There is little understanding on how cell division, expansion and migration drive joint morphogenesis. Mechanical loading is known to affect joint morphogenesis, but the effects of mechanical loading on the cellular level are yet to be explored. Mechanical loading in utero comes mainly from muscle contractions and the resultant movements. However, passive movements may also play a role in skeletal development, particularly when muscle contractions are absent, but the effects of passive movements on the skeleton have not been well characterised. This doctorate aimed (1) to develop a robust method for analysing high-resolution cell-level data of developing joints, (2) to investigate the cell-level processes contributing to joint morphogenesis, (3) to identify the mechanoregulated cell-level processes, and (4) to explore the effects of passive movement on skeletal development.
This research used embryos of wild type mice and “muscleless-limb” mutant mice from the Splotch-delayed line with a mutation in the Pax-3 gene. Aims (1–3) were achieved with ACCESS, an original automated image processing method that qualitatively and quantitatively analysed the properties of cells in murine distal humeri. The cell properties investigated included number, volume, sphericity, orientation and density. Proliferation and ECM contents were also quantified. Aim (4) was achieved by giving pregnant mice wheel exercise and therefore inducing passive movements in the embryos. Forelimbs of the embryos were scanned in 3D and rudiment length and mineralisation were quantified.
For the first time, cell number and volume were reported as being key to the growth of a murine joint. Cell volume, orientation and proliferation were mechanoregulated. Wheel exercise affected forelimb rudiments significantly and could restore the length of some rudiments in muscleless-limb mutants. This research showcased new analytical and experimental methods adaptable for wider purposes, and advanced our knowledge for joint morphogenesis on the cell-level, especially relating to mechanoregulation.