Development of a novel biomaterial model of collagen mediated genetic disease for study of collagen organisation and drug development

成骨不全(OI)是一组17种遗传性结缔组织疾病，由胶原基因突变介导，导致骨骼变形和脆性。目前，体内试验是治疗的标准做法，有20多个动物模型代表OI。这些模型在技术上是有限的，但在胶原排列和组织的可及性和重复性方面也是有限的。一种经过验证的体外方法可以进行准确的临床前建模。间充质干细胞(MSC)可以分化为骨形成细胞，并产生骨样胶原细胞外基质。赖利教授团队的工作表明，胶原蛋白的组织和质量可以用特定于组织的方式建模。我们认为，导致OI的基因突变也将影响细胞正确组织胶原的能力，使用这种支架-细胞系统将为OI的发病机制提供重要的见解。本项目的目的是建立具有特征性和有效性的体外OI胶原病理模型。这将通过使用电纺生产聚己内酯支架来实现，细胞将按照既定的方案在其上生长。支架纤维特征和胶原结构之间的关系将使用光学显微镜和扫描电子显微镜来描述支架的特征，组织学和二次谐波显微镜来研究细胞和细胞外因子。力学测试将被用来确定胶原组织在组织结构中的作用。从患者群体中发现的OI突变将通过CRISPR内源性基因编辑引入MSC细胞，从而能够在健康和OI胶原之间进行比较。这将使分子途径的研究和治疗剂的测试成为可能。将开发一种使用ImageJ的高通量图像分析协议，用于对大数据集进行系统分析。

Osteogenesis imperfecta (OI) is a group of 17 inherited connective tissue disorders mediated by mutations in collagen genes that lead to bone deformation and fragility. Currently, in vivo testing is the standard practice for treatments, with over 20 animal models representing OI. These models are technically limited, but also with regards to accessibility and reproducibility of collagen alignment and organisation. A validated in vitro method would allow for accurate preclinical modelling.Mesenchymal stem cells (MSC) can differentiate into bone forming cells and create a bone-like collagen extracellular matrix. Work in Prof. Reilly's group has demonstrated that the organisation and quality of the collagen can be modelled in a tissue specific manner. We propose that the genetic mutations that cause OI will also affect the ability of cells to organise collagen correctly, and that using this scaffold-cell system will provide important insight into OI pathogenesis. The aim of this project is to develop characterised and validated in vitro model of OI collagen pathology. This will be achieved by using electo-spinning to produce polycaprolactone scaffolds on which cells will be grown following established protocols. The relationship between scaffold fibre characteristics and collagen structure will be mapped using light microscopy and SEM for scaffold characteristics, and histology and second harmonic generation microscopy for cellular and extracellular factors. Mechanical testing will be used to determine the role of collagen organisation in tissue structure. OI mutations identified from patient populations will be introduced to MSC cells using CRISPR endogenous gene editing enabling comparison between healthy vs OI collagen. This will allow for investigation of the molecular pathways and testing of therapeutic agents. A high throughput image analysis protocol using ImageJ will be developed for systematic analysis of large data sets.