Cornea is the most exterior, avascular and transparent layer of the eye and is about 500 &micro / m in thick. It protects the eye from external objects and it is the main optical element of the eye refracting 70 % of the incoming light. After cataract, corneal diseases and wounds are the second leading cause of the blindness that affects more than 4 million people worldwide. For the highly damaged corneas where the corrections with spectacles or contact lenses cannot be achieved, tissue replacement is the only choice, and is done by cornea transplantation or keratoprostheses. However, due to limited number of donor corneas and the risk of infections during transplantation, and development of glaucoma, necrosis and other complications caused by the keratoprostheses, prevent them from meeting expectations. Tissue engineering is a promising field which emerged from biomaterials science and aims to replace, restore or improve the function of the diseased or injured tissues. In this method, after the production of an ideal scaffold that mimics the natural human tissue, cells of the host are isolated, increased in number, and seeded on the scaffold developed to serve as the microenvironment of the cells. In the current study a 3D corneal stroma replacement was designed to mimic the native stroma. It consisted of 4 films of patterned collagen or collagen blended with Elastin Like Recombinamer (ELR) stacked on top of each other and then crosslinked by dehydrothermal (DHT) treatment. The characterization of the films showed that the pattern fidelity was good and they did not deteriorate after crosslinking. Enzymatic and in situ degradation studies showed that the DHT treatment at 150 oC for 24 h (DHT150) was the optimum condition. The transparency of all the films was quite high where uncrosslinked (UXL) films and DHT150 Col:ELR films yielded the best results. The individual films and 3D construct of 4 stacked films were seeded with isolated human corneal keratocytes (HK) and cultured for 21 days. Cells attached and proliferated well on the single Col and Col:ELR films. However, the proliferation was higher on Col multilayer constructs than their Col:ELR counterparts. Cells were aligned along the patterns of the films while no significant alignment was observed for the cells on unpatterned films. Ultimate tensile strength (UTS) and Young&rsquo / s Modulus (E) of Col and Col:ELR films were significantly lower after a 30 day culture than that of unseeded films of Day 1. Transparency of the seeded Col:ELR films was superior to Col films over a 30 days test and quite close to the transmittance of the native human cornea. It was concluded that the Col and Col:ELR patterned films and their 3D constructs have a significant potential for use as a corneal stroma equivalent.

<b>Purpose:</b> Assess the relationship between tear film drying and sensation between blinks. <b>Methods:</b> MATLAB sampled a slitlamp video camera, a potentiometer and a microphone while subjects kept one eye open for as long as possible. 23 subjects rated the intensity of the ocular sensation while video and voice data were collected simultaneously. The tear drying on the cornea was measured. <b>Results:</b> The sensation was triphasic. Two linear functions described the latter 2 parts of the data (r &#8805; 0. 95). The correlation between TBUT and the elbow in the time-discomfort function was 0. 72. Extent of tear film drying was linearly correlated to time (median correlation = 0. 88). The correlation between the discomfort elbow and image elbow was 0. 93 with single data pair for each subject. Analysis of sensation characteristics showed significant differences between itching and burning for both intensity and time (p = 0. 03 and p = 0. 02 respectively). <b>Conclusions:</b> Simultaneous recording of ocular surface appearance, discomfort intensity and attributes of sensation provide novel information about the development of discomfort during ocular surface drying. The rapid increase in discomfort proceeding blinking has been quantified and the relationship between the time course of drying and discomfort is elucidated.

In this study, collagen based scaffolds were prepared for cornea tissue engineering. Three different cell carriers (rat tail collagen foam, insoluble collagen foam and patterned collagen film) were produced using two different collagen sources. Scaffolds were designed to mimic the unique topographical features of the corneal stroma. A novel crosslinking method was developed to achieve constant foam thickness. All scaffolds were tested with the primary cells of the native corneal stroma, human keratocytes. Although both foams promoted cell growth and penetration, rat tail foams were found to be superior for keratocyte proliferation. Their degradation rates were high enough but did not compromise their structural integrity during testing. Transparency studies with the foams revealed a progressive improvement. Collagen films degraded significantly over a one month period / however, the presence of cells increased the tensile strength of the films over a 21 day period to close to that of the native cornea and compensated for the loss of strength due to degradation. The micropatterned films proved to have higher transparency than the unpatterned scaffolds. In this study, it was possible to prepare collagen based micropatterned scaffolds using a silicon wafer and then a silicone template, successively, starting from original designs. The resultant collagen films were able to control cell growth through contact guidance, restricted cells and secreted-ECM within the pattern grooves, resulting in a higher transparency in comparison to unpatterned films. Thus, the tissue engineered constructs revealed a significant potential for use as total artificial corneal substitutes.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Richard J. Braun, Dept. of Mathematical Sciences. Includes bibliographical references.

Cornea is a part of the optical system of the eye, the condition of which is directly related with the eyesight and its quality. Due to the increasing popularity of the correction of refraction defects with the help of excimer laser, central cornea thickness acquires higher prognostic significance for the determination of the success of the operation and probable post-surgical complications. The objective of the survey is to measure the central corneal thickness of adult residents in Lithuania and the relevant factors having impact on it. In total 1650 residents of Lithuania in the age from 18 to 89 years of age were tested. The survey was carried out in the urban and regional primary health care institutions of the 10 counties of Lithuania and at the Centre of Eye Diseases of the Vilnius University Hospital Santariškių klinikos. The average central corneal thickness of men and women in Lithuania was defined in the survey. It was observed that central corneal thickness is neither conditioned by the gender, height or weight of an individual, nor by the eye refraction or the number and the size of corneal endothelia cells. The thickest cornea was observed in persons under 40 years of age, whereas it becomes thinner each decade from 2 to 8 µm after forty. It was identified in the survey that the thicker the cornea is - the smaller is its curvature. On the basis of the survey a table of central corneal thickness was developed for doctors ophthalmologists to be used in their... [to full text] / Ragena – tai akies optinės sistemos dalis, nuo kurios būklės tiesiogiai priklauso regėjimas ir jo kokybė. Populiarėjant refrakcijos ydų korekcijai eksimeriniu lazeriu, ragenos centrinės dalies storis turi vis didesnę prognostinę reikšmę operacijos sėkmės bei pooperacinių komplikacijų galimybės nustatymui. Darbo tikslas – nustatyti Lietuvos suaugusių gyventojų ragenos centrinės dalies storį bei jį veikiančius veiksnius. Ištirta 1650 Lietuvos gyventojų nuo 18 iki 89 metų amžiaus.Tyrimas vykdytas 10 Lietuvos apskričių miestų ir rajonų pirminėse sveikatos priežiūros įstaigose, bei VUL Santariškių klinikos Akių ligų centre. Tyrimo metu nustatytas Lietuvoje gyvenančių vyrų ir moterų vidutinis ragenos centrinės dalies storis. Nustatyta, kad ragenos centrinės dalies storis nepriklauso nuo žmogaus lyties, ūgio, svorio, bei akių refrakcijos ir ragenos endotelio ląstelių skaičiaus ir dydžio. Storiausia ragena nustatoma iki 40 metų amžiaus, vyresniems nei 40 metų ji plonėja kas dešimtmetį nuo 2 iki 8 µm. Nustatyta, kad kuo storesnė ragena, tuo jos gaubtumas yra mažesnis. Tyrimo pagrindu sukurta skirtingo amžiaus žmonių ragenos centrinės dalies storio lentelė, kuria savo praktiniame darbe galės naudotis visi gydytojai oftalmologai.

Ragena – tai akies optinės sistemos dalis, nuo kurios būklės tiesiogiai priklauso regėjimas ir jo kokybė. Populiarėjant refrakcijos ydų korekcijai eksimeriniu lazeriu, ragenos centrinės dalies storis turi vis didesnę prognostinę reikšmę operacijos sėkmės bei pooperacinių komplikacijų galimybės nustatymui. Darbo tikslas – nustatyti Lietuvos suaugusių gyventojų ragenos centrinės dalies storį bei jį veikiančius veiksnius. Ištirta 1650 Lietuvos gyventojų nuo 18 iki 89 metų amžiaus.Tyrimas vykdytas 10 Lietuvos apskričių miestų ir rajonų pirminėse sveikatos priežiūros įstaigose, bei VUL Santariškių klinikos Akių ligų centre. Tyrimo metu nustatytas Lietuvoje gyvenančių vyrų ir moterų vidutinis ragenos centrinės dalies storis. Nustatyta, kad ragenos centrinės dalies storis nepriklauso nuo žmogaus lyties, ūgio, svorio, bei akių refrakcijos ir ragenos endotelio ląstelių skaičiaus ir dydžio. Storiausia ragena nustatoma iki 40 metų amžiaus, vyresniems nei 40 metų ji plonėja kas dešimtmetį nuo 2 iki 8 µm. Nustatyta, kad kuo storesnė ragena, tuo jos gaubtumas yra mažesnis. Tyrimo pagrindu sukurta skirtingo amžiaus žmonių ragenos centrinės dalies storio lentelė, kuria savo praktiniame darbe galės naudotis visi gydytojai oftalmologai. / Cornea is a part of the optical system of the eye, the condition of which is directly related with the eyesight and its quality. Due to the increasing popularity of the correction of refraction defects with the help of excimer laser, central cornea thickness acquires higher prognostic significance for the determination of the success of the operation and probable post-surgical complications. The objective of the survey is to measure the central corneal thickness of adult residents in Lithuania and the relevant factors having impact on it. In total 1650 residents of Lithuania in the age from 18 to 89 years of age were tested. The survey was carried out in the urban and regional primary health care institutions of the 10 counties of Lithuania and at the Centre of Eye Diseases of the Vilnius University Hospital Santariškių klinikos. The average central corneal thickness of men and women in Lithuania was defined in the survey. It was observed that central corneal thickness is neither conditioned by the gender, height or weight of an individual, nor by the eye refraction or the number and the size of corneal endothelia cells. The thickest cornea was observed in persons under 40 years of age, whereas it becomes thinner each decade from 2 to 8 µm after forty. It was identified in the survey that the thicker the cornea is - the smaller is its curvature. On the basis of the survey a table of central corneal thickness was developed for doctors ophthalmologists to be used in their... [to full text]

Precise measurements of corneal layer thickness are required to treat, evaluate risk of, and determine the progression of pathologies within the eye. The thickness measurements are typically acquired as 2d images, known as tomograms, from an optical coherence tomography (OCT) system. With the creation of ultra-high resolution OCT (UHROCT), there is active research in precisely measuring, in vivo, previously unresolvable corneal structures at arbitrary locations within the cornea to determine their relationship with corneal health. In order to obtain arbitrary corneal thickness measurements, existing reconstruction techniques require the cornea to be densely sampled so that a 3d representation can be interpolated from a stack of tomograms. Unfortunately, tomogram alignment relies solely on image properties such as pixel intensity, and does not constrain the reconstruction to corneal anatomy. Further, the reconstruction method cannot properly compensate for eye-motion. The deficiencies due to eye-motion are exacerbated due to the amount of time required in a single imaging session to acquire a sufficient number of tomograms in the region of interest. The proposed methodology is the first to incorporate models of the anatomy and the imaging system to address the limitations of existing corneal reconstruction methods. By constructing the model in such a way as to decouple anatomy from the imaging system, it becomes less computationally expensive to estimate model parameters. The decoupling provides an iterative methodology that can allow additional constraints to be introduced in the future. By combining sparsely sampled UHROCT measurements with a properly designed corneal model, reconstruction allows researchers to determine corneal layer thicknesses at arbitrary positions in both sampled and unsampled regions. The proposed methodology demonstrates an approach to decouple anatomy and physiology from measurements of a cornea, allowing for characterization of pathologies through corneal thickness measurements. Another significant contribution resulting from the corneal model allows five of the corneal layer boundaries to be automatically located and has already been used to process thousands of UHROCT tomograms. Recent studies using this method have also been used to correlate contact-lens wear to hypoxia and corneal layer swelling. While corneal reconstruction represents the main application of this work, the reconstruction methodology can be extended to other medical imaging domains and can even represent temporal changes in tissue with minor modifications to the framework.

ABSTRACT CORNEA ENGINEERING ON BIODEGRADABLE POLYESTERS Zorlutuna, Pinar M. Sc., Department of Biotechnology Supervisor: Prof. Vasif Hasirci Co-Supervisor: Asst. Prof. AySen Tezcaner January 2005, 66 pages Cornea is the outermost layer of the eye and has an important role in vision. Damage of cornea due to injuries or infections could lead to blindness lowering the quality of life of the patient severely. In such cases, transplantation or artificial corneas have been used for treatment but both had drawbacks. The novel approach for corneal replacements is the tissue engineering of the cornea, a promising method which would be free of these drawbacks, if successful. In this study, carriers for tissue engineering of the cornea were designed and tested in vitro. Blends of biodegradable and biocompatible polyesters of natural (PHBV8) and synthetic (PLLA) origin were used to construct these carriers. For the epithelial layer of the cornea, PLLA-PHBV8 micropatterned films were prepared with solvent casting and seeded with D407 (retinal pigment epithelial) cells. In order to achieve proper cell growth, the films were coated with fibronectin. For the stromal layer of the cornea, highly porous foams of PLLA-PHBV8 were prepared by lyophilization and seeded with 3T3 cells (fibroblasts). A new approach was developed to create a combination of the film and the foam to obtain a surface patterned, 3 dimensional cell carrier. These carriers were seeded with Saos-2 cells (osteosarcoma cells) in the preliminary optimization studies and with D407 and 3T3 cells in further studies. The cell numbers on the carriers were quantified by using MTS assay (non-radioactive cell proliferation assay) and the cell proliferation on polymeric carriers was significantly higher than that of control (Tissue culture polystyrene) by the day 14. Characterization of these cells and the carrier was done using a variety of microscopic methods. The micrographs showed that the foam had a highly porous structure and the pores were interconnected. 3T3 cells were found to be distributed quite homogeneously at the seeding site, but due to the high thickness of the foam, the cells could not sufficiently populate the core (central parts of the foam) during the given incubation time. The micropatterned film allowed multilayer formation of D407 cells. The functionality of the cells seeded on the carriers was examined by immunohistochemistry. These analyses proved that the cells retained their phenotype during culturing. D407 cells formed tight junctions characteristic of epithelial cells, and 3T3 cells deposited collagen type I into the foams. Based on the results, it can be concluded that the 3-D PLLA-PHBV8 construct with surface patterns have a serious potential for use as a tissue engineering carrier for the reconstruction of the cornea. Key words: Tissue engineering, cornea, polymeric carrier, biodegradable, polyester.

PURPOSE:The limbus of the cornea is said to be the niche for limbal stem cells (LSCs) and the primary source of corneal epithelial maintenance. In this model, adult corneal epithelial cells are maintained by LSCs that cycle slowly and give rise to transient amplifying (TA) cells. These migrate centripetally, differentiate outwards to the surface, and are then lost by desquamation. This study set out to investigate the stem cell properties of human corneal epithelium and their contribution towards corneal epithelial regeneration after wounding. METHODS: Frozen sections of human corneal tissues were labelled with a number of putative stem cell markers. Human central and limbal corneal epithelial cells were isolated for holoclone formation assay and FACS isolation. Side population (SP) cells were separated based upon cell size and Hoechst dye efflux ability. A human corneal organotypic culture model was used to assess corneal healing in vitro. Injured corneas were analysed using cytokine antibody arrays and immunohistochemical markers for cell proliferation and stem cells. RESULTS: The expression of putative stem cell markers ΔNp63α and ABCG2 was clearly evident in the suprabasal and basal layers of the limbus, but was also observed in central epithelium. Human limbal and central corneal epithelial cells were both capable of forming holoclones in 2:1 ratio respectively. In FACS, central SP and limbal SP cells showed no significant difference based upon size and dye efflux. After wounding, the capacity for epithelial cell proliferation and migration appears to be as active in the central cornea as in the periphery/limbus. Central and peripheral epithelial recovery remains equal even after ablation of the limbus. CONCLUSION: Cells from the central human corneal epithelium have many putative stem cell properties. These results raise questions not only about the distribution and substance of stem cells in the cornea, but also the role of the limbus itself. The central epithelium is able to heal independently of the limbus.

The notion that forces from the eyelids can alter the shape of the cornea has been proposed for many years. In recent times, there has been a marked improvement in our ability to measure and define the corneal shape, allowing subtle changes in the cornea to be measured. These improvements have led to the findings that pressure from the eyelids can cause alterations in corneal shape following everyday visual tasks such as reading. There are also theories to suggest that pressure from the eyelids may be involved in the aetiology of corneal astigmatism. In this program of research, a series of experiments were undertaken to investigate the influence of the eyelids on the shape of the cornea. In the first experiment, an investigation into the diurnal variation of corneal shape was carried out by measuring corneal topography at three different times (approximately 9 am, 1 pm and 5 pm) during the day over three days of the week (Monday, Tuesday and Friday). Highly significant diurnal changes were found to occur in the corneal topography of 15 of the 17 subjects. This change typically consisted of horizontal bands of distortion in the superior, and to a lesser extent, inferior cornea, increasing throughout the day (and returning to baseline the next morning). These changes appeared to be related to forces from the eyelids on the anterior cornea. Some changes were also found in corneal astigmatism. Corneal astigmatism power vector J0 (astigmatism 90/180°) was found to increase slightly over the course of the week. Whilst the changes in astigmatism were small in magnitude, this result leaves open the possibility that pressure from the eyelid may cause changes in corneal astigmatism. If pressure from the eyelids is involved in the aetiology of corneal astigmatism, then one may expect associations to exist between certain characteristics of the eyelids and corneal shape. An experiment was then undertaken to explore these possible associations. We defined the average morphology of the palpebral fissure in different angles of vertical gaze for 100 young normal subjects. This was achieved through analysis of digital images that were captured in primary gaze, 20° downgaze and 40° downgaze. Parameters defining the size, position, angle and contour of the eyelids were determined. Highly significant changes were found to occur in the palpebral fissure with downward gaze. The palpebral aperture narrows in downward gaze, and the angle of the eyelids changes from being slightly upward slanted in primary gaze, to being slightly downward slanted in downward gaze. The eyelid margin contour also flattens significantly in downward gaze. The average topography of the central and peripheral cornea was also defined for this same population. A technique was used that allowed the capture and subsequent combination of topography data from both the central and the peripheral cornea. The use of this technique provided a large corneal topography map, with data extending close to the limbus for each subject. Marked flattening was found to occur in the peripheral cornea and a conic section was found to be a poor descriptor of corneal contour in the periphery (i.e. greater than 6 mm diameter). Corneal astigmatism was also found on average to reduce in the periphery. However a number of distinct patterns of peripheral corneal astigmatism were noted in the population. Corneal astigmatism in the peripheral cornea was either found to remain stable (59% of subjects), increase (10% of subjects) or reduce (31% of subjects) in magnitude in comparison to the amount of central corneal astigmatism. We also investigated associations between the parameters defining the palpebral fissure and parameters describing corneal shape in this population of subjects. A number of highly significant associations were found between the morphology of the palpebral fissure in primary gaze and the shape of the cornea. A general tendency was found for subjects with wider horizontal palpebral fissure widths to exhibit larger corneas and also flatter central corneal powers. There were also highly significant associations found between the angle of the eyelids and the axis of corneal astigmatism, but not the magnitude of corneal astigmatism. The associations found between corneal astigmatism and palpebral fissure morphology is further evidence supporting the hypothesis that pressure from the eyelids is involved in the aetiology of corneal astigmatism. The results of these investigations have shown that corneal changes as a result of eyelid forces occur in the majority of young subjects tested over the course of a normal working day. The average morphology of the palpebral fissure and topography of the central and peripheral cornea has also been defined in detail for a large population of young subjects. Significant associations were found between corneal astigmatism and the morphology of the palpebral fissure. Whilst these results support a model of corneal astigmatism development based on eyelid morphology, they do not prove causation. Further research including measurement of eyelid pressure and corneal rigidity may aid in understanding the exact aetiology of the magnitude and axis of corneal astigmatism.