Compressive stress gradients direct mechanoregulation of anisotropic growth in the zebrafish jaw joint.

Mechanical stimuli arising from fetal movements are critical factors underlying joint growth. Abnormal fetal movements negatively affect joint shape features with important implications for joint health, but the mechanisms by which mechanical forces from fetal movements influence joint growth are still unclear. In this research, we quantify zebrafish jaw joint growth in 3D in free-to-move and immobilised fish larvae between four and five days post fertilisation. We found that the main changes in size and shape in normally moving fish were in the ventrodorsal axis, while growth anisotropy was lost in the immobilised larvae. We next sought to determine the cell level activities underlying mechanoregulated growth anisotropy by tracking individual cells in the presence or absence of jaw movements, finding that the most dramatic changes in growth rates due to jaw immobility were in the ventrodorsal axis. Finally, we implemented mechanobiological simulations of joint growth with which we tested hypotheses relating specific mechanical stimuli to mechanoregulated growth anisotropy. Different types of mechanical stimulation were incorporated into the simulation to provide the mechanoregulated component of growth, in addition to the baseline (non-mechanoregulated) growth which occurs in the immobilised animals. We found that when average tissue stress over the opening and closing cycle of the joint was used as the stimulus for mechanoregulated growth, joint morphogenesis was not accurately predicted. Predictions were improved when using the stress gradients along the rudiment axes (i.e., the variation in magnitude of compression to magnitude of tension between local regions). However, the most accurate predictions were obtained when using the compressive stress gradients (i.e., the variation in compressive stress magnitude) along the rudiment axes. We conclude therefore that the dominant biophysical stimulus contributing to growth anisotropy during early joint development is the gradient of compressive stress experienced along the growth axes under cyclical loading.

Growth orientations, rather than heterogeneous growth rates, dominate jaw joint morphogenesis in the larval zebrafish.

In early limb embryogenesis, synovial joints acquire specific shapes which determine joint motion and function. The process by which the opposing cartilaginous joint surfaces are moulded into reciprocal and interlocking shapes, called joint morphogenesis, is one of the least understood aspects of joint formation and the cell-level dynamics underlying it are yet to be unravelled. In this research, we quantified key cellular dynamics involved in growth and morphogenesis of the zebrafish jaw joint and synthesised them in a predictive computational simulation of joint development. Cells in larval zebrafish jaw joints labelled with cartilage markers were tracked over a 48-h time window using confocal imaging. Changes in distance and angle between adjacent cell centroids resulting from cell rearrangement, volume expansion and extracellular matrix (ECM) deposition were measured and used to calculate the rate and direction of local tissue deformations. We observed spatially and temporally heterogeneous growth patterns with marked anisotropy over the developmental period assessed. There was notably elevated growth at the level of the retroarticular process of the Meckel's cartilage, a feature known to undergo pronounced shape changes during zebrafish development. Analysis of cell dynamics indicated a dominant role for cell volume expansion in growth, with minor influences from ECM volume increases and cell intercalation. Cell proliferation in the joint was minimal over the timeframe of interest. Synthesising the dynamic cell data into a finite element model of jaw joint development resulted in accurate shape predictions. Our biofidelic computational simulation demonstrated that zebrafish jaw joint growth can be reasonably approximated based on cell positional information over time, where cell positional information derives mainly from cell orientation and cell volume expansion. By modifying the input parameters of the simulation, we were able to assess the relative contributions of heterogeneous growth rates and of growth orientation. The use of uniform rather than heterogeneous growth rates only minorly impacted the shape predictions, whereas isotropic growth fields resulted in altered shape predictions. The simulation results suggest that growth anisotropy is the dominant influence on joint growth and morphogenesis. This study addresses the gap of the cellular processes underlying joint morphogenesis, with implications for understanding the aetiology of developmental joint disorders such as developmental dysplasia of the hip and arthrogryposis.

Exposure to hypergravity during zebrafish development alters cartilage material properties and strain distribution.

AIMS: Vertebrates have adapted to life on Earth and its constant gravitational field, which exerts load on the body and influences the structure and function of tissues. While the effects of microgravity on muscle and bone homeostasis are well described, with sarcopenia and osteoporosis observed in astronauts returning from space, the effects of shorter exposures to increased gravitational fields are less well characterized. We aimed to test how hypergravity affects early cartilage and skeletal development in a zebrafish model. METHODS: We exposed zebrafish to 3 g and 6 g hypergravity from three to five days post-fertilization, when key events in jaw cartilage morphogenesis occur. Following this exposure, we performed immunostaining along with a range of histological stains and transmission electron microscopy (TEM) to examine cartilage morphology and structure, atomic force microscopy (AFM) and nanoindentation experiments to investigate the cartilage material properties, and finite element modelling to map the pattern of strain and stress in the skeletal rudiments. RESULTS: We did not observe changes to larval growth, or morphology of cartilage or muscle. However, we observed altered mechanical properties of jaw cartilages, and in these regions we saw changes to chondrocyte morphology and extracellular matrix (ECM) composition. These areas also correspond to places where strain and stress distribution are predicted to be most different following hypergravity exposure. CONCLUSION: Our results suggest that altered mechanical loading, through hypergravity exposure, affects chondrocyte maturation and ECM components, ultimately leading to changes to cartilage structure and function. Cite this article: Bone Joint Res 2021;10(2):137-148.