Testing and validation of an in vitro 3D human chondrocyte model to replace animal use in mechanobiology research

测试和验证体外 3D 人类软骨细胞模型以替代机械生物学研究中的动物模型

• 批准号: NC/X002411/1
• 负责人: Emma Blain
• 金额: $ 25.39万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FX002411%2F1
• 关键词: Testing; validation; vitro; 3D; human

During everyday movement e.g. walking, running, climbing stairs, our knee joints are exposed to mechanical forces arising from body weight. Articular cartilage, which lines the ends of our bones, functions to absorb and dissipate the forces experienced by our joints; the cells - called chondrocytes can sense these alterations in force regulating the production of cartilage to support its function. As we age or when our joints are exposed to trauma i.e. sports injury, cartilage can begin to degrade and joint health is negatively impacted. How do we know so much about this phenomenon? Over the decades, we have relied on the use of animal models to study joint health and to mimic tissue ageing and degeneration. Historically, large animal models including horses and dogs were used, however small animal models, specifically rodents are used for many of these investigative studies today. The rodent models aim to mimic mechanical forces experienced in the joint and by manipulating these forces can determine what happens to the tissue during ageing and disease onset; both surgical and non-surgical approaches are used to alter weight-bearing in the knee, or conversely 'off-loading' where the rear end of a rodent is suspended to prevent weight bearing on the hind legs. Although these models provide information on how the chondrocytes sense and respond to changes in the forces applied, the procedures are considered moderate in severity by the Home Office. Furthermore, fundamental differences between rodents and humans in their anatomy and biomechanics likely contributes to the low success rate for research translation to the clinic. Yet, it has been conservatively estimated that typically 3,708 mice and 486 rats are used per annum worldwide in such experiments. Why can't we use in vitro models to study these responses? Several in vitro alternatives have been developed to obviate the need for in vivo animal research in line with the 3Rs ethos of refinement, reduction and replacement. Unfortunately, these models fall short in replicating the unique features of articular cartilage and are incapable of forming the correct composition and structural features which give the tissue its highly specialised biomechanical function. Importantly, these models fail to support the extensive communication that exists between chondrocytes and the tissue it resides in which are imperative to how the cells can sense and respond accordingly. Is there a suitable non-animal technology alternative? We have developed a novel three-dimensional model system which utilises human chondro-progenitors i.e. the precursor cell type to mature chondrocytes that have actively produced a highly organised tissue that develops into a cartilage-like tissue with the correct molecular composition to support mechanical function. However, this model has not previously been used to investigate mechanical responses and forms the basis of this proposal. We aim to determine whether this model responds to physiological and non-physiological forces in a similar way to in vivo animal models to validate it as a replacement system. We will assess how the cells in this human in vitro model respond to load by mapping the forces experienced by cells through the depth of the tissue followed by analysing changes at the gene level. We will then be able to compare the responses to those detected in two in vivo loading models using our previously acquired data to enable validation and provide evidence of utility of this non-animal technology. Once validated, we will widely publicise the model, invite interested users to our laboratory to learn how to establish the model and overall calculate that we can realistically reduce experimental animal use by at least 40% in this field. Use of this human cell derived model could also provide long-term translational impact in facilitating the identification and screening of new targets to prevent cartilage catabolism and preserve joint health.

在日常运动中，例如步行，跑步，爬楼梯，我们的膝盖接头暴露于体重引起的机械力。关节软骨在我们的骨骼的末端排列，功能吸收和消散关节所经历的力；称为软骨细胞的细胞可以感觉到这些改变的作用，从而调节软骨的产生以支持其功能。随着我们的年龄或关节接触创伤，即运动损伤，软骨可能会开始降解，而联合健康受到负面影响。我们怎么知道这一现象？在过去的几十年中，我们一直依靠使用动物模型来研究关节健康和模仿组织衰老和变性。从历史上看，使用了包括马和狗在内的大型动物模型，但是小型动物模型，特别是当今许多研究性研究的啮齿动物模型。啮齿动物模型的目的是模仿关节中经历的机械力，并通过操纵这些力可以确定在衰老和疾病发作期间组织发生的情况。手术和非手术方法均用于改变膝盖上的体重，或者相反“卸载”啮齿动物的后端被悬挂以防止后腿上的重量轴承。尽管这些模型提供了有关软骨细胞如何感知并应对所施加力的变化的信息，但该程序被内政部视为中等程度。此外，啮齿动物和人类在解剖学和生物力学中之间的根本差异可能会导致研究转化为诊所的成功率低。然而，保守估计，在此类实验中，全球每年使用3,708只小鼠和486只大鼠。为什么我们不能使用体外模型来研究这些反应？已经开发了几种体外替代方法，以消除根据3RS的精致，还原和替代品的精神，消除了体内动物研究的需求。不幸的是，这些模型在复制关节软骨的独特特征方面缺乏，并且无法形成正确的组成和结构特征，从而使组织具有高度专业化的生物力学功能。重要的是，这些模型无法支持软骨细胞与其所在组织之间存在的广泛沟通，这对于细胞如何感知和相应地反应至关重要。是否有合适的非动物技术替代方法？我们已经开发了一种新型的三维模型系统，该系统利用了人软骨前生物，即成熟的软骨细胞的前体细胞类型，该细胞类型积极地产生了高度有条理的组织，该组织将其发展成具有正确分子组成的软骨样组织以支持机械功能。但是，该模型以前尚未用于研究机械响应并构成了该建议的基础。我们旨在确定该模型是否以类似的方式对生理和非生理力作出反应，以验证其作为替代系统。我们将通过映射细胞通过组织深度所经历的力来评估该人类体外模型中的细胞如何响应负载，然后分析基因水平的变化。然后，我们将能够使用我们先前获取的数据来比较两个体内加载模型中检测到的响应，以实现验证并提供了这种非动物技术的效用的证据。一旦得到验证，我们将广泛宣传该模型，邀请感兴趣的用户到我们的实验室学习如何建立模型并进行整体计算，以便在该领域将实验性动物的使用实际减少至少40％。这种人类细胞得出的模型的使用还可以提供长期的翻译影响，以促进对新靶标的识别和筛查，以防止软骨分解代谢并保留关节健康。