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

在日常活动中，例如走路、跑步、爬楼梯，我们的膝关节会受到由体重产生的机械力的影响。关节软骨位于我们骨骼的末端，它的功能是吸收和消散关节所承受的力；这种被称为软骨细胞的细胞可以感知这些变化，从而调节软骨的生成以支持其功能。随着年龄的增长或关节受到外伤（如运动损伤），软骨开始退化，关节健康受到负面影响。我们是如何对这种现象了解这么多的呢？几十年来，我们一直依靠动物模型来研究关节健康，模拟组织老化和退化。从历史上看，包括马和狗在内的大型动物模型被使用，然而，小动物模型，特别是啮齿类动物被用于许多这些调查性研究。啮齿动物模型旨在模拟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了什么；手术和非手术方法都用于改变膝关节的负重，或者相反地“卸载”，即悬挂啮齿动物的后端以防止后腿承受重量。尽管这些模型提供了软骨细胞如何感知和应对施加的力的变化的信息，但内政部认为这些过程的严重程度是中等的。此外，啮齿类动物和人类在解剖学和生物力学方面的根本差异可能导致研究成果转化为临床的成功率较低。然而，据保守估计，全世界每年通常使用3708只小鼠和486只大鼠进行此类实验。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，以消除对体内动物研究的需要，符合3r的精神，即改进，减少和替代。不幸的是，这些模型在复制关节软骨的独特特征方面存在不足，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，这些模型不能支持存在于软骨细胞和它所在的组织之间的广泛交流，而这种交流对于细胞如何感知和做出相应的反应是必不可少的。有没有合适的非动物技术替代品？我们开发了一种新的三维模型系统，它利用人类软骨祖细胞，即成熟软骨细胞的前体细胞类型，积极地产生高度组织的组织，发展成软骨样组织，具有正确的分子组成来支持机械功能。然而，这个模型以前并没有被用来研究机械反应，并形成了本建议的基础。我们的目标是确定该模型是否以与体内动物模型相似的方式响应生理和非生理力，以验证其作为替代系统。我们将通过绘制细胞在组织深处所经历的力，然后分析基因水平上的变化，来评估这个人类体外模型中的细胞如何对负荷作出反应。然后，我们将能够使用我们先前获得的数据将反应与在两种体内加载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性的证据。一旦验证，我们将广泛宣传模型，邀请感兴趣的用户到我们的实验室学习如何建立模型，并总体计算我们可以切实减少该领域至少40%的实验动物使用。使用这种人类细胞衍生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护关节健康方面提供长期的转化影响。

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.