Bone Abstracts

Introduction: Normal mechanical loading potently induces bone formation via effects on osteocytes. Current investigations of mechanical loading of bone do not reflect the interactions of the cells within it, mostly focusing on mechanical loading of osteoblasts in monolayers. Existing 3D models do not elucidate the osteoblast-osteocyte interactions that regulate mechanically-induced bone formation. We developed a novel in vitro 3D co-culture model of bone¹ to investigate osteoblast-osteocyte interactions.

Methods: MLO-Y4 cells (1.5×10⁶ cells per ml) were incorporated into acid-soluble rat tail tendon type I collagen (2 mg/ml in MEM, pH7.4) gels and MC3T3-E1 (1.5×10⁵ cells/well) layered on top and cultured at 37 °C (DMEM 5% dialysed FBS) for 1 week. Co-cultures were fixed with 1% paraformaldehyde, infiltrated with OCT, cryosectioned and labelled with 1) phalloidin and DAPI to assess cell morphology, 2) ethidium homodimer and DAPI to assess cell viability, 3) immunostained using anti-connexin 43 antibody to assess cell connectivity, or 4) immunostained with anti-E11 antibody. Cell phenotype was determined by RT-qPCR of RNA extracted (Trizol) separately from surface osteoblasts (surface zone) and encased osteocytes (deep zone).

Results: Data show co-cultures survive, for at least one week, with osteocyte cell death, within gels, averaging 16.86±3.56% at day 1 and 14.11±2.69% at day 7 comparable to monolayer cultures. MC3T3-E1 and MLO-Y4 cells maintain their morphology, express Runx2, osteocalcin, ColI, ALP mRNA and E11 and connexin 43 protein. 3D MLO-Y4 monocultures released PGE₂ after mechanical loading (preliminary data).

Conclusion: We have established a mouse osteoblast-osteocyte 3D co-culture system where MLO-Y4 cells form a network throughout the gel and respond to loading, overlaid with surface osteoblasts that express type I collagen. We are using this system to investigate mechanically-induced signals in osteocytes and osteoblasts.

References: Mason DJ, Dillingham CH, Evans B, et al. Bio reconstruction de l’os a la peau. Saraumps Medical. 35–39, 2009.