditioned medium that stimulated the growth of cultured corneal epithelial cells. The growth stimulation involved initiation of DNA synthesis (assayed by [3H]-thymidine incorporation) and enhanced cell proliferation (quantified by cell counting). Among the three corneal cell types, only fibroblasts (rabbit and human) released the stimulatory substance, which acted only on epithelial cells. The effect of this stromal fibroblast factor (SFF) was observed after an exposure period of less than 16 hr and persisted as long as it was present. Its action was concentration-dependent and was not a result of improvement in the survival of epithelial cells during culture. Both sparse and confluent epithelial cultures were susceptible to SFF. The release of SFF was correlated with the number of fibroblasts in the culture and appeared to be sensitive to the growth condition of the cells. Both the release and action of SFF did not depend on the presence of serum in the culture medium. The factor was heat resistant and insensitive to proteolytic enzymes. From ultrafiltration studies, the size of SFF was estimated to be in the approximate range of 50-1000 daltons. By direct comparison of the stimulatory effect with other previously studied growth promoting agents, it was concluded that SFF was not epidermal growth factor, fibroblast growth factor, putrescine, cyclic AMP, hydrocortisone or acetate. The implication of SFF in the regulation of epithelial growth by endogenous, intercellular mechanisms is discussed.

title: Corneal cells: chatty in development, homeostasis, wound healing, and disease.
abstract: To provide an overview of cell-cell interactions in the cornea that have a critical role in corneal development, homeostasis, wound healing, and disease.



Review.



Review of the literature.  Cell-cell interactions make critical contributions to development, homeostasis, and wound healing in the cornea. Many of these interactions are mediated by cytokines, growth factors, and chemokines. The best characterized are stromal-epithelial interactions between epithelial cells and stromal cells such as keratocytes, keratoblasts, and myofibroblasts. However, interactions also occur between corneal nerves and epithelial cells and between corneal cells (epithelial cells and stromal cells) and corneal immune cells. Although investigations are limited, it is likely that there are interactions between corneal endothelial cells and keratocytes in the posterior stroma.



Cellular communications in the cornea are critical during development, homeostasis, and wound healing. Disorders of cellular communication likely contribute to many corneal diseases.

title: Co‐culture in cartilage tissue engineering
abstract: For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to observe, measure or manipulate cell behaviour and copying the in situ environment of that cell. Most tissues in the body consist of more than one cell type. The organization of the cells in the tissue is essential for the tissue's normal development, homeostasis and repair reaction. In a co‐culture system, two or more cell types brought together in the same culture environment very likely interact and communicate. Co‐culture has proved to be a powerful in vitro tool in unravelling the importance of cellular interactions during normal physiology, homeostasis, repair and regeneration. The first co‐culture studies focused mainly on the influence of cellular interactions on oocytes maturation to a pre‐implantation blastocyst. Therefore, a brief overview of these studies is given here. Later on in the history of co‐culture studies, it was applied to study cell–cell communication, after which, almost immediately as the field of tissue engineering was recognized, it was introduced in tissue engineering to study cellular interactions and their influence on tissue formation. This review discusses the introduction and applications of co‐culture systems in cell biology research, with the emphasis on tissue engineering and its possible application for studying cartilage regeneration.

title: [The effect of corneal epithelial cells on the collagen gel contraction by keratocytes].
abstract: To understand the mechanism of corneal stromal wound contraction, we investigated the effect of corneal epithelial cells on the collagen gel contraction by keratocytes. Subcultured rabbit keratocytes embedded in type I collagen gel were cultured. Rabbit corneal epithelial cells were also cultured and the cultured medium was collected and used as an epithelial cell conditioned medium (ECCM). The collagen gel contraction by keratocytes was estimated by measuring the diameter of the collagen disc once a day for 5 days. The diameter of the collagen gel decreased in proportion to the number of keratocytes in the presence of fetal calf serum (FCS), but it decreased in inverse proportion to the concentration of collagen. The collagen gel contraction by keratocytes was enhanced by the addition of ECCM in a dose dependent manner. This stimulatory activity was found in ECCM obtained from 3 to 7 day cultivation of corneal epithelial cells. These findings demonstrated that keratocytes contracted collagen gel in the presence of FCS. It also appeared that corneal epithelial cells secrete a factor or factors that stimulate the collagen gel contraction by keratocytes.

title: Modification of proteoglycan synthesis by corneal stromal cells on co-culture with either epithelial or endothelial cells.
abstract: Corneal stromal cells, prepared from 2-day-old chicks and embedded in collagen gel on an insert dish, were co-cultured with corneal epithelial cells or endothelial cells on a fibronectin-coated companion plate. Cell growth and proteoglycan synthesis, as determined as the incorporation of [35S] sulfate and [3H] glucosamine, were examined for each cell type in both combinations. In comparison with single cultures, growth was affected little, while proteoglycan synthesis was appreciably altered in both combinations. Stromal and epithelial cells stimulated proteoglycan synthesis by each other, while endothelial cells stimulated the synthesis by stromal cells, but stromal cells reduced that by endothelial cells to some extent. Endothelial cells alone synthesized proteoglycans much more actively than did epithelial or stromal cells. Analysis of the radiolabeled proteoglycans revealed that endothelial and epithelial cells had different effects on proteoglycan synthesis by stromal cells: the former tended to increase keratan sulfate synthesis by stromal cells, while the latter tended to increase chondroitin sulfate/dermatan sulfate synthesis. Proteoglycan synthesis in the corneal stroma in vivo might thus be controlled by the balance between the antipodal actions of the epithelial and endothelial cell layers.

title: Effect of epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, on proliferation, motility and differentiation of human corneal epithelial cells.
abstract: We sought to determine the effects of exogenous epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), transforming growth factor alpha (TGF-alpha), single-chain precursor hepatocyte growth factor (SC-HGF), double-chain mature HGF (DC-HGF), and keratinocyte growth factor (KGF) on proliferation, motility, and differentiation of first passage cultures of human corneal epithelial cells in serum-free chemically defined medium. The effect of EGF, HB-EGF, TGF-alpha, SC-HGF, DC-HGF, KGF or combinations of the growth factors on proliferation was measured by counting cells present after 3 weeks of culture and by immunostaining for the cell-cycle-specific nuclear proliferation antigen Ki-67. The effect of the factors on epithelial cell motility was assessed by morphometric analysis of photographs of cells migrating from confluent islands of cells. The effect of growth factors on differentiation of epithelial cells were determined by immunostaining epithelial cell islands for the keratin K3 and by Western blotting for keratin K3. EGF, alone or in combination with KGF and SC-HGF, significantly stimulated motility of epithelial cells at the periphery of confluent islands of cells and induced an elongated cell morphology. TGF-alpha, HB-EGF and DC-HGF produced motility effects similar to EGF. There was diminished proliferation of the migrating cells in response to EGF, HB-EGF, TGF-alpha or DC-HGF, while non-migrating epithelial cells in the center of confluent islands continued to proliferate in response to the growth factors. EGF, HB-EGF, TGF alpha or DC-HGF inhibited expression of the differentiation-related marker keratin K3 in epithelial cells, both at the edge and at the center of the islands. KGF stimulated proliferation of corneal epithelial cells at low density and in confluent islands of cells. KGF did not affect expression of keratin K3 or migration of epithelial cells. SC-HGF had no effect on corneal epithelial cells. These results indicate that the effects of EGF, HB-EGF, TGF-alpha and DC-HGF on corneal epithelial cell proliferation, motility and differentiation vary from those of KGF and SC-HGF. EGF, HB-EGF, TGF-alpha and DC-HGF induced changes in epithelial cell morphology and motility in cells plated at low cell density or in cells located at the edge of a confluent island. Thus, these effects appear to be dependent on the extent of cell-cell contact. The inhibitory effect of EGF, HB-EGF, TGF-alpha or DC-HGF on corneal epithelial cell differentiation, however, is independent of cell density.(ABSTRACT TRUNCATED AT 400

title: Paracrine role of keratinocyte growth factor in rabbit corneal epithelial cell growth.
abstract: Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is expressed in stromal fibroblasts and acts specifically on cells of epithelial origin as a paracrine mediator. In this study, we examined KGF and KGF receptor (KGF-R) mRNA expression in rabbit corneal epithelial cells and keratocytes, using a reverse transcription polymerase chain reaction (RT-PCR) method. We also examined the effects of KGF on the proliferation of rabbit corneal cells in vitro. mRNA from cultured rabbit skin fibroblasts, conjunctival fibroblasts, corneal epithelial cells and keratocytes was first reverse-transcribed, and then amplified by PCR using primers specific for KGF and KGF-R. KGF mRNA was expressed in skin fibroblasts, conjunctival fibroblasts and keratocytes, but not in corneal epithelial cells. In contrast, KGF receptor mRNA was expressed in corneal epithelial cells, but not in skin fibroblasts, conjunctival fibroblasts or keratocytes. The results of Southern hybridization experiments of PCR products also showed that KGF mRNA was expressed in skin fibroblasts, conjunctival fibroblasts and keratocytes, whereas KGF receptor mRNA was expressed in corneal epithelial cells. Nucleotide sequencing of the PCR products revealed that their sequences were identical to the expected nucleotide sequences. Using the methylene blue method, we found that KGF enhanced the growth of cultured rabbit corneal epithelial cells, but not keratocytes, at a concentration of 0.01 to 100 ng ml-1. Furthermore, KGF also augmented 3H-thymidine incorporation into the corneal epithelial cells. These results suggest that KGF is a paracrine mediator of the proliferation of corneal epithelial cells as well as of keratinocytes in the skin.

title: Medication use and the risk of Stevens-Johnson syndrome or toxic epidermal necrolysis.
abstract: Toxic epidermal necrolysis and Stevens-Johnson syndrome are rare, life-threatening, drug-induced cutaneous reactions. We conducted a case-control study to quantify the risks associated with the use of specific drugs.



Data were obtained through surveillance networks in France, Germany, Italy, and Portugal. Drug use before the onset of disease was compared in 245 people who were hospitalized because of toxic epidermal necrolysis or Stevens-Johnson syndrome and 1147 patients hospitalized for other reasons (controls). Crude relative risks were calculated and adjusted for confounding by multivariate methods when numbers were large enough.



Among drugs usually used for short periods, the risks were increased for trimethoprim-sulfamethoxazole and other sulfonamide antibiotics (crude relative risk, 172; 95 percent confidence interval, 75 to 396), chlormezanone (crude relative risk, 62; 21 to 188), aminopenicillins (multivariate relative risk, 6.7; 2.5 to 18), quinolones (multivariate relative risk, 10; 2.6 to 38), and cephalosporins (multivariate relative risk, 14; 3.2 to 59). For acetaminophen, the multivariate relative risk was 0.6 (95 percent confidence interval, 0.2 to 1.3) in France but 9.3 (3.9 to 22) in the other countries. Among drugs usually used for months or years, the increased risk was confined largely to the first two months of treatment, when crude relative risks were as follows: carbamazepine, 90 (95 percent confidence interval, 19 to infinity); phenobarbital, 45 (19 to 108); phenytoin, 53 (11 to infinity); valproic acid, 25 (4.3 to infinity); oxicam nonsteroidal antiinflammatory drugs (NSAIDs), 72 (25 to 209); allopurinol, 52 (16 to 167); and corticosteroids, 54 (23 to 124). For many drugs, including thiazide diuretics and oral hypoglycemic agents, there was no significant increase in risk.



The use of antibacterial sulfonamides, anticonvulsant agents, oxicam NSAIDs, allopurinol, chlormezanone, and corticosteroids is associated with large increases in the risk of Stevens-Johnson syndrome or toxic epidermal necrolysis. But for none of the drugs does the excess risk exceed five cases per million users per week.

title: Corneal wound healing: II. The process
abstract: Corneal wound repair is a complex dynamic process in which cells, extracellular matrix, tears, and growth factors interact to bring about the restoration of the integrity of the tissues while maintaining clarity and hydration. This article reviews major recent findings that might affect innovations in therapeutic applications.

title: Basic fibroblast growth factor stimulates corneal endothelial cell growth and endothelial wound healing of human corneas.
abstract: To determine the dose response of human recombinant basic fibroblast growth factor (bFGF) on mitogenic activity, and the supplementary role of serum in cultured bovine and human corneal endothelial cells (BCECs, HCECs). To investigate the effect of bFGF on endothelial wound healing of human corneas in vitro.



In cell culture, DNA synthesis was assessed by 3H-thymidine incorporation. Wound healing was studied using paired human corneas after mechanical damaging of the endothelium. One cornea was treated with bFGF, and the mate served as control. Wound closure was determined after staining with trypan blue. Endothelial cell density (ECD) was assessed in the closed wound area after alizarin red staining. DNA synthesis was assessed using 3H-thymidine autoradiography.



In cell culture, bFGF induced a dose-dependent mitogenic response on BCECs and HCECs. Addition of serum to the culture medium shifted the dose-response curve to considerably lower bFGF concentrations. In organ culture, the time of complete wound closure shortened only marginally (0.5 day) after bFGF treatment (P < 0.01). In the closed wound center, ECD was significantly higher in 1 ng/ml bFGF-treated corneas (686 +/- 134 cells/mm2) than in controls (554 +/- 117 cells/mm2), an increase of +25%. Doses of 0.1 and 10 ng/ml also were effective, but less so than with 1 ng/ml (+11% and +15%, respectively), whereas a dose of 100 ng/ml even had a negative effect (-11%). DNA synthesis was marginally enhanced in bFGF-treated (1 ng/ml) corneas.



The maximal effective dose of bFGF producing a BCEC mitogenic response is dependent on serum. In human senior donor corneas, bFGF promotes endothelial wound healing predominantly by stimulation of cell migration.

title: The Development of a Tissue-Engineered Cornea: Biomaterials and Culture Methods
abstract: The field of corneal tissue engineering has made many strides in recent years. The challenges of engineering a biocompatible, mechanically stable, and optically transparent tissue are significant. To overcome these challenges, researchers have adopted two basic approaches: cell-based strategies for manipulating cells to create their own extracellular matrix, and scaffold-based strategies for providing strong and transparent matrices upon which to grow cells. Both strategies have met with some degree of success. In addition, recent advances have been made in innervating a tissue-engineered construct. Future work will need to focus on further improving mechanical stability of engineered constructs as well as improving the host response to implantation.

title: Keratinocyte growth factor accelerates corneal epithelial wound healing in vivo.
abstract: To examine whether the topical application of keratinocyte growth factor (KGF) can enhance corneal epithelial healing in vivo. In addition, the distribution of S-phase cells in KGF-treated and control corneas was investigated during regeneration and under normal conditions.



A 10-mm diameter epithelial defect was made in the center of rabbit corneas. A 50-microliters aliquot of 10 micrograms/ml human keratinocyte growth factor (hKGF) was then applied topically five times a day. The same volume of phosphate-buffered saline (PBS) vehicle was applied to the contralateral eye as a control. Each corneal epithelial defect was subsequently photographed every 12 hours and was measured by a computer-assisted digitizer. For the S-phase cell analysis, entire corneas were labeled with 3H-thymidine and were subjected to autoradiography at 24 hours after wounding or in the normal cornea at 24 hours after the application of KGF or PBS.



Topical application of 10 micrograms/ml hKGF significantly accelerated corneal epithelial wound healing when compared with controls. In the S-phase cell analysis, KGF did not have any effect on normal corneal epithelial cells. However, in the regenerating cornea, the number of S-phase cells in the KGF-treated limbal epithelium was twofold higher than in the controls.



Topical application of KGF accelerated corneal epithelial wound healing in vivo and increased cell proliferation in the limbal epithelium of the regenerating cornea.