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)影响着全球数千万人。尽管最近发现了一些增加骨关节炎易感性的基因，但目前尚不清楚其中有多少基因会导致关节的病理性变化。然而，我们确实了解到关节形状以及随后形状对关节载荷和应变分布的影响影响了骨关节炎的发病机制。我们的小组已经确定了一个斑马鱼品系，该品系携带一种基因突变，该基因被认为是人类对OA的易感性增加(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的形状；关节的轮廓更平坦，不会形成与正常关节相关的“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会导致大约两周大的鱼的关节故障和死亡，因为它们不能再张开嘴巴进食。这个项目使用斑马鱼的实验，结合计算模型来建立颌骨发育中的软骨如何对压力做出反应，从而使我们能够研究OA基因、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硬度差异，以及正常鱼类和突变鱼类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的颌部肌肉骨骼系统，该系统由肌肉、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)基因的突变鱼相比，颌骨软骨的哪些部分处于压力和应变状态。我们预测，突变鱼关节形状的变化会改变发育中的软骨承受应力和应变的方式。我们还将确定下颌肌肉被固定的突变鱼的颌骨组织的形态，这将使我们能够测试肌肉活动如何影响关节形状，探索肌肉负荷对关节形状的影响。然后，我们将利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，在更高的菌株下，细胞是更有可能分裂、成熟还是通过一种称为凋亡的过程死亡？利用当不同的胶原基因被激活时表达荧光蛋白的斑马鱼品系，我们将测试处于最高应变下的细胞是否改变了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控制软骨细胞行为的主要信号通路(称为WNT通路)活动的数据。我们将利用这一点来预测WNT信号如何参与调节细胞对关节形状变化的反应，然后我们可以在突变鱼身上进行测试。这将帮助我们开始理解机械应变的变化是如何被细胞以一种导致细胞行为改变的方式解释的。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、生物机械学家、进化和发育生物学家、细胞生物学家和生物医学工程师。这些结果将对从疾病的遗传学基础到骨科植入物和替代关节的发展的所有级别的骨性关节炎的研究具有特别的相关性。通过对年轻科学家进行多学科培训和国际合作，英国的科学基地将受益。

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.