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，例如走路、跑步、爬楼梯，我们的膝关节都会受到体重产生的机械力的影响。关节软骨排列在我们骨头的末端，其功能是吸收和消散关节所承受的力；这些细胞（称为软骨细胞）可以感知这些力的变化，从而调节软骨的产生以支持其功能。随着年龄的增长或当我们的关节受到创伤（即运动损伤）时，软骨可能开始退化，关节健康受到负面影响。我们如何对这种现象了解如此之多？几十年来，我们一直依靠动物模型来研究关节健康并模拟组织老化和退化。从历史上看，使用了包括马和狗在内的大型动物模型，但是今天的许多调查研究都使用小型动物模型，特别是啮齿动物。啮齿动物模型旨在模拟关节中经历的机械力，通过操纵这些力可以确定组织在衰老和疾病发作期间发生的情况；手术和非手术方法都用于改变膝盖的承重，或者相反地“减轻负载”，即啮齿动物的后端被悬挂以防止后腿承重。尽管这些模型提供了有关软骨细胞如何感知和响应所施加力的变化的信息，但内政部认为这些程序的严重程度适中。此外，啮齿动物和人类在解剖学和生物力学上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 3,708 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？为了避免体内动物研究的需要，已经开发了几种体外替代方案，符合细化、减少和替代的 3R 精神。不幸的是，这些模型无法复制关节软骨的独特特征，并且无法形成赋予组织高度专业化的生物力学功能的正确成分和结构特征。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通信，而这对于细胞如何感知和做出相应反应至关重要。是否有合适的非动物技术替代方案？我们开发了一种新颖的三维模型系统，该系统利用人类软骨祖细胞（即前体细胞类型）来成熟软骨细胞，这些软骨细胞积极产生高度组织化的组织，该组织发育成具有正确分子组成以支持机械功能的软骨样组织。然而，该模型之前并未用于研究机械响应，并且构成了该提案的基础。我们的目标是确定该模型是否以与体内动物模型类似的方式对生理和非生理力做出反应，以验证其作为替代系统的效果。我们将通过绘制细胞在组织深度所经历的力，然后分析基因水平的变化，来评估该人体体外模型中的细胞如何响应负载。然后，我们将能够使用我们之前获得的数据来比较两个体内负载模型中检测到的响应，以进行验证并提供这种非动物技术的实用性证据。一旦验证通过，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，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。使用这种人类细胞衍生模型还可以提供长期转化影响，促进新靶标的识别和筛选，以防止软骨分解代谢和保持关节健康。