The cornea is the soft, transparent tissue that covers the front of the eye, and has the twin roles of protecting the inner eye and assisting vision. It is a remarkable structure, being almost totally transparent to visible light and providing the majority of the focussing power of the eye, as well as being resistant to external insult and durable enough to last most of us a lifetime. These properties derive from the unique microstructure of the cornea, specifically the arrangement of the fibrillar protein, collagen.
Here, Dr James Bell from Cardiff University's School of Optometry and Vision Sciences, writes about how the Structural Biophysics Research Group have been using Diamond to better understand the mechanics behind keeping the cornea healthy.
The Cardiff team in the I02 beamline. From left to right: Professor Keith Meek, Chair of the Structural Biophysics Research Group at Cardiff University, Dr James Bell, and Dr Sally Hayes, Team Leader, with work experience students Joseff Davies and Thomas Martin. Picture wisely taken at the start of the experiment, before an all-night data acquisition marathon!
Our group was the first to recognise the potential for synchrotron light to answer the many questions surrounding the link between corneal structure and function; questions that could not be ascertained using qualitative imaging methods, such as electron microscopy. In the 1980s we obtained the first synchrotron wide angle scattering (WAXS) patterns at the Daresbury SRS, using exposures of tens of minutes and, as beam intensities increased and exposure times reduced, were later able map the preferential orientations of the collagen throughout the cornea.
Figure 1. A normal cornea compared with a keratoconic cornea. Keratoconus causes the cornea to deform into a conic shape, which distorts vision and causes significant discomfort. Image courtesy of Ellen Hayes and Ken Pullum.
Using one-second exposures at Diamond we can now make quantitative maps of the collagen structure and organisation throughout a whole cornea at a resolution of 250 μm (2700 X-ray patterns) in about one hour. Using this technique we have been able to show that the precise arrangement of collagen in the cornea varies subtly between individuals1-2, and have revealed that in diseases such as keratoconus3-5 where the cornea is weakened (Fig. 1) or following corneal surgery6-7, the arrangement is highly disrupted (Fig. 2). This structural information is vital to increase our understanding of the mechanical behaviour of the cornea in health and disease, and develop new therapies aimed at preventing disease progression.
Figure 2. Orientation of collagen in the human cornea determined using WAXS. Each polar plot indicates the directions and spread of preferred collagen orientations, while the colour represents the relative degree of anisotropy at each point. A) Normal cornea with collagen predominantly aligned in the vertical and horizontal directions; B) keratoconus cornea exhibiting abnormalities in collagen orientation which are indicative of lamellar slippage; C) corneal scar following surgery which is associated with D), disrupted collagen orientation along the wound edge.
Recent estimates suggest that loss of vision due to corneal trauma or disease affects over 19 million individuals worldwide but the supply of quality donor corneas cannot meet demand. This has prompted an intense effort to (i) develop artificial cross-linking therapies aimed at strengthening the cornea and reducing the need for transplantation and (ii) establish suitable corneal donor tissue alternatives. In association with the UK Cross-linking Consortium, which we founded in 2013 to promote cross-disciplinary research between ophthalmologists, opticians and vision scientists, we have successfully used X-ray scattering techniques to gain an insight into the structural mechanism by which the cornea can be strengthened by cross-linking9-10.
Figure 3. A collagen type III biosynthetic corneal substitute.
To date, biosynthetic corneal substitutes appear to be the most promising corneal tissue alternative (Fig. 3). In particular, a corneal construct formed from cross-linked collagen type III scaffolds, developed by Professor May Griffith from Linköping University, saw success in Phase I clinical trials11. Using X-ray scattering data collected on one of Diamond’s MX beamlines (I02) and the Small Angle Scattering & Diffraction beamline (I22) we have been able to characterise the arrangement of collagen within these constructs8 and pave the way for future studies investigating how these constructs integrate with the host cornea following implantation.
The aim of our current work on I02 and I22 is to take our understanding of the relationship between corneal structure and function one step further by elucidating what happens to the collagen network when it is mechanically loaded. Until recently, our maps of corneal micro- and nano-structure have been taken when the sample was at rest, but our current study involves simultaneous loading and X-ray diffraction imaging. We have opted in this first venture to carry out the simplest of mechanical tests - strip testing (extensometry), but intend to develop more sophisticated testing techniques in the future.
Setting up the experiment
To get the experiment working involved a considerable collaborative effort. We first had to design an experiment with our collaborators that was biologically relevant and compatible with existing models. We then worked closely with beamline scientists to design an extensometer that was compatible with the stages on I02 and I22, which required small, lightweight components that could be controlled from outside the hutch. To co-ordinate the experiment required assistance from the local control and GDA teams, who wrote controllers and scripts that enabled synchronised mechanical testing and imaging. The result was an experiment that successfully yielded results for the changes in collagen architecture in healthy corneas following precise static stretches, as well as an exciting pilot study showing the rate at which these changes occur.
Figure 4. Biomechanical testing of the cornea. A) Close-up of a corneal strip mounted on the extensometer in the I02 beamline. B) Sample data taken from the pictured experiment, showing increases in collagen alignment in the direction of stretch.
Results to understand stiffening
The results from our tests showed significant collagen reorientation, even at stretches of less than 3%, which the cornea experiences in vivo (Fig. 4). This is an exciting finding, as it provides a compelling answer for why the cornea gets stiffer as it is stretched – collagen aligns in the direction of stretch to resist the force. As we begin to understand and quantify the mechanisms behind collagen reorientation, we can start to explain some of the more subtle biomechanical phenomena found in the cornea, such as its ability to maintain its focussing curvature when the pressure in the eye changes (such as in glaucoma), and distortions in corneal shape in degenerative diseases such as keratoconus. The next step in this study will be to test corneas that have been weakened by disease, and also those that have been artificially strengthened by crosslinking.
This collaboration would not have been possible without the tireless support of both beamline teams, the GDA team, and the controls team. Several late nights and many hours of frustrating bug-fixing were involved, but with a robust protocol now established we have a tool with which we can further enhance our understanding of the cornea and significantly improve the accuracy of numerical models aimed at predicting the biomechanical response of the cornea to therapies, surgery, and disease12-14.
I’m a Research Associate working in the School of Optometry and Vision Science at Cardiff University. My background is in biomechanics, specifically developing novel techniques for understanding the relationship between structure and mechanical properties of tissue. My work at Cardiff focuses on the cornea, and developing ways of quantifying changes associated with disease and treatment. I am driven by the hope that my work might make a difference to the way disease is treated.
1. Aghamohammadzadeh H et al. X-ray scattering used to map the preferred collagen orientation in the human cornea and limbus. Structure (2004). DOI: 10.1016/j.str.2004.01.002
2. Boote C et al. Mapping collagen organization in the human cornea – left and right eyes are structurally distinct. Invest Ophthalmol Vis Sci. (2006). DOI: 10.1167/iovs.05-0893
3. Meek KM et al. Changes in collagen fibril orientation in keratoconus corneas. Invest Ophthalmol Vis Sci. (2005). DOI: 10.1167/iovs.04-1253
4. Hayes S et al. The relationship between surface corneal topography and stromal collagen organisation in normal and keratoconus corneas. Exp Eye Res. (2007). DOI: 10.1016/j.exer.2006.10.014
5. Hayes S et al. A depth profile study of abnormal collagen orientation in keratoconus corneas. Arch Ophthalmol. (2012). DOI: 10.1001/archopthalmol.2011.1467
6. Hayes S et al. A structural investigation of corneal graft failure in a suspected case of recurrent keratoconus. Eye (2010). DOI: 10.1038/eye.2009.159
7. Boote C et al. Quantification of Collagen Ultrastructure after Penetrating Keratoplasty – Implications for Corneal Biomechanics. PLoS One (2013). DOI: 10.1371/journal.pone.0068166
8. Hayes S et al. The structural and optical properties of type III human collagen biosynthetic corneal substitutes. Acta Biomater. (2015). DOI:10.1016/j.actbio.2015.07.009
9. Hayes S et al. Riboflavin/UVA collagen cross-linking-induced changes in normal and keratoconus corneal stroma. PloS One (2011). DOI:10.1371/journal.pone.0022405
10. Hayes S et al. The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma. PloS One (2013). DOI: 10.1371/journal. pone.0052860
11. Fagerholm P et al. A Biosynthetic Alternative to Human Donor Tissue for Inducing Corneal Regeneration: 24-Month Follow-Up of a Phase 1 Clinical Study. Sci Transl Med. (2010). DOI: 10.1126/scitranslmed.3001022
12. Petsche SJ and Pinsky PM. The role of 3-D collagen organization in stromal elasticity: a model based on X-ray diffraction data and second harmonic-generated images. Biomech Model Mechanobiol. (2013). DOI: 10.1007/s10237-012-0466-8
13. Koster M et al. Inter- and intra-lamellar slippage of collagen fibrils as a potential mechanism of keratoconus progression. Invest Ophthalmol Vis Sci. (2013). 54 (15):1642
14. Whitford C et al. Biomechanical model of the human cornea: considering shear stiffness and regional variation of collagen anisotropy and density. J Mech Behav Biomed Mater. (2015). DOI: 10.1016/j.jmbbm.2014.11.006
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