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

背景资料：随着人口老龄化和增加，与软骨组织相关的疾病，如椎间盘（IVD）变性（腰痛[LBP]的主要原因），构成了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通过允许因疾病受损的组织再生而提供了巨大的潜力。这包括恢复患病关节的活动性，并提供长期的疼痛缓解。成体干细胞，称为间充质干细胞（MSC），存在于骨髓中，为组织工程提供了巨大的潜力，因为它们可以容易地分离，生长迅速，并且可以形成（分化成）软骨组织中发现的细胞。事实上，曼彻斯特研究小组已经证明，MSC可以形成IVD细胞，使其成为IVD再生的理想选择。然而，组织工程需要生物材料来支持细胞和帮助组织再生。IVD的中心是凝胶状的;因此水凝胶（含水量高的凝胶）是最合适的。水凝胶也可以注射到IVD中，从而避免侵入性手术。曼彻斯特研究小组先前已经证明，将MSC接种到壳聚糖/甘油磷酸盐水凝胶中成为IVD细胞并产生IVD样组织。然而，目前这些凝胶缺乏在人类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。开发新型的基于壳聚糖的水凝胶，其可以承受人类脊柱内经历的负荷，同时允许MSC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（室温下为液体，体温下为凝胶），其可以注射到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，或添加因子来防止未来疾病对组织的进一步损害，从而为机械耐用的热敏水凝胶添加特定功能。为了实现这一目标，沃里克团队将采用最先进的化学方法，以先前评估的相同生物相容性材料为基础，生产机械稳定的水凝胶。为此，将研究高效的共价交联化学。在接近人体的温度下经历凝胶化的机械稳健材料的开发将通过使用活性凝胶化位点的选择性可逆保护来解决，并且将导致用于组织工程的水凝胶材料的范式转变。曼彻斯特团队将在每个阶段筛选这些水凝胶，以确保它们支持MSC存活和分化为IVD细胞。他们已经确定了IVD细胞的特异性基因，这些基因可以用来确保MSC确实成为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）中获得的人类IVD组织，将MSC接种的水凝胶注入其中。然后可以在“生物反应器”中培养注射的细胞接种水凝胶，该生物反应器准确地模拟了人类脊柱中的条件，包括日常运动期间经历的机械负荷，使其成为比当前动物模型更相关的系统。所获得的数据将允许鉴定用于IVD再生的最合适的水凝胶。预期结果和潜在临床受益：我们将化学和基于MSC的组织工程专业知识与我们独特的测试系统相结合的能力，将有助于推动基于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，从而改善数百万人的生活质量，并为医疗保健系统和地球仪的更广泛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.