OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading

Abstract

Osteocytes' mechano-regulation of bone formation and resorption is key to maintaining appropriate bone health. Although extensive in vitro studies have explored osteocyte mechanobiology using the well-established MLO-Y4 cell model, the low amount of sclerostin secreted by this cell line renders it inadequate for studying cross-talk between osteocytes and osteoblasts under mechanical loading. Here, we investigated the potential of the sclerostin-expressing OCY454 osteocyte cell model in fulfilling this role. Fully differentiated OCY454 cells were tested for mechano-sensitivity by measuring changes in protein secretion, total adenosine triphosphate (ATP) content, and intracellular calcium in response to oscillatory fluid flow. Increases in sclerostin expression and total ATP content were observed. However, very low levels of receptor activator of the nuclear factor κ-B ligand were detected, and there was a great inconsistency in calcium response. Conditioned medium (CM) from OCY454 cells was then used to culture osteoblast and osteoclast precursors. Osteoblast activity was quantified with alkaline phosphatase (ALP) and Alizarin Red S stain, while osteoclast differentiation was quantified with tartrate-resistant acid phosphatase (TRAP) staining. We demonstrated that mechanically stimulated OCY454 cells released soluble factors that increased osteoblasts' ALP activity (p < 0.05) and calcium deposition (p < 0.05). There was also a significant decrease of large-sized TRAP-positive osteoclasts when osteoclast precursors were treated with CM from flow-stimulated OCY454 cells (p < 0.05). Results from this study suggest that OCY454 cells respond to mechanical loading with the release of key factors such as sclerostin to regulate downstream bone cells, thus demonstrating its potential as a novel cell model for in vitro osteocyte mechanobiology studies.

Scl-Ab reverts pro-osteoclastogenic signaling and resorption in estrogen deficient osteocytes

“…Mechanical loading has been shown to downregulate sclerostin expression by TGF-β dependant mechanisms [27,28,53,54]. However, here we report that estrogen de cient osteocytes following loading failed to downregulate SOST expression compared to estrogen treated osteocytes.…”

“…Secondly, here all osteocytes were cultured in a monolayer and mechanically stimulated using a parallel plate ow chamber to mimic the in vivo environment more accurately, rather than culturing the osteocytes under static conditions, this is an oversimpli cation of the stresses experienced by osteocytes in vivo. However, parallel plate ow chambers have been widely used to study osteocyte mechanobiology as they allow downstream chemical events to be easily studied [22,23,28,47]. In the clinical setting, Scl-Ab is administered systemically, therefore we treated both OCY454 and BMM cells with Scl-Ab in vitro.…”

“…Following mechanical loading, SOST mRNA and sclerostin protein expression are downregulated both in vivo [27] and in vitro in the osteocyte cell line OCY454 [28]. Estrogen has been observed to negatively affect SOST mRNA and sclerostin protein expression in human postmenopausal bone and ovariectomised mice respectively [29,30].…”

Scl-Ab reverts pro-osteoclastogenic signalling and resorption in estrogen deficient osteocytes&nbsp;

“…Secondly, here all osteocytes were cultured in a monolayer and mechanically stimulated using a parallel plate ow chamber to mimic the in vivo environment more accurately, rather than culturing the osteocytes under static conditions, this is an oversimpli cation of the stresses experienced by osteocytes in vivo. However, parallel plate ow chambers have been widely used to study osteocyte mechanobiology as they allow downstream chemical events to be easily studied [26,27,35,52]. In the clinical setting, Scl-Ab is administered systemically, therefore we treated both OCY454 and BMM cells with Scl-Ab in vitro.…”

“…The Wnt antagonist sclerostin (encoded by the SOST gene), produced by mature osteocytes, binds to LRP5/6 Wnt co-receptors, negatively regulates osteoblast proliferation and differentiation via inhibition of the Wnt/β-catenin signalling pathway, and also promotes osteocyte and osteoblast apoptosis [33]. Following mechanical loading, SOST mRNA and sclerostin protein expression are downregulated both in vivo [34] and in vitro in the osteocyte cell line OCY454 [35]. Estrogen has been observed to negatively affect SOST mRNA and sclerostin protein expression in human postmenopausal bone and ovariectomised mice respectively [36,37].…”