Searchable abstracts of presentations at key conferences on calcified tissues
Bone Abstracts (2013) 1 PP246 | DOI: 10.1530/boneabs.1.PP246

ECTS2013 Poster Presentations Cell biology: osteocytes (10 abstracts)

Development of a novel 3D mineralising culture system to investigate the differentiation of osteoblasts to osteocytes

Nicole E E Scully 1, , Sam L Evans 3, , Deborah J Mason 2, & Bronwen A J Evans 1,


1School of Medicine, Institute of Molecular and Experimental Medicine, Cardiff University, Cardiff, UK; 2Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Cardiff, UK; 3School of Engineering, Institute of Mechanical and Manufacturing Engineering, Cardiff, UK; 4Arthritis Research UK Biomechanics and Bioengineering Centre, Cardiff University, Cardiff, UK.


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.

Volume 1

European Calcified Tissue Society Congress 2013

Lisbon, Portugal
18 May 2013 - 22 May 2013

European Calcified Tissue Society 

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