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Endocrine Abstracts (2013) 31 P6

SFEBES2013 Poster Presentations Bone (34 abstracts)

Retinoic acid and IGF1 stimulate the differentiation of human primary osteoblasts to osteocytes in 3D collagen gels

Sarah Goring 1 , Nicole Scully 1, , Deborah Mason 2, & Bronwen Evans 1,

School of Medicine, Cardiff University, Cardiff, UK, 2School of Biosciences, Cardiff University, Cardiff, UK, 3Centre for Biomechanics and Bioengineering, Cardiff, UK.

Osteocytes differentiate from osteoblasts, are embedded in mineralised matrix and are critical regulators of bone remodelling. In vitro osteocyte models are currently limited to cell lines in monolayer, but these do not represent their 3D environment in vivo. We have recently shown that osteoblasts in 3D gels differentiate along the osteocytic pathway. Since retinoic acid (RA) has been shown to stimulate monolayer osteoblast/osteocyte differentiation, we have investigated the effects of RA on cells in 3D. We have also hypothesised that IGF1 modulates osteocyte differentiation and function.

We maintained osteoblasts in 3D type I collagen gels (up to 15 days) +/− RA (5×10−6M) or IGF1 (5 μg/ml). We measured cell number and viability (trypan blue), VEGF and IL6 secretion (ELISA), and expression and secretion of osteocyte related proteins (e.g. DMP1, RANKL, FGF-23; qRTPCR, ELISA).

RA significantly (P<0.001) reduced cell numbers (HOBS, MC-3T3s), whereas IGF1 had no effect on either cell type. Cell viability was high throughout. In HOBS, IL6 secretion was decreased at day 7 (P<0.001), whereas IGF1 had no effect. In MC-3T3s both compounds decreased IL6 secretion. Interestingly, both RA and IGF1 significantly increased RANKL expression and stimulated FGF-23 expression and secretion in MC3T3 cells, confirming that the cells had differentiated to osteocytes. Both compounds also increased VEGF secretion in these cells.

RA and IGF1 modulate mouse and human osteoblast/osteocyte number and function in 3D gels, with broadly similar results obtained with the two cell types tested. The production of FGF-23 in the presence of IGF1 highlights the possible role of IGF1 in osteocyte differentiation and function. Since we have also recently developed a method of applying load to these gels, this study provides a novel 3D in vitro system to further study the role of IGF1 in osteocyte differentiation and function, especially those related to mechano-sensing signalling pathways.

Declaration of funding: Yes.

Details: Society for Endocrinology

Arthritis Research UK

Cardiff University.

DOI: 10.1530/endoabs.31.P6

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