Bone Abstracts

Osteocytes make up >90% of bone cells, are embedded in mineralised matrix where they form a communication network. Osteocytes differentiate from osteoblasts, and are mechano-sensitive. They are very difficult to isolate with a dependence on cell lines for in vitro studies of osteocyte biology. Therefore new methods to study these cells are essential. Recent publications indicate that osteoblasts maintained in in vitro 3D collagen gels may differentiate to osteocytes.

We maintained osteoblasts (MC-3T3; human primary) in 3D type I collagen gels (250 μl; 48-well plates; 15 days) in either α-MEM (basal medium) or mineralising medium (basal medium, dexamethasone, and β-glycerophosphate). Cell number, viability and phenotype (IHC, qRTPCR, and confocal microscopy), gel stiffness (Losenhausen machine), and VEGF and IL6 secretion (ELISA) were quantified.

Cells appeared more dendritic over time and formed connecting cellular networks (H&E, Phalloidin). Cell viability was similar in both media (>85% MC-3T3 s; >95% human primary), but cell numbers were significantly higher (P<0.001) in mineralising conditions. Mineralisation was confirmed from day 7 (calcein). DMP-1 was not expressed (IHC) at day 3 but then gradually increased in expression (days 7–14). E11 was low at day 3 (IHC, qRTPCR), peaked at day 10 (P<0.001), and returned to lower levels by day 14. Gel stiffness significantly increased over 11 days (P<0.01) and the mineralised gels were stiffer than those in basal medium (P<0.01). VEGF and IL6 secretion also changed significantly with time and culture conditions. Mechanical loading conditions for these 3D osteocyte cultures are currently being optimized.

Osteoblasts maintained in 3D gels differentiate along the osteocytic pathway. It is possible to mineralise these cultures thus mimicking further their in vivo environment. This methodology provides a novel model to study osteocyte differentiation and function, and will enable important studies relating to bone loading, repair and regeneration.