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Development of a novel in vitro 3d osteocyte-osteoblast co-culture model to investigate mechanically-induced signalling

Vazquez, Marisol 2013. Development of a novel in vitro 3d osteocyte-osteoblast co-culture model to investigate mechanically-induced signalling. PhD Thesis, Cardiff University.
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Normal mechanical loading potently induces bone formation mediated by osteocyte effects on osteoblasts. Current in vitro bone models do not reflect these cellular interactions, either focusing on mechanical loading of osteoblasts in monolayers or in 3D and therefore not elucidating the osteocyte-osteoblast interactions that regulate mechanically-induced bone formation. Adenosine, calcium-sensing and glutamate signalling have been shown to influence bone biology, with both adenosine precursors and glutamate having been implicated in mechanotransduction. The aims were to develop a novel in vitro 3D co-culture model of bone to investigate mechanically-induced signalling, and to determine the expression of adenosine, calcium-sensing and glutamate signalling components within the 3D model and their contribution to the regulation of mechanically-induced bone formation markers. A 3D model was developed as a two-phase culture system where MLO-Y4 osteocytes were embedded within type I collagen gels and MC3T3-E1osteoblasts were layered on top. In this model, cells were viable over 7 days (100 % osteoblasts, 87 % osteocytes), maintained appropriate morphology and contacted neighbouring cells through CX43 labelled projections. RT-qPCR revealed Runx2, OCN and E11 mRNA expression in both osteoblasts and osteocytes. COL1A1 mRNA expression was significantly higher in the osteoblasts (P=0.0001), whereas ALP mRNA was higher in the osteocytes (P=0.001). RT-PCR revealed expression of adenosine receptors A2A and A2B and glutamate transporter GLAST1 in osteoblasts and osteocytes, as well as glutamate receptors AMPAR2 and KA1 in osteocytes. Immunostaining confirmed expression of A2A, GLAST1 and KA1, and revealed expression of CaSR, in both osteoblasts and osteocytes. A novel mechanical loading device was developed which was used to apply osteogenic loads (5 min, 10 Hz, 2.5 N) to 3D osteocyte mono-cultures and 3D osteocyte-osteoblast co-cultures. A minimum of 48 hr pre-load time was required for a reliable load response. 3D osteocyte mono-cultures cultured for 48-72 hr or 7 days pre-load, remained viable, significantly increased PGE2 0.5 hr after load (48-72 hr: P=0.0249, 7 days: P=0.041) and decreased their IL-6 synthesis. RT-qPCR revealed a load-induced decrease in E11 (P=0.018) and RANKL (P=0.0486) mRNA, in 48-72 hr cultures. In 7 day cultures, E11 mRNA (P=0.041) increased as a result of loading. Preliminary data showed that the same loading conditions increased PINP synthesis, a bone formation marker, in 3D co-cultures (P=0.022). The AMPA/KA receptors antagonist NBQX increased PINP synthesis by 2-fold over 5 days, similar levels induced by loading in untreated cultures, suggesting that NBQX has similar anabolic effects as mechanical stimuli. Similarly, the A2A receptor antagonist SCH 442416 increased osteoblast ALP mRNA expression by 3.5-fold at day 1 post-load and increased PINP synthesis by 1.9-fold, in co-cultures after 5 days. This 3D osteocyte-osteoblast co-culture model represents a useful in vitro model for the investigation of the osteocyte-osteoblast interactions that lead to mechanically-induced signalling and regulation of bone formation markers. Adenosine, calcium-sensing and glutamate signalling components are expressed within the model, facilitating future investigations of their roles in mechanically-induced signalling. Preliminary experiments indicated that adenosine and glutamate signalling may each contribute individually to the regulation of mechanically-induced bone formation markers.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > QH Natural history > QH301 Biology
R Medicine > R Medicine (General)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 04 Jun 2017 06:08

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