Development of mechanically robust functionalised chitosan-based hydrogels for tissue engineering applications in cartilaginous tissues

背景：随着人口老龄化和人口增长，与年龄相关的软骨组织疾病，如椎间盘（IVD）退变（腰痛[LBP]的主要原因），成为日益严重的全球社会经济问题。目前还没有成功的长期治疗方法，尽管基于细胞的组织工程提供了巨大的潜力，允许因疾病而受损的组织再生。这包括恢复患病关节的活动能力和提供长期的疼痛缓解。成体干细胞，又称间充质干细胞（MSCs），存在于骨髓中，因其易于分离、生长迅速且能形成（分化为）软骨组织中的细胞，为组织工程提供了巨大的潜力。事实上，曼彻斯特大学的研究小组已经证明，间充质干细胞可以形成IVD细胞，使其成为IVD再生的理想选择。然而，组织工程需要一种生物材料来支持细胞和帮助组织再生。IVD中心呈凝胶状；因此水凝胶（高含水量的凝胶）是最合适的。水凝胶也可以注射到IVD中，从而避免了侵入性手术。曼彻斯特大学的研究小组先前已经证明，将间充质干细胞植入壳聚糖/甘油磷酸水凝胶中，可以成为IVD细胞，并产生类似IVD的组织。然而，目前这些凝胶缺乏在人体脊柱的负载环境中存在所需的机械强度。因此，我们的目标是：1。开发新型壳聚糖水凝胶，能够承受人体脊柱内的负荷，同时允许间充质干细胞分化为IVD细胞并再生功能组织；2. 将这些新的机械坚固的水凝胶转化为热敏水凝胶（室温下的液体和体温下的凝胶），可以注射到IVD中，从而最大限度地减少复杂的手术和术后护理；3. 通过添加营养物质（如葡萄糖）来增强细胞存活率，或添加因子来防止未来疾病对组织的进一步损害，为机械坚固的热敏水凝胶添加特定功能。为了实现这一目标，华威团队将采用最先进的化学技术，以先前评估的相同生物相容性材料为基础，生产机械坚固的水凝胶。要做到这一点，将研究化学的高效共价交联。在接近人体的温度下进行凝胶化的机械坚固材料的开发将通过使用选择性可逆的活性凝胶位点保护来解决，并将导致组织工程水凝胶材料的范式转变。曼彻斯特大学的研究小组将在每个阶段对这些水凝胶进行筛选，以确保它们支持MSC存活并向IVD细胞分化。他们已经鉴定出了IVD细胞特有的基因，这些基因可以用来确保MSCs确实变成了IVD细胞。此外，他们还开发了一种“生物反应器”，该反应器使用从尸体（已捐赠用于研究）中获得的人体IVD组织，并将msc种子水凝胶注入其中。注射的细胞种子水凝胶可以在“生物反应器”中培养，它精确地模拟了人类脊柱的条件，包括日常运动中经历的机械负荷，使其成为比目前的动物模型更相关的系统。所获得的数据将允许确定最适合IVD再生的水凝胶。预期结果和潜在临床益处：我们将化学和基于msc的组织工程专业知识结合起来的能力，以及我们独特的测试系统，将有助于推进基于msc的组织工程治疗IVD变性的临床转化。这样，我们就有能力根除椎间盘退变，从而提高数百万人的生活质量，为医疗保健系统和全球更广泛的经济节省数十亿英镑。

Background: Age-related diseases of cartilaginous tissues, such as intervertebral disc (IVD) degeneration (a major cause of low back pain [LBP]), pose an increasing global socioeconomic problem as the population ages and increases. There are currently no successful long-term treatments, although cell-based tissue engineering offers huge potential, by allowing regeneration of tissues damaged through disease. This includes restoring mobility to diseased joints and offering long-term pain relief. Adult stem cells, known as mesenchymal stem cells (MSCs), present in bone marrow offer great potential for tissue engineering as they can be isolated easily, grow quickly and can form (differentiate into) the cells found in cartilaginous tissues. Indeed the Manchester team have shown that MSCs can form IVD cells, making them ideal for IVD regeneration. However, tissue engineering requires a biomaterial to support cells and aid tissue regeneration. The centre of the IVD is gel-like; therefore hydrogels (gels with a high water content) are the most suitable. Hydrogels can also be injected in to the IVD, thus avoiding invasive surgery. The Manchester team have previously shown that MSCs seeded into chitosan/glycerophosphate hydrogels become IVD cells and produce an IVD-like tissue. However, currently these gels lack the mechanical strength required to exist within the loaded environment of the human spine. Therefore our objectives are to: 1. Develop novel chitosan-based hydrogels which can withstand the loads experienced within the human spine, whilst allowing MSCs to differentiate into IVD cells and regenerate a functional tissue; 2. To convert these new mechanically robust hydrogels into thermosensitive hydrogels (liquids at room temperature and gel at body temperature) which can be injected into the IVD thus minimising complex surgery and aftercare; 3. Add specific function to the mechanically robust, thermosensitive hydrogels through the addition of nutrients (such as glucose) to enhance cell survival, or the addition of factors to prevent further damage to the tissue through future disease. To achieve this the Warwick team will employ state-of-the-art chemistry to produce mechanically robust hydrogels, based around the same biocompatible materials previously evaluated. To do this highly efficient covalent cross-linking chemistries will be investigated. The development of mechanically robust materials that undergo gelation at temperatures close to that of the human body will be addressed with the use of selectively reversible protection of the active gelation sites and will result in a paradigm shift in hydrogel materials for tissue engineering. The Manchester team will screen these hydrogels at each stage to ensure they support MSC survival and differentiation to IVD cells. They have identified genes specific to IVD cells, which can be used to ensure MSCs have indeed become IVD cells. Additionally, they have also developed a 'bioreactor', which uses human IVD tissue obtained from cadavers (which have been donated for research) into which MSC-seeded hydrogels are injected. The injected cell-seeded hydrogels can then be cultured within the 'bioreactor', which accurately mimics conditions in the human spine, including the mechanical loads experienced during daily movements, making this a more relevant system than current animal models. The data obtained will allow identification of the most suitable hydrogel for IVD regeneration. Expected outcomes and potential clinical benefit: Our ability to combine expertise in chemistry and MSC-based tissue engineering, together with our unique testing system, will help advance the translation of MSC-based tissue engineering therapies for IVD degeneration to clinic. In so doing we have the ability to eradicate disc degeneration and thus improve the quality of life for millions of people and save billions of pounds for healthcare systems and the wider economies around the globe.