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

结膜是一层覆盖并保护眼睛表面的薄膜。它排列在上下眼睑的内表面，在眼睑和眼球之间形成被称为结膜穹窿的解剖空间。在化学烧伤、严重感染或自身免疫性疾病等损伤后，结膜可能会受到不可逆的损伤，穹窿可能会被疤痕所覆盖。严重时，它会阻止眼睑闭合，限制运动，并导致眼睑畸形，当角膜（眼睛表面的光学透明结构）由于逐渐磨损和瘢痕而变得不透明时，会导致痛苦的失明。这类患者占到专科眼表诊所就诊患者的10%。到目前为止，替代结膜的技术由于复发性疤痕或移植物尺寸不够而失败。这些患者总是因角膜疾病而导致视力丧失，除非首先恢复眼表，否则不能通过透明角膜移植来解决。我将开发一种新的生物和合成材料，在这种材料上，病人自己的结膜细胞将被培养成更大的移植移植物。结膜将从尸体上取出，细胞（活的成分）被移除，留下一个“生物支架”。最终，这项研究将导致患者自身的细胞被植入已开发的基质上，并将由此产生的移植物移植到相同的个体中，这样就不会发生免疫反应。成功移植到人体的生物支架包括皮肤、心脏瓣膜和气管。合成基质将在一种耐受性良好的生物材料（ePTFE）上开发，这种材料通常被称为Gore-Tex，也用于医疗设备，如血管修复的移植物。这已经被证明支持穹窿重建，但结膜在其表面生长将是新的。我计划在ePTFE表面加入化学基团和蛋白质，使其表面有利于细胞生长。我的初步工作表明，在气等离子体处理的ePTFE上结膜生长，但需要进一步发展以达到最佳结果。目的：开发具有新型表面化学成分的脱细胞人结膜和ePTFE材料，使结膜扩展用于未来的移植。研究将如何进行人类结膜组织将从皇家利物浦大学医院的已故患者身上获得，并在利物浦大学的实验室进行研究。结膜脱细胞的新方案将通过与NHS血液和移植合作开发，利物浦。ePTFE将通过改变表面化学（气体等离子体处理）和蛋白质结合的过程进行化学修饰。一旦这两种基质都被开发出来，尸体结膜活检将在两种新的表面上培养，从而产生两种结膜结构。工程结构的物理和生物特性将被测试，并与天然人类结膜进行比较。预期结果本研究将使因青光眼手术、结膜生长切除和穹窿重建等原因需要结膜置换的患者受益。这项研究的最大影响将是严重眼表疾病的患者，从自身免疫性疾病如粘膜类天疱疮到化学烧伤。这些新的移植物将使眼科医生能够开发新的手术策略来重建眼表面。这将大大减轻疼痛，改善严重结膜疾病患者的视力。通过这项研究开发的新材料也可能导致细胞替代疗法在未来治疗其他无法治愈的眼病。

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.