Abstract
Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.
Highlights
Endochondral growth, which occurs in all skeletal limb elements, involves early mesenchymal cell condensation, as well as sequential proliferation, differentiation into cartilage-forming chondrocytes with linked extracellular matrix (ECM) production, and later, hypertrophy
Data are presented in intervals that represent the mean and Results were analysed mTOR modulators, rapamycin, and leucine both somewhat overcome the hydrogel-mediated arrest with a linear mixed effect model and ANOVA test according to normality test distribution
Our findings show: (i) that E17 metatarsi preserved a scope for ex vivo growth equivalent to 50% of the in vivo rate and showed sensitivity to known endochondral growth inhibitors (LY294002/PD98059); (ii) that neither mTOR nor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) regulators affected growth under basal conditions; (iii) that quasi-static load via hydrogel exerted growth arrest, which was reversed by mTOR but not by NF-kB modulators; and, (iv) that hydrogel-related growth restraint was linked to an S-G2/M cell cycle arrest, itself linked to ECM (Col2, Acan, Col10) transcriptional changes—with those linked to hypertrophy (Col10, Mmp13) exhibiting a corresponding reversal by mTOR modulators
Summary
Endochondral growth, which occurs in all skeletal limb elements, involves early mesenchymal cell condensation, as well as sequential proliferation, differentiation into cartilage-forming chondrocytes with linked extracellular matrix (ECM) production, and later, hypertrophy. In this sequence, the cartilage ECM is calcified and invaded by blood vessels and is primed for subsequent ossification. The cartilage ECM is calcified and invaded by blood vessels and is primed for subsequent ossification These endochondral processes are critical at the expanding epiphyses, where long bone lengthening continues until maturity, and are recapitulated in pathology during bone fracture healing and osteoarthritis. Resting chondrocytes are more distant, constituting a reservoir of skeletal stem cells expressed under parathyroid
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