Investigation and treatment of trabecular meshwork fibrosis using 3D glaucoma models

背景：开发安全有效的治疗纤维化的方法是青光眼患者的首要任务。这种眼病的特征是眼内眼压（IOP）升高，这是由于房水引流无效造成的。部分原因是由于小梁网（TM）细胞外基质沉积增加导致房水流出受阻1。随着时间的推移，压力的增加会损害眼睛的结构，导致视力丧失。目前降低IOP的治疗策略包括不定期滴眼液，这可能会引起副作用，或者复杂的滤过手术。手术时用抗代谢物如丝裂霉素C治疗组织可减少手术时的纤维化，但这与局部毒性有关，易发生渗漏、组织破裂和感染2。缺乏安全有效的抗纤维化治疗是一个重要的临床挑战。因此，确定药物开发的新靶点非常重要。青光眼的瘢痕和纤维化与小梁网的显著结构和功能重组有关，小梁网是眼部抵抗液体流出的主要部位。青光眼的病理生理学尚未完全阐明，研究人员主要依靠啮齿动物体内模型。这部分是由于缺乏有意义的组织工程和合适的离体模型。为了确定新的靶点和开发新的治疗方法，我们一直与伯明翰和米德兰兹眼科中心合作，成功地从患有和不患有青光眼的患者身上收集TM组织。因此，这一人类数据来源不仅将为我们提供有关该疾病潜在机制的新见解，而且还将为我们提供作为治疗策略的新途径。目的：我们的目标是设计一种新的体外“芯片上器官”模型，以了解青光眼TM中发生的病理，并确定治疗青光眼的新型抗瘢痕化合物。我们将通过研究控制人类小梁网（TM）纤维化的机制以及与施莱姆管细胞的相互作用来实现这一目标。我们将开发模拟人类小梁网/施勒姆管的3D模型共培养系统，然后使用该模型测试和筛选新的抗疤痕治疗方法。此外，我们将在体外实验中开发动态灌注模型，该实验来源于外植体人体组织（伦理和来源材料），以研究眼部疤痕的压力诱导效应。通过对“芯片”模型的对齐光学相干断层扫描（OCT），我们还将定义工程人类TM机械特性的变化以及治疗的细胞和分子结果。培训结果：博士候选人将由眼科生物学家（Hill博士）、生物材料科学家（Grover教授）和组织工程师（a El Haj教授）指导，并将得到临床眼科医生（Masood先生，青光眼顾问）和细胞指导系统有限公司（Michael Jones博士）的工业支持。总的目标是减少使用我们的啮齿动物青光眼模型来评估新的抗疤痕治疗的需要。在这个项目中，学生将接受人体组织处理（来自患者的样本）、细胞培养技术的培训，用于开发3D体外模型重建胶原蛋白和弹性蛋白支架（建模TM），并培养建立离体猪和人类模型的技能，以了解青光眼病理并评估候选治疗方法。学生将学习常规的分子生物学技术（免疫细胞化学、免疫印迹、PCR），以表征模型和评估抗疤痕治疗的效果，并有机会在工业和国际上实习。

Background: The development of safe and effective therapies to treat fibrosis is a major priority for patients with glaucoma. This ocular disease is characterised by elevated intraocular eye pressure (IOP), resulting from ineffective drainage of the aqueous humour. This in part is caused by the blockage of the aqueous humour outflow due to increased extracellular matrix deposition in the trabecular meshwork (TM)1. Over time the increased pressure can damage structures in the eye resulting in vision loss. Current therapeutic strategies to lower the IOP include indefinite eye-drops, which can cause side effects, or complex filtration surgery. Fibrosis at the time of surgery is reduced by treatment of tissue with anti-metabolites such as mitomycin C at the time of surgery, but this is associated with local toxicity predisposing to leaks, tissue breakdown and infections2. The lack of safe and effective anti-fibrotic treatments presents an important clinical challenge. It is therefore important to identify novel targets for drug development. Scarring and fibrosis of the eye in glaucoma is associated with marked structural and functional reorganisation of the trabecular meshwork, the main site of resistance to fluid outflow in the eye. The pathophysiology of glaucoma, has not yet been fully elucidated and investigators are mainly reliant on in vivo rodent models3. This in part has been due to the lack of meaningful tissue engineered and suitable ex vivo models. In order to identify novel targets and develop new treatments, we have been collaborating with Birmingham and Midlands Eye Centre to successfully collect TM tissue from patients with and without glaucoma. Therefore, this human data source will not only provide us with novel insights into the underlying mechanisms of the disease but also provide us with new pathways to target as a therapeutic strategy.Aims: Our aim is design a novel in vitro 'organ on a chip' model to understand the pathology which occurs in the TM in glaucoma and to identify novel anti-scarring compounds for the treatment of glaucoma. We will achieve this by investigating the mechanisms that control fibrosis in human trabecular meshwork (TM) and the interaction with Schlemm's Canal cells. We will develop co-culture systems in 3D models4 which mimic the human trabecular meshwork/Schlemm's canal and then use this model for testing and screening new anti-scarring treatments. In addition, we will develop dynamic perfusion models within ex vivo assays derived from explant human tissues (Ethics and sourced material in place) to investigate the pressure inducing effects of scarring in the eye. Using aligned optical coherence tomography (OCT) to the 'chip' models we will also define changes in mechanical properties of the engineered human TM alongside cell and molecular outcomes of treatments. Training outcomes: The PhD Candidate will be supervised by an ocular biologist (Dr Hill), a biomaterial scientist (Prof Grover) and tissue engineer (Prof A El Haj) and will have close input from a clinical ophthalmologist (Mr Masood, Glaucoma Consultant) and industrial support from the Cell Guidance Systems Ltd (Dr Michael Jones). The overall aim is to reduce the need to use our rodent glaucoma models to assess new anti-scarring treatments. Within this project the student will expect to receive training on human tissue processing (samples derived from patients), cell culture techniques for developing 3D in vitro models reconstructing collagen and elastin scaffolds (to model the TM) and to develop skills in setting up ex vivo porcine and human models for understanding glaucoma pathology and to assess candidate treatments. Students would learn routine molecular biology techniques (immunocytochemistry, western blots, PCR) in order to characterize the models and assess effects of anti-scarring treatments and have the opportunity for both industrial and international placements.