Bone Abstracts

Osteocytes differentiate from osteoblasts, are embedded in mineralised matrix and are critical regulators of bone remodelling. In vitro osteocyte models are limited to cell lines in monolayer, which do not represent their 3D environment in vivo. We have shown that osteoblasts in 3D gels differentiate along the osteocytic pathway. Since IGF1 regulates osteoblasts, and is involved in osteocyte response to mechanical loading, we hypothesised that IGF1 modulates osteocyte differentiation and function.

We maintained osteoblasts (MC-3T3; HOBS) in 3D type I collagen gels (250 μl; 48-well plates; 15 days) in α-MEM±IGF1 (5 μg/ml). Cell number, viability and phenotype (IHC, confocal), IL6, VEGF, and FGF23 secretion (ELISA), and the expression of osteocyte-related genes (DMP1, RANKL, E-11, and FGF23; qRTPCR) were measured.

Cells with IGF1 appeared more dendritic than untreated cells. Cell viability was high both±IGF1 (>85% MC-3T3 s; >90% HOBS) but MC-3T3 numbers were decreased with IGF1. From day 11 onwards, the expression of DMP-1, Cx43, RANKL (P<0.01), and FGF23 (P<0.001) were significantly increased in MC-3T3 s with IGF1, compared to untreated, also confirming differentiation to osteocytes. There was no change in MC-3T3 IL6 secretion, but VEGF was higher with IGF1 on all days (P<0.01), and FGF23 secretion only detected with IGF1 treatment from day 11. In HOBS, and with IGF1, VEGF, and IL6 (P<0.05) secretion on all days were significantly reduced when compared to untreated controls.

IGF1 modulated cell number and function, with, generally similar results obtained with both cell types tested. Modulation of VEGF secretion, however, was different between the two cell types. FGF23 production with IGF1 treatment highlights the possible role of IGF1 in osteocyte differentiation and function. This novel 3D in vitro system provides a tool to further study the role of IGF1 in osteocyte differentiation and function, especially those related to the mechano-sensing signaling pathways.