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

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.

Recently, several genes conferring increased risk of osteoarthritis (OA) have been identified. Currently, the mechanisms by which OA genes exert their effects on joints are largely unknown. We have data demonstrating that one of these genes, Chst11, alters gross joint morphology and chondrocyte maturation in developing zebrafish. Changes to joint shape are progressive; leading to joint failure at day 14. We will test the relationship between genotype and biomechanics in joint development.
Using confocal microscopy to visualise muscle, cartilage and bone, we will develop finite element (FE) models of the jaw cartilages, their joints and associated muscles in wild type (wt) and chst11 mutant fish. FE-models are commonly used to deduce functional strain in engineering contexts and have increasing utility in animal biology. Our FE-models will map the positions of strain within the joint.
We will then use the FE-models twinned with in vivo experiments to establish the extent and mechanism by which chondrocytes (in wild type and mutants) are influenced by mechanical strain. We predict that differing joint morphologies will generate different strain responses between wt and mutant fish. We will assay chondrocyte proliferation, maturation and matrix secretion in wt, active and anaesthetised mutants. We predict that changes to mechanical environment (e.g. decreased strain in anesthetised mutants) will lead to changes to local growth factor signalling. In vitro data implicates Chst11 in Wnt signalling; we also have preliminary data that Wnt signalling in the joint is suppressed following jaw immobilisation. We will use transgenic reporter lines to test the relationship between Wnt signalling and strain in wt and mutant fish.
Utilising computational modelling in concert with genetics and live cell imaging we aim to shed light on how OA susceptibility genes affect joint shape and loading, and how chondrocytes respond to strain under normal and mutant conditions.

WIDER PUBLIC: We plan to hold a 1-day meeting in year 3, bringing together researchers from different disciplines to discuss joint function at a wider level. While much of the day will be aimed at academics we plan to hold a keynote talk aimed at a more lay audience open to the general public, we anticipate that this will have broad appeal. This model is one that we have successfully used in the past. Our work is very visual with public appeal; in the past both of our groups have worked successfully with museums and galleries. Such outlets provide opportunities for non-academic target groups to engage with science.
SCHOOLS AND EDUCATION. CLH is one of the Bristol co-ordinators (along with Prof. Paul Martin) of the Wellcome Trust-funded Authentic Biology programme, in which University research is introduced to a local school with ongoing involvement between 6th form students and researchers over a number of years. The programme that we have introduced into Cotham School is based around osteoarthritis and cancer genetics using zebrafish as a model organism. We have already engaged 28 students in the programme of research and we plan to increase numbers over the next few years. CLH's PhD students are involved in this initiative and we envisage that the PDRA would also become actively involved.
CHARITIES: CLH is also funded by, and maintains close links to, Arthritis Research UK (ARUK), who have media and outreach groups who help to disseminate information on arthritis research to government, patients and carers and to the wider public.
PATIENTS: CLH is involved with PEP-R a group of patients with musculoskeletal conditions who meet with scientists involved in relevant research to discuss their findings and pathways to translation. CLH also collaborates with a number of consultant rheumatologists who can help disseminate relevant information directly to their patients and suggest directions which are of the highest clinical priority.
GOVERNMENT POLICY: CLH is a member of the EuFishBioMed COST Action, which lobbies governments on behalf of the European zebrafish research community for example to unify European regulation, to ease transport restrictions on zebrafish and to maintain/increase funding for biomedical research. We will also continue to support ARUK's work with policy makers to ensure that government policy meets the need of patients with OA.
ANIMAL RESEARCH AND THE 3Rs. The computational modelling approaches developed and used in this project fit well with the RCUK 3Rs strategy (replacement, refinement and reduction) for animal experimentation. These modelling techniques, validated by our own in vivo work, will provide an approach for computationally modelling jaw and vertebral movements in larval fish, allowing us to better predict outcomes and therefore reduce the number of in vivo experiments. The dissemination of these simulation methods will be of interest to groups working in mechanical simulation in a wide variety of systems and taxa which have a wide variety of impacts including health (particularly joint health and orthopaedic research) and animal welfare.
SKILL SETS This project is, by its nature highly interdisciplinary, bringing together experts from a number of fields. By training the PDRA in these fields, genetics/molecular biology, material properties and biomechanics/modelling we will be enhancing the knowledge economy, as well as benefiting the individual by providing them with a highly desirable skill set which will benefit their future career.
INTERFACE WITH INDUSTRY We are currently working with Perkin Elmer to develop software allowing us to integrate data from the Finite Elements models we produce with cell tracking data from confocal microscopy, this software will be made freely available to academics with existing Velocity licences (see letter of support). We will investigate any commercial applications for our research with the University's Research and Development team.