Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway
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...
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MDPI AG
2021-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/24/13220 |
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| author | Soraia Caetano-Silva Bigboy H. Simbi Neil Marr Andrew Hibbert Steve P. Allen Andrew A. Pitsillides |
| author_facet | Soraia Caetano-Silva Bigboy H. Simbi Neil Marr Andrew Hibbert Steve P. Allen Andrew A. Pitsillides |
| author_sort | Soraia Caetano-Silva |
| collection | DOAJ |
| description | 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 <i>ex vivo</i> 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 <i>Col2</i> and <i>Acan</i> 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. |
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| last_indexed | 2024-03-10T03:58:04Z |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f7be30201b6a47b0a4c85c8b38234fc42023-11-23T08:42:59ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-12-0122241322010.3390/ijms222413220Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR PathwaySoraia Caetano-Silva0Bigboy H. Simbi1Neil Marr2Andrew Hibbert3Steve P. Allen4Andrew A. Pitsillides5Comparative Biomedical Sciences Department, Royal Veterinary College, London NW1 0TU, UKComparative Biomedical Sciences Department, Royal Veterinary College, London NW1 0TU, UKComparative Biomedical Sciences Department, Royal Veterinary College, London NW1 0TU, UKComparative Biomedical Sciences Department, Royal Veterinary College, London NW1 0TU, UKComparative Biomedical Sciences Department, Royal Veterinary College, London NW1 0TU, UKComparative Biomedical Sciences Department, Royal Veterinary College, London NW1 0TU, UKMechanical 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 <i>ex vivo</i> 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 <i>Col2</i> and <i>Acan</i> 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.https://www.mdpi.com/1422-0067/22/24/13220endochondral ossificationmTORNF-ĸBquasi-staticloadinghydrogel |
| spellingShingle | Soraia Caetano-Silva Bigboy H. Simbi Neil Marr Andrew Hibbert Steve P. Allen Andrew A. Pitsillides Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway International Journal of Molecular Sciences endochondral ossification mTOR NF-ĸB quasi-static loading hydrogel |
| title | Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway |
| title_full | Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway |
| title_fullStr | Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway |
| title_full_unstemmed | Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway |
| title_short | Restraint upon Embryonic Metatarsal <i>Ex Vivo</i> Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway |
| title_sort | restraint upon embryonic metatarsal i ex vivo i growth by hydrogel reveals interaction between quasi static load and the mtor pathway |
| topic | endochondral ossification mTOR NF-ĸB quasi-static loading hydrogel |
| url | https://www.mdpi.com/1422-0067/22/24/13220 |
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