The development of culture conditions to promote the differentiation of hyaline chondrocytes from mesenchymal stem cells

骨关节炎（OA）是一种退行性关节疾病所造成的损失透明关节软骨。鉴于OA在老年患者中的传播以及避免广泛手术的期望，人们对开发微创细胞疗法以替代受损的关节软骨有浓厚的兴趣。骨髓间充质干细胞（MSC）在体外可向软骨细胞分化，是骨关节炎细胞治疗的理想来源。然而，基于MSC的疗法面临两个主要挑战：首先，难以引导MSC分化为透明软骨，其次，未来的疗法将需要开发能够在移植后维持MSC衍生的软骨细胞表型的生物相容性材料支架。透明软骨细胞的分化：MSC体外软骨形成的可控性较差，产生的软骨细胞类似于作为骨形成模板的短暂肥大软骨细胞，而不是关节正常功能所需的永久透明软骨细胞。首席学术主管（PM）的实验室最近表明，含有特定纤连蛋白基序的新型仿生材料基质可以诱导骨髓来源的MSC分化为新生软骨细胞，而不需要额外的生长因子（见上文，PM研究经验）。在这些条件下形成的软骨细胞表达早期分化软骨细胞的标记物，例如N-钙粘蛋白、Sox 9和胶原II，其在肥大和透明软骨细胞的祖细胞中表达。最近，已经取得了很大的进展，对阐明的机制，调节这两种软骨细胞类型的分化在体内。有趣的是，透明软骨的形成不仅依赖于具有软骨形成活性的因子，如TGF-β家族成员Gdf 5，而且还依赖于抗软骨形成因子的活性，如Wnt 9a。用于软骨细胞移植的生物材料：尽管在开发用于移植原代透明软骨细胞的生物材料支架方面取得了一些进展，但主要问题是随着时间的推移，移植的软骨细胞不能保持其表型，并且倾向于形成纤维软骨。一个可能的原因是，移植后，软骨细胞不再暴露于有助于维持其体外表型的培养基成分。已经采取了各种方法来改善生物材料支架的性能，其中许多方法涉及将信号分子或肽基序掺入支架基质中。然而，已经证明难以实现引发所需细胞应答所需的配体/基序的正确密度。学术联合主管（OM）的小组开发了一种新型自组装蛋白质共聚物（称为ZT），具有经证实的自下而上的功能化能力，有望克服许多这些问题（见上文，OM研究经验）。项目目标：[1]建立能够引导源自MSC的新生软骨细胞分化为透明软骨而不是肥大软骨的培养条件。[2]测试1中鉴定的生长因子是否可以被小分子量模拟物或肽基序取代。[3]制造ZT生物材料支架的分子工程变体以并入2中鉴定的关键基序/肽基序。[4]确定3中制备的分子工程生物材料是否能够在体外保持MSC衍生的透明软骨细胞的表型。[5]实施培养条件、培养基成分和源自本工作的工程生物材料的商业化策略，这些材料能够维持透明软骨细胞的表型。

Osteoarthritis (OA) is a degenerative joint disease caused by loss of hyaline articular cartilage. Given the spread of OA in elderly patients and the desirability of avoiding extensive surgery, there is strong interest in developing minimally-invasive cell-based therapies to replace damaged articular cartilage. It is well-recognised that bone marrow-derived mesenchymal stem cells (MSC) can readily differentiate to chondrocytes in vitro, and are thus a promising source for OA cell therapy. However, there are two major challenges facing MSC-based therapies: firstly, it is difficult to direct MSC to differentiate to hyaline cartilage, and secondly, future therapies would require the development of a biocompatible material scaffold capable of maintaining the phenotype of MSC-derived chondrocytes following transplantation. Differentiation of hyaline chondrocytes: MSC chondrogenesis in vitro is poorly controllable, with the resulting chondrocytes resembling the transient hypertrophic chondrocytes that serve as a template for bone formation, rather than the permanent hyaline chondrocytes required for the normal functioning of joints. The laboratory of the lead academic supervisor (PM) has recently shown that novel biomimetic material substrates containing specific fibronectin-based motifs can induce the differentiation of bone marrow-derived MSC to nascent chondrocytes, without the need for additional growth factors (see above, PM research experience). The chondrocytes that form under these conditions express markers of early differentiating chondrocytes, such as N-cadherin, Sox9 and collagen II, which are expressed in the progenitors of both hypertrophic and hyaline chondrocytes. Recently, much progress has been made towards elucidating the mechanisms that regulate the differentiation of these two chondrocytic cell types in vivo. Interestingly, the formation of hyaline cartilage is not only dependent on factors with chondrogenic activity, such as the TGF-b family member, Gdf5, but is also dependent on the activity of anti-chondrogenic factors, such as Wnt9a. Biomaterials for chondrocyte transplantation: Although some progress has been made towards the development of biomaterial scaffolds for transplantation of primary hyaline chondrocytes, a major problem is that over time, the transplanted chondrocytes fail to maintain their phenotype and tend to form fibrocartilage. A likely reason for this is that following transplantation, the chondrocytes are no longer exposed to the culture medium components that help maintain their phenotype in vitro. Various approaches have been taken to improve the performance of biomaterial scaffolds, many of which involve incorporating signalling molecules or peptidic motifs into the scaffold matrix. However, it has proved difficult to achieve the correct density of ligands/motifs needed to elicit the required cellular response. The group of the academic co-supervisor (OM) has developed a novel self-assembling protein co-polymer (termed ZT) with proven bottom-up functionalization capabilities that holds high promise to overcome many of these problems (see above, OM research experience). Project Aims: [1] To establish culture conditions capable of directing the differentiation of nascent chondrocytes derived from MSC to hyaline, rather than hypertrophic cartilage. [2] To test if the growth factors identified in 1 can be replaced by small molecular weight mimetics or peptidic motifs. [3] To fabricate molecularly engineered variants of the ZT biomaterial scaffold to incorporate the key motifs/peptide motifs identified in 2. [4] To determine if the molecularly engineered biomaterials fabricated in 3 are able to maintain the phenotype of MSC-derived hyaline chondrocytes in vitro. [5] To implement a commercialisation strategy for culture conditions, media compositions and engineered biomaterials derived from this work that are capable of maintaining the phenotype of hyaline chondrocytes.