Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion. © 2013 Bottos et al.

Evaluation of Eph receptor and ephrin expression within the human cornea and limbus

Eph receptor tyrosine kinases and their ligands, the ephrins, regulate the development and maintenance of multiple organs but little is known about their potential role within the cornea. The purpose of this study was to perform a thorough investigation of Eph/ephrin expression within the human cornea including the limbal stem cell niche. Initially, immunohistochemistry was performed on human donor eyes to determine the spatial distribution of Eph receptors and ephrins in the cornea and limbus. Patterns of Eph/ephrin gene expression in (1) immortalised human corneal endothelial (B4G12) or corneal epithelial (HCE-T) cell lines, and (2) primary cultures of epithelial or stromal cells established from the corneal limbus of cadaveric eye tissue were then assessed by reverse transcription (RT) PCR. Limbal epithelial or stromal cells from primary cultures were also assessed for evidence of Eph/ephrin-reactivity by immunofluorescence. Immunoreactivity for ephrinA1 and EphB4 was detected in the corneal endothelium of donor eyes. EphB4 was also consistently detected in the limbal and corneal epithelium and in cells located in the stroma of the peripheral cornea. Expression of multiple Eph/ephrin genes was detected in immortalised corneal epithelial and endothelial cell lines. Evidence of Eph/ephrin gene expression was also demonstrated in primary cultures of human limbal stromal (EphB4, B6; ephrinA5) and epithelial cells (EphA1, A2; ephrinA5, B2) using both RT-PCR and immunofluorescence. The expression of Eph receptors and ephrins within the human cornea and limbus is much wider than previously appreciated and suggests multiple potential roles for these molecules in the maintenance of normal corneal architecture.

Non-contact laser-scanning confocal microscopy of the human cornea in vivo

PURPOSE To investigate the utility of using non-contact laser-scanning confocal microscopy (NC-LSCM), compared with the more conventional contact laser-scanning confocal microscopy (C-LSCM), for examining corneal substructures in vivo. METHODS An attempt was made to capture representative images from the tear film and all layers of the cornea of a healthy, 35 year old female, using both NC-LSCM and C-LSCM, on separate days. RESULTS Using NC-LSCM, good quality images were obtained of the tear film, stroma, and a section of endothelium, but the corneal depth of the images of these various substructures could not be ascertained. Using C-LSCM, good quality, full-field images were obtained of the epithelium, subbasal nerve plexus, stroma, and endothelium, and the corneal depth of each of the captured images could be ascertained. CONCLUSIONS NC-LSCM may find general use for clinical examination of the tear film, stroma and endothelium, with the caveat that the depth of stromal images cannot be determined when using this technique. This technique also facilitates image capture of oblique sections of multiple corneal layers. The inability to clearly and consistently image thin corneal substructures - such as the tear film, subbasal nerve plexus and endothelium - is a key limitation of NC-LSCM.

The Use of Mesenchymal Stromal Cells in the Treatment of Diseases of the Cornea

As a key component of the ocular surface required for vision, the cornea has been extensively studied as a site for cell and tissue-based therapies. Historically, these treatments have consisted of donor corneal tissue transplants, but cultivated epithelial autografts have become established over the last 15 years as a routine treatment for ocular surface disease. Ultimately, these treatments are performed with the intention of restoring corneal transparency and a smooth ocular surface. The degree of success, however, is often dependent upon the inherent level of corneal inflammation at time of treatment. In this regard, the anti-inflammatory and immuno-modulatory properties of mesenchymal stromal cells (MSC) have drawn attention to these cells as potential therapeutic agents for corneal repair. The origins for MSC-based therapies are founded in part on observations of the recruitment of endogenous bone marrow-derived cells to injured corneas, however, an increasing quantity of data is emerging for MSC administered following their isolation and ex vivo expansion from a variety of tissues including bone marrow, adipose tissue, umbilical cord and dental pulp. In brief, evidence has emerged of cultured MSC, or their secreted products, having a positive impact on corneal wound healing and retention of corneal allografts in animal models. Optimal dosage, route of administration and timing of treatment, however, all remain active areas of investigation. Intriguingly, amidst these studies, have emerged reports of MSC transdifferentiation into corneal cells. Clearest evidence has been obtained with respect to expression of markers associated with the phenotype of corneal stromal cells. In contrast, the evidence for MSC conversion to corneal epithelial cell types remains inconclusive. In any case, the conversion of MSC into corneal cells seems unlikely to be an essential requirement for their clinical use. This field of research has recently become more complicated by reports of MSC-like properties for cultures established from the peripheral corneal stroma (limbal stroma). The relationship and relative value of corneal-MSC compared to traditional sources of MSC such as bone marrow are at present unclear. This chapter is divided into four main parts. After providing a concise overview of corneal structure and function, we will highlight the types of corneal diseases that are likely to benefit from the anti-inflammatory and immuno-modulatory properties of MSC. We will subsequently summarize the evidence supporting the case for MSC-based therapies in the treatment of corneal diseases. In the third section we will review the literature concerning the keratogenic potential of MSC. Finally, we will review the more recent literature indicating the presence of MSC-like cells derived from corneal tissue.

On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein

Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Corneal sensitivity and slit scanning in vivo confocal microscopy of the subbasal nerve plexus of the normal central and peripheral human cornea

Purpose: To determine the subbasal nerve density and tortuosity at 5 corneal locations and to investigate whether these microstructural observations correlate with corneal sensitivity. Method: Sixty eyes of 60 normal human subjects were recruited into 1 of 3 age groups, group 1: aged ,35 years, group 2: aged 35–50 years, and group 3: aged .50 years. All eyes were examined using slit-lamp biomicroscopy, noncontact corneal esthesiometry, and slit scanning in vivo confocal microscopy. Results: The mean subbasal nerve density and the mean corneal sensitivity were greatest centrally (14,731 6 6056 mm/mm2 and 0.38 6 0.21 millibars, respectively) and lowest in the nasal mid periphery (7850 6 4947 mm/mm2 and 0.49 6 0.25 millibars, respectively). The mean subbasal nerve tortuosity coefficient was greatest in the temporal mid periphery (27.3 6 6.4) and lowest in the superior mid periphery (19.3 6 14.1). There was no significant difference in mean total subbasal nerve density between age groups. However, corneal sensation (P = 0.001) and subbasal nerve tortuosity (P = 0.004) demonstrated significant differences between age groups. Subbasal nerve density only showed significant correlations with corneal sensitivity threshold in the temporal cornea and with subbasal nerve tortuosity in the inferior and nasal cornea. However, these correlations were weak. Conclusions: This study quantitatively analyzes living human corneal nerve structure and an aspect of nerve function. There is no strong correlation between subbasal nerve density and corneal sensation. This study provides useful baseline data for the normal living human cornea at central and mid-peripheral locations

Unilateral pseudogerontoxon

Peripheral corneal opacities are a common clinical finding. In this case report we describe the routine presentation of a young adult male patient with unilateral opacities in the peripheral cornea resembling a corneal arcus. These opacities were confined to the level of the anterior corneal stroma. The patient also exhibited bilateral signs of mild keratoconus and reported a history of vernal keratoconjunctivitis as a child. A diagnosis of unilateral pseudogerontoxon was made. Pseudogerontoxon is an opacity of the peripheral cornea resembling corneal arcus that typically occurs in patients with a history of allergic eye disease. The clinical features and differential diagnoses of this relatively uncommon cause of peripheral corneal opacity are discussed.

Eyelid pressure on the cornea

The eyelids play an important role in lubricating and protecting the surface of the eye. Each blink serves to spread fresh tears, remove debris and replenish the smooth optical surface of the eye. Yet little is known about how the eyelids contact the ocular surface and what pressure distribution exists between the eyelids and cornea. As the principal refractive component of the eye, the cornea is a major element of the eye’s optics. The optical properties of the cornea are known to be susceptible to the pressure exerted by the eyelids. Abnormal eyelids, due to disease, have altered pressure on the ocular surface due to changes in the shape, thickness or position of the eyelids. Normal eyelids also cause corneal distortions that are most often noticed when they are resting closer to the corneal centre (for example during reading). There were many reports of monocular diplopia after reading due to corneal distortion, but prior to videokeratoscopes these localised changes could not be measured. This thesis has measured the influence of eyelid pressure on the cornea after short-term near tasks and techniques were developed to quantify eyelid pressure and its distribution. The profile of the wave-like eyelid-induced corneal changes and the refractive effects of these distortions were investigated. Corneal topography changes due to both the upper and lower eyelids were measured for four tasks involving two angles of vertical downward gaze (20° and 40°) and two near work tasks (reading and steady fixation). After examining the depth and shape of the corneal changes, conclusions were reached regarding the magnitude and distribution of upper and lower eyelid pressure for these task conditions. The degree of downward gaze appears to alter the upper eyelid pressure on the cornea, with deeper changes occurring after greater angles of downward gaze. Although the lower eyelid was further from the corneal centre in large angles of downward gaze, its effect on the cornea was greater than that of the upper eyelid. Eyelid tilt, curvature, and position were found to be influential in the magnitude of eyelid-induced corneal changes. Refractively these corneal changes are clinically and optically significant with mean spherical and astigmatic changes of about 0.25 D after only 15 minutes of downward gaze (40° reading and steady fixation conditions). Due to the magnitude of these changes, eyelid pressure in downward gaze offers a possible explanation for some of the day-to-day variation observed in refraction. Considering the magnitude of these changes and previous work on their regression, it is recommended that sustained tasks performed in downward gaze should be avoided for at least 30 minutes before corneal and refractive assessment requiring high accuracy. Novel procedures were developed to use a thin (0.17 mm) tactile piezoresistive pressure sensor mounted on a rigid contact lens to measure eyelid pressure. A hydrostatic calibration system was constructed to convert raw digital output of the sensors to actual pressure units. Conditioning the sensor prior to use regulated the measurement response and sensor output was found to stabilise about 10 seconds after loading. The influences of various external factors on sensor output were studied. While the sensor output drifted slightly over several hours, it was not significant over the measurement time of 30 seconds used for eyelid pressure, as long as the length of the calibration and measurement recordings were matched. The error associated with calibrating at room temperature but measuring at ocular surface temperature led to a very small overestimation of pressure. To optimally position the sensor-contact lens combination under the eyelid margin, an in vivo measurement apparatus was constructed. Using this system, eyelid pressure increases were observed when the upper eyelid was placed on the sensor and a significant increase was apparent when the eyelid pressure was increased by pulling the upper eyelid tighter against the eye. For a group of young adult subjects, upper eyelid pressure was measured using this piezoresistive sensor system. Three models of contact between the eyelid and ocular surface were used to calibrate the pressure readings. The first model assumed contact between the eyelid and pressure sensor over more than the pressure cell width of 1.14 mm. Using thin pressure sensitive carbon paper placed under the eyelid, a contact imprint was measured and this width used for the second model of contact. Lastly as Marx’s line has been implicated as the region of contact with the ocular surface, its width was measured and used as the region of contact for the third model. The mean eyelid pressures calculated using these three models for the group of young subjects were 3.8 ± 0.7 mmHg (whole cell), 8.0 ± 3.4 mmHg (imprint width) and 55 ± 26 mmHg (Marx’s line). The carbon imprints using Pressurex-micro confirmed previous suggestions that a band of the eyelid margin has primary contact with the ocular surface and provided the best estimate of the contact region and hence eyelid pressure. Although it is difficult to directly compare the results with previous eyelid pressure measurement attempts, the eyelid pressure calculated using this model was slightly higher than previous manometer measurements but showed good agreement with the eyelid force estimated using an eyelid tensiometer. The work described in this thesis has shown that the eyelids have a significant influence on corneal shape, even after short-term tasks (15 minutes). Instrumentation was developed using piezoresistive sensors to measure eyelid pressure. Measurements for the upper eyelid combined with estimates of the contact region between the cornea and the eyelid enabled quantification of the upper eyelid pressure for a group of young adult subjects. These techniques will allow further investigation of the interaction between the eyelids and the surface of the eye.

Development of a method to measure upper eyelid pressure on the cornea

Purpose: The cornea is known to be susceptible to forces exerted by eyelids. There have been previous attempts to quantify eyelid pressure but the reliability of the results is unclear. The purpose of this study was to develop a technique using piezoresistive pressure sensors to measure upper eyelid pressure on the cornea. Methods: The technique was based on the use of thin (0.18 mm) tactile piezoresistive pressure sensors, which generate a signal related to the applied pressure. A range of factors that influence the response of this pressure sensor were investigated along with the optimal method of placing the sensor in the eye. Results: Curvature of the pressure sensor was found to impart force, so the sensor needed to remain flat during measurements. A large rigid contact lens was designed to have a flat region to which the sensor was attached. To stabilise the contact lens during measurement, an apparatus was designed to hold and position the sensor and contact lens combination on the eye. A calibration system was designed to apply even pressure to the sensor when attached to the contact lens, so the raw digital output could be converted to actual pressure units. Conclusions: Several novel procedures were developed to use tactile sensors to measure eyelid pressure. The quantification of eyelid pressure has a number of applications including eyelid reconstructive surgery and the design of soft and rigid contact lenses.

Overview of methods used in cross-cultural comparisons of menopausal symptoms and their determinants : Guidelines for Strengthening the Reporting of Menopause and Aging (STROMA) studies

This paper reviews the methods used in cross-cultural studies of menopausal symptoms with the goal of formulating recommendations to facilitate comparisons of menopausal symptoms across cultures. It provides an overview of existing approaches and serves to introduce four separate reviews of vasomotor, psychological, somatic, and sexual symptoms at midlife. Building on an earlier review of cross-cultural studies of menopause covering time periods until 2004, these reviews are based on searches of Medline, PsycINFO, CINAHL and Google Scholar for English-language articles published from 2004 to 2010 using the terms “cross cultural comparison” and “menopause.” Two major criteria were used: a study had to include more than one culture, country, or ethnic group and to have asked about actual menopausal symptom experience. We found considerable variation across studies in age ranges, symptom lists, reference period for symptom recall, variables included in multivariate analyses, and the measurement of factors (e.g., menopausal status and hormonal factors, demographic, anthropometric, mental/physical health, and lifestyle measures) that influence vasomotor, psychological, somatic and sexual symptoms. Based on these reviews, we make recommendations for future research regarding age range, symptom lists, reference/recall periods, and measurement of menopausal status. Recommendations specific to the cross-cultural study of vasomotor, psychological, somatic, and sexual symptoms are found in the four reviews that follow this introduction.

A dual-layer silk fibroin scaffold for reconstructing the human corneal limbus

Membranes prepared from Bombyx mori silk fibroin have shown potential as a substrate for human limbal epithelial (L-EC) and stromal cell cultivation. Here we present fibroin as a dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. We have compared the growth and phenotype of L-EC on non-porous versus porous fibroin membranes. Furthermore, we have compared the growth of limbal mesenchymal stromal cells (L-MSC) in either serum-supplemented medium or the MesenCult-XF® culture system within fibroin fibrous mats. The co-culture of L-EC and L-MSC in fibroin dual-layer constructs was also examined. L-EC on porous membranes displayed a squamous monolayer; in contrast, L-EC on non-porous fibroin appeared cuboidal and stratified. Both constructs maintained evidence of corneal phenotype (cytokeratin 3/12) and distribution of ΔNp63+ progenitor cells. L-MSC cultivated within fibroin fibrous mats in serum-supplemented medium contained less than 64% of cells expressing the characteristic MSC phenotype of CD73+CD90+CD105+ after two weeks, compared with over 81% in MesenCult-XF® medium. Dual-layer fibroin scaffolds consisting of L-EC and L-MSC maintained a similar phenotype as on the separate layers. These results support the feasibility of a 3D engineered limbus constructed from B. mori silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration.

Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Identification of GABA receptors in chick cornea

Purpose: The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea. Methods: Corneal tissues were collected from 25, 12-day-old chicks. Real time PCR, western blot, and immunohistochemistry were used to determine whether alpha1 GABAA, GABAB, and rho1 GABAC receptors were expressed and located in chick cornea. Results: Corneal tissue was positive for alpha1 GABAA and rho1 GABAC receptor mRNA (PCR) and protein (western blot) expression but was negative for GABAB receptor mRNA and protein. Alpha1 GABAA and rho1 GABAC receptor protein labeling was observed in the corneal epithelium using immunohistochemistry. Conclusions: These investigations clearly show that chick cornea possesses alpha1 GABAA, and rho1 GABAC receptors, but not GABAB receptors. The purpose of the alpha1 GABAA and rho1 GABAC receptors in cornea is a fascinating unexplored question.