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，例如行走、跑步、爬楼梯，我们的膝关节会受到体重所产生的机械力的影响。关节软骨排列在我们骨骼的两端，起到吸收和分散关节所受力量的作用；被称为软骨细胞的细胞可以感觉到这些力量的变化，调节软骨的产生以支持其功能。随着我们年龄的增长，或当我们的关节暴露在创伤中，即运动损伤时，软骨可能会开始退化，关节健康会受到负面影响。我们怎么知道这么多关于这种现象的信息呢？几十年来，我们一直依赖动物模型来研究关节健康，并模拟组织衰老和退化。在历史上，包括马和狗在内的大型动物模型被使用，然而，今天许多此类调查研究都使用了小型动物模型，特别是啮齿动物。啮齿动物模型旨在模拟关节中所经历的机械力，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了什么；手术和非手术方法都被用来改变膝盖的负重，或者相反地，在啮齿动物的后端被悬挂以防止后腿负重的情况下，改变其负重。尽管这些模型提供了有关软骨细胞如何感知和响应外力变化的信息，但内政部认为这些程序的严重性中等。此外，啮齿动物和人类在解剖学和生物力学方面的根本差异可能是导致研究转化为临床的成功率较低的原因。然而，据保守估计，全世界每年通常有3708只小鼠和486只大鼠用于此类实验。为什么我们不能使用体外模型来研究这些反应呢？已经开发了几种体外替代方案，以消除对体内动物研究的需要，以符合3R提纯、减少和替代的精神。不幸的是，这些模型不能复制关节软骨的独特特征，也不能形成正确的组成和结构特征，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及其所在组织之间存在的广泛交流，而这种交流对细胞如何感知和做出相应的反应至关重要。有没有合适的非动物技术替代方案？我们开发了一种新的三维模型系统，它利用人类软骨前体细胞，即成熟软骨细胞的前体细胞类型，活跃地产生高度组织化的组织，发育成具有正确分子组成的软骨样组织，以支持机械功能。然而，这个模型以前没有被用来研究机械响应，并形成了这个建议的基础。我们的目标是确定该模型是否以类似于活体动物模型的方式对生理和非生理力量做出反应，以验证其作为替代系统的有效性。我们将通过绘制细胞在组织深度中感受到的力，然后在基因水平上分析变化，来评估这个人类体外模型中的细胞如何对负荷做出反应。然后，我们将能够使用我们之前获得的数据，将这些反应与在两个活体负荷模型中检测到的反应进行比较，以验证并提供这种非动物技术实用的证据。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该模型，并总体计算我们可以在该领域实际减少至少40%的实验动物使用量。使用这种人类细胞衍生的模型还可以在促进识别和筛选新的靶点以防止软骨分解代谢和保持关节健康方面提供长期的翻译影响。