Bone in vitro 3D osteoblast-osteocyte co-culture model

Abstract

Event Abstract Back to Event Bone in vitro 3D osteoblast-osteocyte co-culture model M Vazquez1*, BAJ Evans2, J Ralphs3, D Riccardi3 and DJ Mason3 1 Cardiff University, Arthritis Research UK Biomechanics and Bioengineering Centre, School of Biosciences, United Kingdom 2 Cardiff Universit, Department of Child Health, School of Medicine, United Kingdom 3 Cardiff University, Division of Pathophysiology and Repair, School of Biosciences, United Kingdom Background: Osteoporosis occurs when the activities of bone cells become unbalanced leading to increased bone remodelling and weakening of bones. Anabolic therapies are limited, necessitating further investigation of the mechanisms that regulate bone formation. 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, with most focusing on mechanical loading of osteoblasts in monolayers. Of the 3D models that do exist, none elucidate the osteoblast-osteocyte interactions that regulate mechanically-induced bone formation. We are developing a novel in vitro 3D co-culture model of bone to investigate osteoblast-osteocyte interactions (1) and the role of glutamate, calcium and adenosine signalling in bone mechanotransduction.{BR}Methods: MLO-Y4 cells (1.5x10^6 cells/ml) were incorporated into acid-soluble rat tail tendon type I collagen (2 mg/ml in alpha MEM, pH7.4) gels and MC3T3-E1(1.0x10^5 cells/well) layered on top and cultured at 37C in SMEM (1 mM CaCl2, 2 mM Glutamax, 50 microgram/ml ascorbic acid, 5 % dialysed FBS) for 1 week. Co-cultures were fixed with 1 % paraformaldehyde, infiltrated with OCT, cryosectioned and labelled with phalloidin and DAPI to assess cell morphology, ethidium homodimer and DAPI to assess cell viability or immunostained using anti-connexin 43 antibody to assess cell connectivity. Osteoblast and osteocyte phenotype were determined by RT-PCR of RNA extracted (Trizol) separately from surface osteoblasts and encased osteocytes.{BR}Results: Preliminary data show co-cultures survive, for at least one week, with osteocyte cell death within gels (18 +/- 2.16 %, n=3) comparable to monolayer cultures (2). MLO-Y4 and MC3T3-E1 cells maintain their morphology, form a network through connexin 43, and express osteocyte and osteoblast phenotypic markers respectively, and components of the glutamate, calcium and adenosine signalling pathways.{BR}Conclusion: We have established a mouse osteoblast-osteocyte 3D co-culture system which can be used to investigate co-ordination of mechanical signalling through glutamate, calcium and adenosine delineating their roles in mechanically-induced bone formation.{BR}