Structural and biochemical investigations of the cornea and the trabecular meshwork

The experiments which comprise this thesis focused on structure-function relationships in two distinct collagen-rich connective tissues of the eye, the cornea and the trabecular meshwork. The cornea is the transparent tissue that covers the front of the eye and has the ability to transmit and refract light. Corneal transparency is the result of the unique organisation of collagen fibrils in the corneal stroma matrix, of which sulphated proteoglycans are key regulators, owing to the presumed importance of the sulfation pattern of corneal proteoglycans. The trabecular meshwork is the sponge-like tissue located around the cornea through which the bulk of the aqueous humor flows towards the juxtacanalicular tissue and the inner wall of Schlemm’s canal to exit the eye and control intraocular pressure. First part of the current research examined the chemical composition and sulphur speciation during corneal embryogenesis in order to elucidate important changes in the biochemical signature of the corneal matrix associated with the acquisition of transparency. It also investigated the content and distribution of distinct sulphur species through the depth of the mature corneal stroma and assessed biochemical-functional relationships that ultimate render tissue transparency. The research also studied the three-dimensional ultrastructure of the human trabecular meshwork, particularly the ultrastructure of the juxtacanalicular tissue that lies adjacent to the inner wall of Schlemm’s canal and the three-dimensional assembly of collagen type VI in the trabecular meshwork itself. X-ray fluorescence microscopy revealed key differences in the chemical composition of the cornea of the developing chick. In particular, the chemical signature of phosphorus, chlorine, sulphur, potassium and calcium were observably different during the developmental period from day 12 to day 16. S k-edge x-ray near edge structure spectroscopy showed that the main sulphur species present in the embryonic cornea were thiols, organic monosulfides, ester sulphate and inorganic sulphate. The chemical signature of these sulphur species was also noticeably different during embryonic corneal development. The changes in the chemical signature of phosphorus with development are believed to underline changes in the presumptive keratocyte population within the embryonic corneal stroma. Chlorine, potassium and calcium are important elements involved in the regulation and balance of the net negative or positive charge of the embryonic cornea and may influence the interactions of corneal matrix molecules. The changes in the sulphur speciation character amongst different developmental corneas are associated with changes in the sulphation status of corneal proteoglycans which play a fundamental role in governing tissue structure and function, and thus transparency. With regards to the sulphur speciation across the depth of the mature corneal stroma, it was found that there is an inconsistency in the sulphur content and distribution throughout the depth of the tissue, from the stromal region closest to the epithelium against the deeper stromal regions near the endothelium. The heterogeneity of the sulphur species in the most anterior part of the mature corneal stroma, at the interface with the Bowman’s layer supports the view that the differentiation and the transition between these two corneal layers is not very abrupt. The rest of the mature corneal stroma depth does not show any differences regarding its content in the sulphur-containing compounds indicating that the distribution and sulfation status of the corneal glycosaminoglycans have very little impact on the overall sulfur speciation. The three-dimensional ultrastructure of the human trabecular meshwork in large volumes and at high resolution identified giant vacuoles in the endothelial cell layer of the inner wall of Schlemm’s canal and these were grouped into four categories based on whether they formed pores, basal and apical, or not. Interestingly, the distribution of these vacuoles was found to be non-uniform. It was discovered that the juxtacanalicular tissue was not homogenous with respect to the proportion of the electron lucent, matrix free spaces throughout the tissue’s depth away from the inner wall of Schlemm’s canal. Three-dimensional reconstructions of collagen type VI in the trabecular meshwork showed that there is no structural regularity in the organisation of type VI collagen assemblies, or their association with sulphated proteoglycans, suggesting a role in aqueous humor outflow. This data allow us to propose a model of aqueous humor outflow and how this is funneled through the juxtacanalicular tissue towards the lumen of Sclemm’s canal

The hierarchical response of human corneal collagen to controlled inflation

Purpose : To elucidate the hierarchical deformation mechanisms of human corneal collagen under controlled inflation of physiological and pathological magnitudes. Methods : 6 human donor corneas with scleral rim were mounted onto a bespoke sealed cell, which was connected via two pressure regulators and an expansion vessel to a source of compressed nitrogen. The cell included a window transparent to X-rays and was connected to translation and rotation stages inside beamline I22 at the Diamond Light Source synchrotron, UK. Images were acquired naso-temporally across the cornea from limbus to limbus at intraocular pressures of 5.5 mmHg, 17.5 mmHg and 40 mmHg. These increments were chosen for comparison with previous static results in the literature and to mimic the normal physiological state and ocular hypertension. Results : The inflation apparatus was able to maintain pressure to an accuracy better than 0.1 mmHg. Features corresponding to collagen fibril diameter, spacing, D-period and orientation as well as tropocollagen spacing and orientation were measured simultaneously. Preliminary analysis revealed a radial distribution of strain (manifested as a change in D-period) across the cornea, which was accentuated in the outer periphery and limbus. In general, the interfibrillar spacing in the periphery and limbus (where collagen fibrils are arranged circumferentially) was found to increase with pressure. Conclusions : The trends in D-period strain are indicative of bulging under increased pressure, which is pronounced at the limbus and the outer periphery. This agrees with the general consensus that the outer periphery and limbus act as a buffer zone, which takes up most of the strain under changes in intraocular pressure, and thus minimises changes in focussing power of the cornea. Further in-depth analysis of this data will allow us to calculate the supramolecular twist (which gives rise to a spring-like stretch in collagen fibrils) and examine in more detail the hierarchical response of corneal collagen to changes in intraocular pressures

Developmental changes in patterns of distribution of fibronectin and tenascin-C in the chicken cornea: evidence for distinct and independent functions during corneal development and morphogenesis

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior–posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman’s layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles

Genipin delays corneal stromal enzymatic digestion

Purpose: To evaluate the use of genipin in delaying enzymatic digestion of corneal stroma. Methods: Human corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy. Results: Genipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis. Conclusions: Genipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging. Translational Relevance: This study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity

Template curvature influences cell alignment to create improved human corneal tissue equivalents

To accurately create corneal stromal equivalents with native‐like structure and composition, a new biofunctionalized, curved template is developed that allows the precise orientation of cells and of their extracellular matrix. This template is the first demonstration that curvature alone is sufficient to induce the alignment of human corneal stromal cells, which in turn are able to biofabricate stromal tissue equivalents with cornea‐like shape and composition. Specifically, tissues self‐released from curved templates show a highly organized nanostructure, comprised of aligned collagen fibrils, significantly higher expression of corneal stroma‐characteristic markers keratocan, lumican, decorin, ALDH3, and CHST6 (p = 0.012, 0.033, 0.029, 0.003, and 0.02, respectively), as well as significantly higher elastic modulus (p = 0.0001) compared with their planar counterparts. Moreover, curved tissues are shown to support the growth, stratification, and differentiation of human corneal epithelial cells in vitro, while maintaining their structural integrity and shape without any supporting carriers, scaffolds, or crosslinking agents. Together, these results demonstrate that corneal stromal cells can align and create highly organized, purposeful tissues by the influence of substrate curvature alone, and without the need of additional topographical cues. These findings can be important to further understand the mechanisms of corneal biosynthesis both in vitro and in vivo

Cell–cell and cell–matrix interactions at the presumptive stem cell niche of the chick corneal limbus

(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell–cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea

Evolution of the vertebrate corneal stroma

Although the cornea is the major refractive element of the eye, the mechanisms controlling corneal shape and hence visual acuity remain unknown. To begin to address this question we have used multiphoton, non-linear optical microscopy to image second harmonic generated signals (SHG) from collagen to characterize the evolutionary and structural changes that occur in the collagen architecture of the corneal stroma. Our studies show that there is a progression in complexity of the stromal collagen organization from lower (fish and amphibians) to higher (birds and mammals) vertebrates, leading to increasing tissue stiffness that may control shape. In boney and cartilaginous fish, the cornea is composed of orthogonally arranged, rotating collagen sheets that extend from limbus to limbus with little or no interaction between adjacent sheets, a structural paradigm analogous to 'plywood'. In amphibians and reptiles, these sheets are broken down into broader lamellae that begin to show branching and anastomosing with adjacent lamellae, albeit maintaining their orthogonal, rotational organization. This paradigm is most complex in birds, which show the highest degree of lamellar branching and anastomosing, forming a 'chicken wire' like pattern most prominent in the midstroma. Mammals, on the other hand, diverged from the orthogonal, rotational organization and developed a random lamellar pattern with branching and anastomosing appearing highest in the anterior stroma, associated with higher mechanical stiffness compared to the posterior stroma

Recapitulation of normal collagen architecture in embryonic wounded corneas

Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure

Chondroitin sulphate/dermatan sulphate proteoglycans: potential regulators of corneal stem/progenitor cell phenotype in vitro

Chondroitin sulphate (CS) proteoglycans with variable sulphation-motifs along their glycosaminoglycan (GAG) chains are closely associated with the stem cell niche of articular cartilage, where they are believed to influence the characteristics of the resident stem cells. Here, we investigated the immunohistochemical distribution of hybrid CS/dermatan sulphate (DS) GAGs in the periphery of the adult chicken cornea, which is the location of the cornea’s stem cell niche in a number of species, using a monoclonal antibody, 6C3, that recognises a sulphation motif-specific CS/DS GAG epitope. This revealed positive labelling that was restricted to the subepithelial corneal stroma, as well as nearby bony structures within the sclera, called ossicles. When cultivated on cell culture dishes coated with 6C3-rich CS/DS, corneal stromal cells (keratocytes) that had been isolated from embryonic chicken corneas formed circular colonies, which took several days to reach confluency. A flow cytometric analysis of these keratocytes revealed changes in their expression levels of the indicative stem cell markers, Connexin 43 (Cx43), Paired Box 6 (PAX6), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1), and C-X-C Chemokine Receptor 4 (CXCR4) suggestive of a less-differentiated phenotype compared with expression levels in cells not exposed to CS/DS. These findings support the view that CS/DS promotes the retention of a stem cell phenotype in corneal cells, much as it has been proposed to do in other connective tissues