Biomechanical characterisation of joints in osteoarthritis mutant zebrafish; studying interactions between genotype and biomechanics in osteoarthritis

退行性关节疾病骨关节炎（OA）影响着全世界数千万人。虽然最近已经确定了一些增加骨关节炎易感性的基因，但目前尚不清楚这些基因中有多少导致关节的致病性改变。然而，我们确实了解关节形状以及关节形状对载荷和应变分布的后续影响会影响OA的发病机制。我们的研究小组已经鉴定出一种斑马鱼系，该系携带一种基因突变，该基因被鉴定为增加人类对OA的易感性（CHST11）。我们有初步证据表明，携带这种突变基因的斑马鱼改变了关节形状；关节有一个更平坦的轮廓，而不是与正常关节相关的“球和杯”形状。这种形状的变化似乎是渐进的，最终这种形状的变化导致鱼在大约2周龄时关节衰竭和死亡，因为它们不能再张开下巴进食。本项目以斑马鱼为实验对象，结合计算模型建立发育中的下颌软骨对应变的反应，使我们能够研究OA基因与关节形状和关节功能之间的关系。我们将使用最先进的高分辨率显微镜和材料测试来确定颌骨组织不同部分之间的刚度差异，以及正常和突变鱼之间是否存在材料特性差异。我们将使用显微镜观察斑马鱼颚肌肉骨骼系统，它由肌肉、软骨、骨骼和结缔组织组成。从我们生成的图像中，我们将开发三维计算模型，以可视化正常（野生型）鱼与携带OA （CHST11）基因的“突变”鱼的下颌软骨的哪些部分处于压力和应变下。我们预测，突变鱼类关节形状的变化会改变发育中的软骨经历压力和应变的方式。我们还将确定颌骨肌肉固定的突变鱼的颌骨组织形态，这将使我们能够测试肌肉活动如何影响探索肌肉负荷对确定关节形状的影响。然后，我们将使用这些生物力学数据，研究生物力学环境的变化对软骨细胞（称为软骨细胞）的影响，测试应变的变化是否可以预测这些细胞表现出的行为变化。例如，较高菌株下的细胞是否更容易分裂、成熟或通过称为细胞凋亡的过程死亡？当各种胶原蛋白基因被打开时，我们将使用表达荧光蛋白的斑马鱼系，测试在最高应变下的细胞是否会改变它们分泌的软骨基质的类型。最后，我们将使用这些模型以及我们积累的关于主要信号通路活动的数据，称为控制软骨细胞行为的wnt通路。我们将利用这一点来预测wnt信号如何参与介导细胞对关节形状变化的反应，然后我们可以在突变的鱼身上进行测试。这将帮助我们开始理解机械应变的变化是如何被细胞解释的，从而导致细胞行为的变化。这项研究本质上是高度跨学科的，因此来自不同学科的研究人员将受益，包括解剖学家、生物力学家、进化和发育生物学家、细胞生物学家和生物医学工程师。研究结果将对骨性关节炎各个层面的研究具有特别的相关性，从疾病的遗传学基础到骨科植入物和置换关节的发展。通过对年轻科学家的多学科培训和国际合作，英国的科学基础将受益。

The degenerative joint condition osteoarthritis (OA) affects tens of millions of people worldwide. Although a number of genes have recently been identified that increase susceptibility to osteoarthritis, it currently remains unclear how many of these genes lead to pathogenic changes to the joint. We do, however, understand that joint shape and the subsequent effects of shape on loading and distribution of strain in the joint affects the pathogenesis of OA. Our group has identified a zebrafish line which carries a mutation in a gene identified as increasing susceptibility to OA in humans (CHST11). We have preliminary evidence showing that zebrafish carrying this mutant gene have altered joint shape; the joints have a more flattened profile that doesn't form the 'ball and cup' shape associated with a normal joint. This change to the shape appears to be progressive, ultimately this shape change leads to joint failure and death of the fish at around 2 weeks of age as they can no longer open their jaws to feed.This project uses experiments in zebrafish, combined with computational modelling to establish how the developing cartilages of the jaw respond to strains, allowing us to study the relationship between OA genes, joint shape and joint function. We will use state of the art high resolution microscopy with materials testing to determine the differences in stiffness between different parts of the jaw tissues, and whether there are material property differences between the normal and mutant fish. We will use microscopy to visualise the zebrafish jaw musculoskeletal system, which is comprised of muscle, cartilage, bone and connective tissue. From the images that we generate we will develop 3-dimensional computational models to visualise which parts of the jaw cartilages are under stress and strain in normal (wild type) fish, compared to 'mutant' fish carrying the OA (CHST11) gene. We predict that the change in shape of the joints in mutant fish changes how the developing cartilages experience stress and strain. We will also determine the morphology of jaw tissues from mutant fish that have their jaw muscles immobilised, this will allow us to test how muscle activity influences explore the influence of muscle loads on determinin joint shape. We will then, using this biomechanical data, study the effect of changes to the biomechanical environment on the cartilage cells (called chondrocytes) testing whether changes to strain can predict the changes in behaviour that these cells exhibit. For example, are cells under higher strains more or less likely to divide, to mature or to undergo death by a process called apoptosis? Using lines of zebrafish that express fluorescent proteins when various collagen genes are switched on, we will test whether the cells under the highest strain change the types of cartilage matrix which they secrete. Finally, we will use the models along with data that we have accumulated about the activity of a major signalling pathway, known as the wnt pathway which controls cartilage cell behaviour. We will use this to predict how wnt-signalling is involved in mediating the cell's response to the change in joint shape, which we can then test in the mutant fish. This will help us start to understand how the changes in mechanical strain are interpreted by the cell in a way that leads to a change in cell behaviour. This research is highly interdisciplinary in nature, therefore researchers from a variety of disciplines will benefit including anatomists, biomechanists, evolutionary and developmental biologists, cell biologists and biomedical engineers. The results will be of particular relevance to the study of OA at all levels, from the genetics underpinning the disease to the development of orthopaedic implants and replacement joints. There will be benefits to the UK science base through multidisciplinary training of young scientists and through international collaborations.