Development of biological and synthetic substrates for the ex-vivo expansion of conjunctival epithelium for ocular surface reconstruction

Background

The conjunctiva is a thin membrane that covers and protects the surface of the eye. It lines the inner surfaces of the upper and lower eyelids creating anatomical spaces between the eyelids and eyeball known as conjunctival fornices. The conjunctiva may become irreversibly damaged and the fornices obliterated by scarring following injuries such as chemical burns, severe infections or autoimmune disease. When severe, this prevents eyelid closure, restricts movement and causes lid deformity leading to painful blindness when the cornea (optically clear structure on the eye's surface) becomes opaque due to progressive abrasion and scarring. Such patients comprise up to ten percent of patients attending specialist ocular surface clinics. So far techniques to replace conjunctiva have failed as a result of recurrent scarring or because the graft has been insufficient in size. These patients invariably suffer visual loss due to corneal disease which cannot be addressed with clear corneal grafts unless the ocular surface is restored first.

I will develop a novel biological and synthetic material on which the patient's own conjunctival cells will be cultivated to create larger grafts for transplantation. Conjunctiva will be retrieved from a cadaver and the cellular (living components) removed leaving behind a 'biological scaffold'. Eventually this research will lead to a patient's own cells being seeded on the developed substrate and the resulting graft transplanted into the same individuals so that an immune reaction should not occur. Examples of biological scaffolds successfully transplanted in humans include skin, heart valves and trachea.

The synthetic substrate will be developed on a well tolerated biomaterial (ePTFE), commonly known as 'Gore-Tex', also used in medical devices such as grafts for blood vessel repair. This has been previously shown to support fornix reconstruction but growth of conjunctiva on its surface would be novel. I plan to render the surface conducive to cell growth by incorporating chemical groups and proteins on the ePTFE surface. My preliminary work has shown conjunctival growth on gas plasma treated ePTFE but further development is required to achieve optimal results.


Aim: To develop decellularised human conjunctiva and ePTFE with novel surface chemistry to enable conjunctival expansion for future use as grafts.


How research will be conducted

Human conjunctival tissue will be obtained from deceased patients at the Royal Liverpool University Hospital and the research carried out in laboratories in the University of Liverpool. A novel protocol for the decellularisation of conjunctiva will be developed through collaboration with NHS Blood and Transplant, Liverpool. The ePTFE will undergo chemical modification by a process that changes surface chemistry (gas plasma treatment) and binding of proteins. Once both substrates have been developed, cadaveric conjunctival biopsies will be cultured on the two novel surfaces leading to the production of two conjunctival constructs. The physical and biological properties of the engineered constructs will be tested and compared to natural human conjunctiva.


Expected outcomes

This research will benefit patients who require conjunctival replacement for reasons including glaucoma surgery, excision of conjunctival growths and fornix reconstruction. The greatest impact of this research will be in patients with severe ocular surface disease ranging from autoimmune conditions such as mucous membrane pemphigoid to those with chemical burns. These novel grafts will enable ophthalmologists to develop new surgical strategies to reconstruct the surface of the eye. This will profoundly reduce pain and improve visual outcomes for patients with severe conjunctival disease. The novel materials developed through this fellowship could also lead to cell replacement therapies to treat other incurable eye diseases in the future.

Aim: To develop a novel tissue-derived and synthetic substrate with novel surface chemistry to support the ex-vivo expansion of conjunctival epithelium.

Objectives

1. Develop a range of expanded polytetrafluoroethylene (ePTFE) substrates with novel surface chemistry through gas plasma treatment and adsorption of
proteins including fibronectin and laminin IV. Determine the surface chemistry through x-ray photoelectron spectroscopy and contact angle analysis.
2. Optimise the decellularisation process for human conjunctiva by determining acellularity using a quantitative DNA assay.
3. Determine the optimal substrates by propagating a human conjunctival cell line and determine adhesion/density (quantitative LDH assay),
morphometric analysis, and phenotype of the resulting cell populations.
4. Propagate primary conjunctival cells on the optimised substrates and characterise their phenotype. Perform more detailed analysis including
immunohistochemistry, histology and confocal analysis.
5. Determine tensile strength of the synthetic and biological cultured constructs.

Cell phenotype will be analysed by immunocytochemistry and flow cytometry to quantify cell populations using the following cell markers: CK4/13/8/19 (conjunctival epithelial surface markers); connexin 43 (gap junctional protein); involucrin (terminally differentiated cells); beta integrins; caspase 3 (apoptotic cells); MUC 5AC/CK7 (goblet cells); Ki67, P63alpha, ABCG2 (progenitor cells).


Scientific and medical opportunities

This study will lead to biological and synthetic cultivated constructs for use as grafts for ocular surface reconstruction. This could profoundly improve clinical outcomes in patients with blinding ocular surface diseases. These developments will also represent novel developments in the field of regenerative medicine with applicability to many diseases in which cellular replacement therapies may hold the only hope for cure.

Health and quality of life

Conditions resulting in significant destruction of conjunctiva are uncommon but for those affected have a severe impact on their quality of life due to pain and visual loss. Visual rehabilitation with corneal grafts cannot be undertaken without first restoring conjunctival function. Given the acute shortage of treatment options the development of tissue constructs capable of regenerating the ocular surface will be of immense clinical value. Patients whose conditions include those listed below are often the most severely affected.

- Injury: Chemical, thermal and radiation including battlefield injuries. Ocular burns are associated with 17.3% of battlefield injuries in Iraq and
Afghanistan. Visual disability occurs in 33% and blindness in 15% of patients.

- Immune disease: These conditions include mucous membrane pemphigoid (1.16/million/year incidence; ocular involvement in 60-95%) and
Stevens Johnson syndrome (2-3/million/year incidence; 43-81% ocular involvement, 35% have permanent visual disability)

- Infections: Severe infections such as trachoma, the second commonest cause of blindness in developing nations (up to 25.7% prevalence reported).

- Ocular surgery: Conjunctival replacement is also required in surgical procedures associated with conjunctival loss such as conjunctival neoplasia,
pterygium and glaucoma surgery.

Novel methods for ocular surface regeneration could be a major breakthrough in the management of patients with severe conjunctival disease. Vision and quality of life could be recovered by improving potential for successful corneal transplantion, reducing pain and dependence on medications.


Commercialisation

The 'Business Gateway' at the University will be consulted to determine if the novel substrates can be protected and exploited. If the study proves successful we would seek a collaborative partnership with industry to develop ePTFE substrates. The industrial sector could initiate pre-clinical and clinical studies from 12-18 months following completion of this fellowship and Human Tissue Authority approval. The University and the involved industrial beneficiaries will directly benefit from the increased knowledge, skills and research capacity that this development would generate. This fellowship is in direct partnership with NHSBT who, along with our department, is a key beneficiary from knowledge arising from this research. Decellularised conjunctiva could be produced at NHSBT within two years and transplantation trials within three years following completion of this fellowship.


Economic and social impact

Successful development of novel biomaterial technology could be applicable to a broad range of tissue engineering applications and therefore be relevant to commercialisation by multiple biotechnology firms. If decellularisation and expansion of conjunctival tissue on the tissue-derived substrate is successful, this too may have wider economic and social impacts including creation of wealth and employment at NHSBT and potentially other biotechnology industrial collaborators. These outcomes could lead to creation of a greater skilled workforce through new training and employment opportunities in these industries. Both these could have potential economic impact for the University in the form of a new spinout company(ies) seeking to exploit these commercial opportunities in associated applications of this technology.

Visual impairment in the UK is a major societal challenge in the face of an already over-burdened healthcare system and is also a serious financial drain on the economy (through lost working days to employers, lost wages for individuals and the expensive on-going care). This fellowship has the potential to significantly reduce these burdens and increase both the quality of life for patients, and could enable significant cost savings across healthcare providers in the long term.