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

背景：随着人口的老龄化和增长，与年龄相关的软骨组织疾病，如椎间盘退行性变(下腰痛的主要原因)，构成了一个日益严重的全球性社会经济问题。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，可以使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，并提供长期的疼痛缓解。骨髓中存在的成体干细胞，即间充质干细胞(MSCs)，由于其易于分离、生长迅速，并能形成(分化为)软骨组织中的细胞，因此为组织工程提供了巨大的潜力。事实上，曼彻斯特团队已经证明，骨髓间充质干细胞可以形成IVD细胞，使它们成为IVD再生的理想细胞。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的中心是凝胶状的；因此水凝胶(高水分含量的凝胶)是最合适的。还可以将水凝胶注入IVD，从而避免侵入性手术。曼彻斯特团队此前已经证明，种植在壳聚糖/甘油磷酸水凝胶中的MSCs会成为IVD细胞，并产生IVD样组织。然而，目前这些凝胶缺乏在人体脊柱负载环境中存在所需的机械强度。因此，我们的目标是：1.开发新型的壳聚糖基水凝胶，它可以承受人体脊柱内的负荷，同时允许MSCs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可以注入IVD的温敏性水凝胶(室温下的液体和体温下的凝胶)，从而最大限度地减少复杂的手术和后续护理；3.通过添加营养物质(如葡萄糖)来增加机械坚固的温敏性水凝胶的特定功能，以提高细胞存活，或添加因子以防止未来疾病对组织的进一步损害。为了实现这一目标，华威团队将使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，生产机械强度高的水凝胶。为了做到这一点，将研究高效的共价交联化学。在接近人体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存活和向IVD细胞分化。他们已经确定了IVD细胞特有的基因，这可以用来确保MSCs确实成为IVD细胞。此外，他们还开发了一种“生物反应器”，它使用从身体(已被捐赠用于研究)中获得的人类IVD组织，并将MSC种子水凝胶注入其中。然后，注入的细胞种子水凝胶可以在“生物反应器”中培养，生物反应器准确地模拟人类脊椎的条件，包括日常运动中经历的机械负荷，使这一系统比目前的动物模型更相关。获得的数据将有助于确定最适合IVD再生的水凝胶。预期结果和潜在的临床益处：我们将化学和基于MSC的组织工程方面的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的组织工程疗法在IVD变性方面的临床应用。通过这样做，我们有能力根除椎间盘退变，从而提高数百万人的生活质量，并为全球医疗体系和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.