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

促进透明软骨细胞向间充质干细胞分化的培养条件的开发

• 批准号: BB/I01666X/1
• 金额: $ 11.71万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI01666X%2F1
• 关键词: development; culture; conditions; promote; differentiation

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.

骨关节炎是一种由透明关节软骨丢失引起的退行性关节疾病。鉴于骨性关节炎在老年患者中的传播以及避免广泛手术的需要，人们对开发基于细胞的微创疗法来替代受损的关节软骨产生了浓厚的兴趣。众所周知，骨髓间充质干细胞(MSC)在体外可以很容易地分化为软骨细胞，因此是骨关节炎细胞治疗的一个有前途的来源。然而，基于MSC的治疗面临着两大挑战：第一，很难指导MSC分化为透明软骨；第二，未来的治疗需要开发一种生物相容性的材料支架，能够在移植后保持MSC来源的软骨细胞的表型。透明软骨细胞的分化：MSC在体外的软骨形成难以控制，所产生的软骨细胞类似于作为骨形成模板的短暂肥大的软骨细胞，而不是关节正常功能所需的永久性透明软骨细胞。首席学术导师(PM)的实验室最近表明，含有特定纤维连接蛋白基序的新型仿生材料底物可以诱导骨髓来源的MSC向新生软骨细胞分化，而不需要额外的生长因子(见上文，PM研究经验)。在此条件下形成的软骨细胞表达早期分化的软骨细胞标志物，如N-钙粘蛋白、Sox9和II型胶原，它们在肥大软骨细胞和透明软骨细胞的前体细胞中都有表达。最近，在阐明这两种软骨细胞在体内分化的机制方面取得了很大进展。有趣的是，透明软骨的形成不仅依赖于具有成软骨活性的因子，如转化生长因子-β家族成员Gdf5，而且还依赖于抗成软骨因子的活性，如Wnt9a。软骨细胞移植的生物材料：尽管用于原代透明软骨细胞移植的生物材料支架的开发已经取得了一些进展，但一个主要的问题是随着时间的推移，移植的软骨细胞不能保持其表型，并倾向于形成纤维软骨。一个可能的原因是，在移植后，软骨细胞不再暴露在有助于保持其体外表型的培养介质成分中。人们已经采取了各种方法来改善生物材料支架的性能，其中许多方法涉及在支架基质中加入信号分子或多肽基序。然而，事实证明，很难获得引起所需细胞反应所需的正确的配体/基序密度。学术联合导师(OM)小组开发了一种新型的自组装蛋白质共聚物(称为ZT)，具有经过证实的自下而上的功能化能力，有望克服其中的许多问题(见上文，OM研究经验)。项目目标：[1]建立能够引导MSC来源的新生软骨细胞分化为透明软骨的培养条件，而不是肥大的软骨。[2]测试1中确定的生长因子是否可以被小分子量模拟物或多肽基序取代。[3]制备ZT生物材料支架的分子工程变体，以整合2.[4]中确定的关键基序/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来源的透明软骨细胞的表型。[5]对能够保持透明软骨细胞表型的培养条件、培养基组合物和由本工作衍生的工程生物材料实施商业化战略。