Quasi-static loading reveals interaction between mechanics and mtor and NF-ĸB pathways in the control of endochondral ossification in mouse metatarsals

Abstract

Purpose: Osteoarthritis (OA), a chronic disease characterized by articular cartilage degradation, osteophyte formation and subchondral sclerosis, is the most common cause of pain and disability in adults. Whilst genetic and mechanical factors are known to be the most significant determinants of such cartilage-to-bone transition involved in OA development, its pathophysiology remains incompletely resolved. The unmet clinical need this represents would clearly benefit from the identification of the underpinning signalling pathways as new drug targets. Recent data indicate that the mTOR and NF-ĸB pathways can regulate cartilage-to-bone transition recapitulated in OA. The aim of the current study is to determine whether regulators of mTOR and NF-ĸB pathway signalling interact with mechanical factors to control the endochondral ossification (EO) underpinning cartilage-to-bone transition. Mammalian target of rapamycin (mTOR) is composed of two protein complexes (mTORC1 and mTORC2) with distinct functions. Activation of mTORC1 is achieved in response to nutrients, amino acids and growth factor levels, whereas mTORC2 is related with cytoskeletal organization and cell survival. NF-ĸB is an important pathway for drug discovery in inflammation, autoimmune diseases and cancer, which is a novel activator of mTOR signalling, in turn regulated by crosstalk with both mTOR complexes. Both mTOR and NF-ĸB signalling pathways can be pharmacologically modulated by selective activators and inhibitors. To explore whether regulators of mTOR and NF-ĸB pathway signalling interact with mechanical factors to control the endochondral ossification (EO) we have used mouse embryonic metatarsal cultures maintained either in aqueous conditions, in which longitudinal growth occurs in the absence of any extraneous mechanical perturbation or in the presence of quasi-static mechanical loading conferred by their maintenance within a stiff hydrogel matrix. This metatarsal organ culture system is an elegant and reproducible method to explore developmental cartilage-to-bone transition. Methods: Murine metatarsi were carefully collected from the paws of mouse pups at day E17 of gestation from pregnant C57BL/6 mice and placed in culture in α-MEM medium supplemented with 0.2% BSA, 5 μg/mL L-ascorbic acid phosphate, 0.05 mg/mL and 1.25 μg amphotericin B and maintained for 2 weeks. Treatment to modify mTOR and NF-ĸB signalling started at day 0 of culture in medium supplemented with selective inhibitors and activators of either mTOR or NF-ĸB using rapamycin (100 nM) and leucine (10 mM) or betulinic acid (BA, 2.5 μM) and SC-514 (20 μM), respectively (n=10-17). To explore whether such mTOR and NF-ĸB pathway regulation may interact with mechanical factors to control EO, we have also cultured identical metatarsals in the presence of quasi-static mechanical loads achieved, for the first time, by maintenance in commercial hydrogel (VitroGel 3DTM) for the entire duration of the culture. Total element length, cartilaginous zone and mineralised tissue zone lengths were measured in images collected at the start and end of the 14 day incubation period. Data were statistically analysed using a linear mixed effect model. Results: Metatarsi grown under control conditions showed ∼2.5 fold expansion in their total, as well as mineralised tissue and cartilage length during the 14 day incubation period. Under these basal conditions, treatment with inhibitors/activators of mTOR, namely rapamycin/leucine and NF-kB using BA/SC-514 failed to significantly modify total metatarsal or mineralised tissue and cartilage length. The most significant modification in this very marked potential for EO-driven expansion was, however, achieved when metatarsi were cultured in the presence of hydrogel, where total longitudinal growth was almost completely stagnated; metatarsal length (and mineralised and cartilage tissue length) was not different between day 0 and day 14 under these quasi-static mechanical loading conditions. Intriguingly, culture under such hydrogel-related, quasi-static loading conditions, revealed a growth-promoting influence for both activators and inhibitors of the mTOR or NF-kB pathways, which all tend to reverse the growth-restricting effects of culture in the presence of hydrogel. Conclusions: These data reveal interaction between mechanical factors and the contribution of mTOR and NF-ĸB pathway signalling in the control of the EO. They suggest that the role for these pathways in OA development requires interpretation in the context of the modifications in mechanical loading conditions that underpin pathological bone-to-cartilage transition. In addition, these data show the potential to modify loading conditions in embryonic metatarsals that will allow the mechanical control of EO to be studied in isolation of systemic influences in vitro.