Abstract
Fetal immobilization affects skeletal development and can lead to severe malformations. Still, how mechanical load affects embryonic bone formation is not fully elucidated. This study combines mechanobiology, image analysis and developmental biology, to investigate the structural effects of muscular loading on embryonic long bones. We present a novel approach involving a semi-automatic workflow, to study the spatial and temporal evolutions of both hard and soft tissues in 3D without any contrast agent at micrometrical resolution. Using high-resolution phase-contrast-enhanced X-ray synchrotron microtomography, we compare the humeri of Splotch-delayed embryonic mice lacking skeletal muscles with healthy littermates. The effects of skeletal muscles on bone formation was studied from the first stages of mineral deposition (Theiler Stages 23 and 24) to just before birth (Theiler Stage 27). The results show that muscle activity affects both growth plate and mineralized regions, especially during early embryonic development. When skeletal muscles were absent, there was reduced mineralization, altered tuberosity size and location, and, at early embryonic stages, decreased chondrocyte density, size and elongation compared to littermate controls. The proposed workflow enhances our understanding of mechanobiology of early bone formation and could be implemented for the study of other complex biological tissues.
Highlights
Fetal immobilization can have dramatic effects on the skeleton [1,2]
Most of the mineralization occurred after TS24, and by TS27 the size difference between controls and mutants were reduced (Fig. 3A, B)
The quantitative analysis indicates that at all stages the bone volume fraction (BV/TV) was similar in controls and mutants, this could suggest that lack of muscular contraction primarily affects growth rates but not the relative bone formation (Fig. 4C)
Summary
Fetal immobilization can have dramatic effects on the skeleton [1,2]. Reduced intrauterine movements can lead to smaller, thinner and weaker long bones that in some cases even fracture before birth, as well as to craniofacial and limb deformities and abnormal joint contractures [3,4,5]. Modified mice with absent or non-contractile musculature are suitable to study the specific effects of an altered mechanical environment on mammalian bone development [8,9,10,11,12,13]. It has been shown that long bones of mice lacking functional muscles were significantly less miner alized than control bones and showed an irregular morphology [10]. This raises interesting questions such as: does mechanical contraction affect the bone morphology overall and bone internal microstructure, and how is the primary bone organized in the bone volume when muscular contractions have been absent?
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