Mechanical Influences on Morphogenesis of the Knee Joint Revealed through Morphological, Molecular and Computational Analysis of Immobilised Embryos

Abstract

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.

A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation

“…The effect of mechanical signals during chondrogenesis Studies of paralyzed chick and mouse embryos have revealed that various cartilaginous skeletal elements were shorter and bone eminences were significantly smaller (Hamburger and Waugh, 1940;Hosseini and Hogg, 1991;Rot-Nikcevic et al, 2006;Blitz et al, 2009;Nowlan et al, 2010), and that the size of the proliferative zone (and number of proliferating chondrocytes) in the growth plates of long bones is reduced relative to control embryos (Germiller and Goldstein, 1997;Roddy et al, 2011). Further support for the involvement of mechanical stimulation in chondrocyte proliferation is provided by a study in which cyclic mechanical stimulation of rabbit premaxillae accelerated the rate of chondrocyte proliferation (Wang and Mao, 2002).…”

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

“…There is evidence that application of mechanical stimuli can determine the developmental fate of mesenchymal stem cells (MSCs) [6]. In particular, appropriate physical stimuli are reported to be essential for limb development: synovial joint and articular cartilage formation in chicken and murine embryos require proper mechanical forces by muscle contractions [7,8]. Optimal intermittent biomechanical activation is also essential for cartilage regeneration during osteoarthritis (reviewed by Sun [9]).…”

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

“…Finite Element models have been used to relate the areas of skeletal elements that are under strain with those undergoing bone formation 10 , as well as to map the areas under strain during endochondral ossification and joint morphogenesis 8,12,21 . Other studies have also been able to apply theoretical growth models to replicate changes during joint development 11,12 .…”