Manipulating and functionalising personalised hydrogels to promote spinal stem cell migration and differentiation

存在各种脊髓的3D模型，包括利用胶原作为基质的先进的组织工程结构。这些3D模型目前被用于模拟脊髓细胞对脊柱内全盘置换所产生的金属磨损颗粒的反应，以及对与脊髓运动和损伤相关的机械力的反应。胶原蛋白模型中细胞的反应很容易监测，环境在一定程度上可以控制，但使用胶原蛋白作为基质有一定的局限性。我们建议开发一种替代的3D高级水凝胶基质，它可以在构建中枢神经系统模型中用作均质的、化学定义的和可持续的胶原凝胶的替代品。一个具体的目标是能够开发一种来自接受者自身组织的个性化凝胶1，并调整基质的物理和化学性质，以便研究并随后指导干细胞向神经细胞的分化。据设想，通过了解个性化基质硬度和功能化对干细胞分化的影响，该项目将导致神经组织修复和再生方面的进展。主题区域脊髓再生和修复大纲我们将从供体组织1合成不同的水凝胶，这些水凝胶将被脱细胞并加工成温度响应性水凝胶。这些凝胶将与生物分子一起功能化，以帮助细胞黏附，例如胺和多肽。脊髓干细胞是室管膜细胞，将从啮齿动物脊髓中分离出来，种植到水凝胶中，水凝胶的硬度变化来确定硬度对室管膜细胞分化的影响。在该项目中可能使用的实验方法包括细胞培养、免疫荧光、免疫细胞化学、细胞活力分析、电子和共聚焦显微镜、3D图像分析、ELISA法、聚合物合成、流变学和使用AFM的纳米压痕。本项目的目的是研究不同个性化水凝胶的使用，这些水凝胶具有明确的基质硬度，并带有生物添加剂以增强细胞附着，以建立干细胞分化的3D脊髓细胞模型。凝胶结构将被用来研究基质硬度对干细胞分化、神经细胞表型和神经元-神经胶质细胞相互作用的影响。

Various 3D models of the spinal cord exist, including an advanced tissue engineering construct utilising collagen as matrix. These 3D models are currently being used to model spinal cord cell responses to metal wear particles from total disc replacements in the spine, and to the mechanical forces associated with spinal cord movement and injury. The responses of cells in the collagen model are readily monitored and the environment can be controlled to some extent, but there are limitations with using collagen as a matrix. We propose to develop an alternative 3D advanced hydrogel matrix that can be used as a homogenous, chemically defined and sustainable alternative to collagen gels in the construction of CNS models. A specific goal is to be able to develop a personalised gel, derived from the recipient's own tissues1, and tune the matrix physical and chemical properties in order to investigate and subsequently direct the differentiation of stem cells into neural cells. It is envisioned that by understanding the effect of personalised matrix stiffness and functionalisation on stem cell differentiation this project will lead to advances in neural tissue repair and regeneration.Topic Area Spinal cord regeneration and repairOutlineWe will synthesise different hydrogels from donor tissues1, which will be decellularised and processed to generate a thermoresponsive hydrogel. These gels will be functionalised with biological molecules to aid cell adhesion e.g. amines and peptides. Spinal cord stem cells, which are ependymal cells (EC), will be isolated from rodent spinal cord, seeded into hydrogels and the stiffness of the hydrogels varied to determine the effect of stiffness on EC differentiation. Compounds influencing stem cell production and differentiation will be added to the cultures.Experimental methodologies likely to be utilised during the project include cell culture, immunofluorescence, immunocytochemistry, cell viability assays, electron and confocal microscopy, 3D image analysis, ELISA, polymer synthesis, rheology and nano-indentation using AFM .ObjectivesThe aims of this project are to investigate the use of different personalised hydrogels, with defined matrix stiffness and functionalised with biological additives in order to enhance cell attachment, in stem cell differentiation to create a 3D spinal cord cell model. Gel constructs will be used to investigate the effect of matrix stiffness on stem cell differentiation, neural cell phenotype and neuron-glial interactions.