To date, joint shape has been studied based on linear measurements of the humerus bone rudiment. Analysing shape using 2D measurements means important information of the 3D structure such as volume or growth distribution might be lost. 3D measurements have not been possible until recently, due to limitations in the existing imaging techniques. Based on our new 3D whole limb microscopy images, we have developed a methodology to map the humerus surface onto a standardized reference surface. We then normalize the mapping and perform statistical analyses on the computed areas and volumes that we extract from each limb’s surface map. Our data analysis method is the first to allow for full 3D quantification of bone shape.
Our poroelastic computational model incorporates, for the first time, the effect of dynamic loading on local tissue growth. Previous models of joint morphogenesis used elastic materials, which cannot account for the fluid flow and, hence, the dynamic pressure maps that change over a loading cycle. These pressure maps are the ones dictating growth in our model. In addition, for the first time, we have developed a joint morphogenesis model informed by and validated with experimental data.
We also started to analyse the results of the latest experiments in which we used a new technique to image the expression of morphogenetic biomolecules in healthy regenerating axolotl forelimbs around the time that the joint appears (cavitation). Studies to date have been limited because techniques to quantify molecular expression loose spatial patterning, while methods that allow visualizing their expression in space have been, thus far, exclusively in 2D sections and non-quantifiable. Our new technique will allow mapping in 3D the location of these biomarkers. We will use the experimental results to develop in parallel a new computational model to predict joint cavitation. We have started to develop this model, which uses reaction-diffusion equations to predict the emergence of Turing patterns in 3D space.
Next steps beyond the end of the CompLimb project will include incorporating a growing domain as well as the potential mechanical stimuli influencing the expression of these morphogens. Combining experiments and modelling will provide a framework for exploration with the aim of better understanding the exact biochemical conditions required for healthy limb formation and, more specifically, how the biophysical environment influences these molecular components responsible for growth. The findings derived from the CompLimb project will contribute to a better understanding of vertebrate joint formation. This knowledge might ultimately have an impact on the development of therapies to treat joint deformities or approaches to prevent congenital malformations.
