β-glycerophosphate, not low magnitude fluid shear stress, increases osteocytogenesis in the osteoblast-to-osteocyte cell line IDG-SW3

Owen, Robert; Wittkowske, Claudia; Lacroix, Damien; Perrault, Cecile M.; Reilly, Gwendolen C.

Abstract

Aim
As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3.

Methods
Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (β-glycerophosphate (βGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker.

Results
βGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. βGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing βGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition.

Conclusions
This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.

Citation

Owen, R., Wittkowske, C., Lacroix, D., Perrault, C. M., & Reilly, G. C. (2024). β-glycerophosphate, not low magnitude fluid shear stress, increases osteocytogenesis in the osteoblast-to-osteocyte cell line IDG-SW3. Connective Tissue Research, https://doi.org/10.1080/03008207.2024.2375065

• Journal Article Type: Article
• Acceptance Date: Jun 26, 2024
• Online Publication Date: Jul 10, 2024
• Publication Date: Jul 10, 2024
• Deposit Date: Jul 10, 2024
• Publicly Available Date: Jul 10, 2024
• Journal: Connective Tissue Research
• Print ISSN: 0300-8207
• Electronic ISSN: 1607-8438
• Publisher: Taylor and Francis
• Peer Reviewed: Peer Reviewed
• DOI: https://doi.org/10.1080/03008207.2024.2375065
• Keywords: Matrix mineralization; mechanobiology; biomechanics; bone tissue engineering; extracellular matrix
• Public URL: https://nottingham-repository.worktribe.com/output/37149683
• Publisher URL: https://www.tandfonline.com/doi/full/10.1080/03008207.2024.2375065
• Additional Information: Peer Review Statement: The publishing and review policy for this title is described in its Aims & Scope.; Aim & Scope: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=icts20; Received: 2024-01-29; Accepted: 2024-06-26; Published: 2024-07-10