The ultrastructural basis of corneal dysfunction and the development and optimization of novel therapeutic strategies

Lead Research Organisation: Cardiff University
Department Name: Optometry and Vision Sciences

Abstract

The cornea is the transparent window at the front of the eye and is its main focussing element. To fulfil its role it has to be very transparent, very strong and precisely shaped. Transparency, strength and shape are all controlled by the collagen fibrils that make up the cornea, and by the small molecules between them. This happens at different structural levels from the molecular level upwards: collagen molecules form fibrils, which in turn form larger structures called lamellae, which are then stacked up to form the tissue itself. From our previous work and work done by others, we know a lot about why the cornea is transparent and are beginning to understand the arrangement of collagen lamellae that gives rise to the cornea's shape and thus its focusing abilities. However, the precise details are still not known and, until they are, it will not be possible to understand why, in numerous diseases of the cornea, or after different types of surgery on the cornea - including laser surgery - transparency, strength and/or shape are abnormal and vision is lost or very blurred. We will use several new and exciting 3-D biological imaging and powerful X-ray measuring techniques, to explain how collagen fibrils and cells are arranged in the cornea to make it transparent, and how other proteins control this arrangement. We will also explain at a higher structural level how lamellae are arranged to provide form and strength. This will allow us to construct computer models from which to predict changes in corneal shape following given surgical incisions or other treatments to help inform surgeons. We will then explain what goes wrong in several important corneal diseases and investigate methods of preventing or correcting these changes, for example by using stem cell therapy or protein crosslinking. The methods will also allow us to explain why, when the cornea is wounded, tissue strength and transparency are compromised. Again, methods to improve this wound healing such as those mentioned above will be investigated, with the aim of strengthening the cornea whilst preserving transparency. Finally, several research groups in different countries are trying to develop a biological artificial cornea as there is, and is likely to continue to be, a worldwide shortage of donor corneal tissue for graft surgery. Synthetic biology depends on understanding how nature utilises the constituents of a tissue to achieve its vital properties. In the case of the cornea, the knowledge that we will obtain by elucidating the exact relationship between structure and function will be invaluable, and will allow us to collaborate with these groups to drive their constructs more quickly towards a fully functioning artificial cornea.

Technical Summary

The cornea of the eye is a uniquely transparent, exquisitely shaped tissue whose functionality depends heavily on the hierarchical ultrastructures and complex microanatomy of its cells and extracellular matrix. Despite the importance of the cornea for vision, the basis of corneal transparency and shape, and their compromise due to injury or disease, are only now starting to be understood in any detail. Our ultimate objective is to build upon the success of our previous studies and relate loss of transparency and changes in corneal shape/astigmatism to tissue micro- and ultra-structure. This will be achieved by taking advantage of recent advances in microfocus synchrotron x-ray scattering, electron tomography, multiphoton laser scanning microscopy and volume scanning electron microscopy to study human foetal corneas, normal and pathological adult corneas, post-surgical corneas and artificial corneal constructs. Our aims are to (i) model corneal transparency at the cellular and fibrillar level, and to extend this to explain the loss of transparency in corneal wounds and diseases; (ii) characterise the full three-dimensional structure of the cornea and explain the structural basis of astigmatism, and demonstrate how surgical treatments can be modeled to predict the cornea's macroscopic behaviour by finite element analysis; (iii) develop and better understand new methodologies for treating or stabilizing corneas, such as cell-based therapies for improving transparency and mechanical strength in corneal wounds and crosslinking methods to stabilize corneas to prevent ectasia; (iv) use the new three-dimensional structural information to assess and improve different approaches being used to develop biological artificial corneal replacements.

Planned Impact

The main non-academic beneficiaries will be the patients themselves. Our work on corneal pathologies will aid understanding of the disease mechanisms and drive the development of better treatments from which patients will benefit. Our work on refractive surgery should improve the outcome of these procedures (stronger flap adherence, less likelihood of post-LASIK ectasia or induced astigmatism) and lead to safer treatments, more predictable clinical outcome and better patient satisfaction. We anticipate our work on corneal crosslinking extending the number of keratoconus patients who can undergo the treatment, while our research into microwave therapy may offer new hope to keratoconus who cannot undergo LASIK and whose only current treatment option is corneal transplant.
Other beneficiaries will include our anticipated commercial partners, who would be involved in clinical trials prior to marketing the methods for stabilizing the post-surgical wounds; this would lead to commercial benefit for the company and economic benefit for the UK. Refractive surgery has been carried out on millions of people worldwide, not always without post-operative complications. If we can develop better methods to carry out such surgery, or find methods of improving the outcomes of the current surgery, there will be a huge worldwide benefit to public health. Finally, there is a pressing need for the development of artificial corneal tissue whose properties mimic the natural tissue. Our work to develop a biological artificial cornea will be in collaboration with groups in other countries who are leading the field in this area. If we succeed, it will have a lasting impact on global quality of life, particularly in developing countries where the shortage of donor tissue is already critical.
Timescales are different for different projects within the proposal. For example, our work on methods of epithelial removal prior to corneal crosslinking had an immediate impact on practice, and this should be the case when this work is continued, with the aim of finding a method that does not require epithelial removal. Developing a suitable method to stabilize LASIK flaps, for example by using oral mucosal cells, will take 5 years, including animal trials, and clinical trials would follow. Investigating alternative methods of refractive correction, such as the use of microwaves, may have an impact within a few years, as clinical trials are already underway. Similarly, the corneal constructs created by Professor Griffith are also in early clinical trials, and the optimized constructs arising from our joint research will follow into trials. Finally, developing computer models to predict the outcome of corneal surgeries is a longer-term outcome with widespread potential benefit and may take up to 10 years.

Publications

10 25 50
 
Description St Thomas'/Cardiff protocol for corneal crosslinking
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Since the advent of corneal-crosslinking (CXL) in 1999, as a therapy for halting keratoconus progression, over 30 modifications to the standard therapy are now in existence. The modified protocols aim to improve patient comfort, reduce patient treatment times and reduce the likelihood of infection, however their efficacy in many cases not been validated prior to use. Modifications to the technique include non-removal of the corneal epithelium, the use of a wide variety of riboflavin solutions and the application of different Ultraviolet-A irradiation intensities, modes (pulsed or continuous) and exposure times. Since 2005, we have been working with Prof David O'Brart at St Thomas's Hospital to test the efficacy of new (St Thomas's/Cardiff Protocol) and existing CXL protocols in vitro. This led to a successful clinical trial of the St Thomas'/Cardiff protocol (ISRCTN04451470). In addition to our published research influencing surgical practice, our investigations into the structural basis of corneal cross-linking have led to a greater understanding of exactly how the therapy works. Such information is vital for the development of computer models aimed at predicting the outcome of CXL protocols associated with refractive surgery. Such models have recently become an essential component in the quest to gain FDA approval for the use of CXL in America.
 
Description UK Crosslinking Consortium
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
Impact n 2013 we established a UK CXL Consortium to bring together ophthalmologists and vision scientists with the aim of standardising CXL in the UK and developing a code of best practice. The consortium now boasts over 50 members. With the support of the consortium and the Medical Research Council, Cardiff University are now funding the development of a national CXL database which will enable ophthalmologists to collect clinical information on patients being considered for CXL and track their treatment and outcomes. It is envisaged that the database will be used as a basis for clinical care, outcome analysis, clinical audit, revalidation and research. NHS England has recently been pitched for specialised services funding to start CXL in approximately 10 regional UK centres to address the postcode lottery of CXL provision in the NHS. A key feature of this pitch was our database which is considered essential for auditing outcomes.
 
Description BBSRC Japan Partnering Award
Amount £46,000 (GBP)
Funding ID BB/R021244/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Follow on Fund
Amount £144,900 (GBP)
Funding ID BB/N022106/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2017 
End 04/2018
 
Description Newton Fund
Amount £31,500 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 07/2017
 
Description Pathfinder
Amount £7,203 (GBP)
Funding ID BB/P011969/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 11/2016
 
Description Research Contract
Amount £828,093 (GBP)
Funding ID PO 1000101930 
Organisation Defence Science & Technology Laboratory (DSTL) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2019
 
Description Research Leave Fellowship
Amount £15,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 09/2018 
End 08/2019
 
Description Responsive mode
Amount £840,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2015 
End 07/2018
 
Description Sparking Impact
Amount £6,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2013 
End 02/2014
 
Title x-ray analysis GUI 
Description A new graphical user interface was developed for the analysis of x-ray diffraction data 
Type Of Material Biological samples 
Provided To Others? No  
Impact Paper in preparation. 
 
Title Elsheik modelling of the healthy human cornea 
Description Our quantitative x-ray data have been used to model the biomechanical behaviour of the human cornea. 
Type Of Material Computer model/algorithm 
Year Produced 2012 
Provided To Others? Yes  
Impact Data helped explain the relationship between human corneal structure and shape. This work has been published. 
 
Title Grytz modelling of keratoconus cornea 
Description Our quantitative x-ray data have been used to model the development of keratoconus. 
Type Of Material Computer model/algorithm 
Year Produced 2012 
Provided To Others? Yes  
Impact Data helped explain the progression of keratoconus involving a mechanism of enzymatic digestion, strain and tissue slippage. The has been presented at conferences. 
 
Title Keratoconus e-registry 
Description A national database collecting patient data relating to keratoconus progression and crosslinking outcomes. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact Data collection is ongoing and when sufficient data is gathered it will be analysed. 
 
Title Pinsky modelling of keratoconus development 
Description Our quantitative x-ray data have been used to model the development of keratoconus.. 
Type Of Material Computer model/algorithm 
Year Produced 2015 
Provided To Others? Yes  
Impact Data helped the development of a computer modelling system which converts discrete data points to mathematical functions which will allow better predictive response of corneal behaviour during the development of keratoconus. 
 
Description Arie Markovich 
Organisation Weizmann Institute of Science
Country Israel 
Sector Academic/University 
PI Contribution Ultrastructural analysis of cross-linked tissue
Collaborator Contribution Carried out cross-linking in rabbits
Impact Manuscript in preparation
Start Year 2013
 
Description BJ Dupps 
Organisation Cleveland Clinic
Country United States 
Sector Charity/Non Profit 
PI Contribution Supply of corneal x-ray scattering data
Collaborator Contribution Incorporation of corneal x-ray scattering data into biomechanical models
Impact Our data is being used to enhance the accuracy of computer models aimed at predicting the response of the cornea to surgery.
Start Year 2015
 
Description Collaboration with Bristol Zoo Gardens 
Organisation Bristol Zoo Gardens
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We have collected x-ray scattering data from these corneas with the aim of learning more about the relationship between corneal structure, shape and function.
Collaborator Contribution Our collaborators provided post-mortem corneas from primates and other mammals.
Impact Meeting abstracts and publications. Multidisciplinary: Zoology, Biophysics, Biology
Start Year 2007
 
Description Collaboration with Farhad Hafezi (Geneva) 
Organisation Geneva University Hospitals
Country Switzerland 
Sector Hospitals 
PI Contribution We have collected x-ray scattering data from normal and keratoconus corneas that have been previously cross-linked using riboflavin and UVA.
Collaborator Contribution Our collaborators have been involved in the concept and design of collagen cross-linking studies involving keratoconus corneas.
Impact Publications. Multidisciplinary: Ophthalmology, Biophysics, Biology
Start Year 2010
 
Description Collaboration with Invitrogen 
Organisation Life Technologies
Department Invitrogen
Country United States 
Sector Private 
PI Contribution Based on our world renowned expertise in electron microscopy we were approached by Invitrogen to do consultancy work involving the use of 3D SEM to characterise potential artificial corneal constructs.
Collaborator Contribution Provided type III recombinant human collagen hydrogels
Impact The company have gone on to develop constructs.
Start Year 2014
 
Description Collaboration with May Griffith 
Organisation Linkoping University
Department Regenerative Medicine
Country Sweden 
Sector Academic/University 
PI Contribution Prof Griffith has developed a biological artificial cornea which is in clinical trial. We will be investigating the ultrastructure of the constructs and what changes occur in vivo.
Collaborator Contribution They will provide the constructs. Joint collaborators in Aberdeen will implant the constructs into mouse eyes.
Impact We have obtained the first x-ray diffraction patterns from tese constructs.
Start Year 2012
 
Description Collaboration with Prof. J. Funderburgh in University of Pittsburgh Medical Centre 
Organisation University of Pittsburgh Medical Center (UPMC)
Country United States 
Sector Academic/University 
PI Contribution collagen ultrastructural x-ray studies of scarred corneas treated with stromal stem cells
Collaborator Contribution Provided wounded mouse corneas for stem cell baseline studies
Impact International Collaboration Award-Early Career Researcher 2007/08, Cardiff University. Funding was allocated to Dr. Christina Kamma-Lorger to visit collaborative lab in in Pittsburgh University for 4 weeks, to receive training in corneal stem cell techniques and establishing a new collaboration (2300GBP)+ publications (see list). Multidisciplinary: Ophthalmology, Biophysics, Biology, Molecular Biology
Start Year 2007
 
Description Dr Ahmed ElSheikh 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Modelling corneal structure and biomechanics. Our structural data is being used to model corneal biomechannical behaviour. We are also working together to simultaneously monitor changes in corneal structure and stress-strain behaviour by covering corneas with a fine-particle speckle pattern and then collecting x-ray scattering data at the same time as collecting images of the corneal surface on spatially oriented cameras.
Collaborator Contribution Allowed us to progress the biomechanical implications of our structural data
Impact These publications are listed separately under MRC grant G0600755
Start Year 2008
 
Description Elena Lanchares 
Organisation University of Zaragoza
Country Spain 
Sector Academic/University 
PI Contribution Provided advice and facilities
Collaborator Contribution During a study visit to Cardiff, Elena carried out a number of biomechanical measurements on cross-linking
Impact Named author on a publication
Start Year 2012
 
Description Fibrillin knock-out mouse 
Organisation University of Sao Paulo
Country Brazil 
Sector Academic/University 
PI Contribution We instigated a collaboration with Prof Lygia Pereira, Department of genetics, Sao Paulo University, Brazil who developed a mouse model for Marfans syndrome, where FBN1 gene is knocked out. We undertook biophysical studies of corneas from these mice which have helped us to understand the role of fibrillin microfibrils in maintaining corneal shape.
Collaborator Contribution Developed the mouse model and supplied the mouse eyes
Impact Paper submitted for publication and further studies with a more severe phenotype planned.
Start Year 2016
 
Description Hatch Mukherjee 
Organisation Colchester Hospital University NHS Foundation Trust
Department Ophthalmology
Country United Kingdom 
Sector Public 
PI Contribution Provided laboratory facilities and animal tissue
Collaborator Contribution Carried out the surgery and data analysis
Impact One paper published
Start Year 2013
 
Description Morio and Keiko 
Organisation Kyoto Prefectural University of Medicine
Country Japan 
Sector Academic/University 
PI Contribution Provision of facilities and guidance in the use of the 3D Scanning electron microscope
Collaborator Contribution Carried out the research
Impact one paper published (Mol Vis. 2015; 21: 1328-1339
Start Year 2014
 
Description Mouse implant study 
Organisation University of Aberdeen
Country United Kingdom 
Sector Academic/University 
PI Contribution Ultrastructural analysis of RCH III corneal implants
Collaborator Contribution Performing the surgery
Impact Paper in presentation
Start Year 2012
 
Description Nanosome network 
Organisation Cardiff University
Department School of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Developing correlative EM methods to identify exosome secretion by corneal endothelial cells
Collaborator Contribution Expertise in the field of exosome biology
Impact None as yet.
Start Year 2017
 
Description Open Eyes and CXL database 
Organisation The Open Eyes Foundation
PI Contribution A keratoconus e-registry was developed as part of OpenEyes electronic patient records system: we developed content
Collaborator Contribution A keratoconus e-registry was developed as part of OpenEyes electronic patient records system: they developed the web presence
Impact Following a trial period at London Moorfields the e-registry went live in 2017 and is now available to any hospital with OpenEyes access. The database allows keratoconus patients progression and treatment outcomes to be monitored.
Start Year 2015
 
Description Peter Winlove 
Organisation University of Exeter
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided facilities to carry out non-linear microscopy and contributed to resulting publication
Collaborator Contribution Carried out the experimental research and data analysis
Impact Exp Eye Research paper Dr James Bell from this group has moved to Cardiff and joined our group
Start Year 2012
 
Description Raphael Grytz 
Organisation University of Alabama at Birmingham
Country United States 
Sector Academic/University 
PI Contribution Supply of x-ray scattering data obtained from normal and keratoconus corneas
Collaborator Contribution Incorporation of x-ray scattering data into computational models aimed at predicting keratoconus progression
Impact The work has been presented at several international conferences
Start Year 2013
 
Description Veni Vidi 
Organisation Veni Vidi
Country United Kingdom 
Sector Private 
PI Contribution Corneal cross-linking
Collaborator Contribution supply of riboflavin and one travel grant
Impact several publications on cross-linking
Start Year 2012
 
Description heard collaboration 
Organisation Cardiff University
Department School of Pharmacy and Pharmaceutical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of novel topical drug delivery systems to the eye and determining their biological effects
Collaborator Contribution expertise in the field of drug delivery systems
Impact None as yet
Start Year 2017
 
Title CXL e-registry 
Description The creation of a CXL e-registry within Open Eyes software to facilitate the tracking of keratoconus progression and treatment outcomes following NICE recommendations 
Type Of Technology Software 
Year Produced 2017 
Impact Significant take-up by the community 
 
Title SAXS4COL 
Description A software package to analyse x-ray scattering data from fibrous biomaterials was developed and published 
Type Of Technology Software 
Year Produced 2017 
Impact The software has been used extensively in our publications and is being used by beamline scientists at the Alba synchrotron in Barcelona. Details have been published Abass A, Bell JS, Spang MT, Hayes S, Meek KM, Boote C. SAXS4COLL: an integrated software tool for analysing fibrous collagen-based tissues. Journal of Applied Crystallography2017; 50: 1235-1240. doi.org/10.1107/S1600576717007877 
 
Description British Science Association Science Cafe talk on Developments in corneal research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Dr Craig Boote was invited by the British Science Association to speak in a public house about our research on the cornea and the meeting was attended by approximately 20 people, which sparked questions and discussion.
Year(s) Of Engagement Activity 2016
 
Description Doutch S4C 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Dr James Doutch, graduate of our group and current collaborator, appeared on an S4C science programme which focused on science at the Diamond synchrotron in Oxfordshire. James enthusiastically explained, in Welsh, the structural changes in the cornea when keratoconus develops.
Year(s) Of Engagement Activity 2015
URL http://www.bbc.co.uk/iplayer/episode/p02rv29x/dibendraw-clir-fel-crisial
 
Description Hosting work experience students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Work experience students spent one week shadowing our research at Cardiff University and Diamond Light Source
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Invitation to contribute a feature article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Invitation to contribute a guest feature on our research to Diamond News Magazine (2016): Visualising the Dynamic Behaviour of corneal collagen: Diamond News: http://www.diamond.ac.uk/Home/Corporate-Literature/newsletter/Spring2016/User-story.html
This piece shows evidence of the work experience students with us at the Diamond synchrotron as part of our STEM programme..
Year(s) Of Engagement Activity 2016
 
Description Invitation to speak to Diamond Users and staff 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dr James Bell received an invitation to speak at the DIAMONDS seminar series on Jan 19th 2017 about his ongoing research at Diamond - seminar open to all scientific staff, guests and users. The was plenty of discussion after the presentation.
Year(s) Of Engagement Activity 2017
 
Description Invited to contribute guest feature for Science Parks 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Dr Phil Lewis contributed an article on the use of 3D SEM to the Spring issue of Innovation into Success, a magazine Ten Alps Publishing produce for the United Kingdom Science Park Association www.scienceparks.co.uk (the article starts on page 90). The publication is distributed three times each year across the UKSPA membership, taking in approximately 70 science parks, 3,000 businesses and 70,000 professionals working in science, technology and R&D.
Year(s) Of Engagement Activity 2015
 
Description James OBCS 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Dr James Bell presented research findings on corneal biomechanics to an audience of clinicians, research scientists and graduate students
Year(s) Of Engagement Activity 2017
 
Description Phil CITER presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Dr Phil Lewis, on behalf of the group, gave a presentation on the capabilities of 3D EM to an audience of academic tissue engineers
Year(s) Of Engagement Activity 2018
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Dr Sally Hayes visited a local primary school to run a Mad Science session which involved teaching children about light perception. The session was well received and she has been invited back to run another session in the near future.
Year(s) Of Engagement Activity 2017
 
Description cornea stroma wikipedia 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The generation of a wikipedia page describing the biology of the corneal stroma
Year(s) Of Engagement Activity 2015
URL https://en.wikipedia.org/wiki/Stroma_of_cornea