Osteocytes are critical in bone maintenance, adaptation and have important endocrine functions including mineral homeostasis through osteocyte-specific factors such as fibroblast growth factor 23 (fgf-23), a regulator of serum phosphate. MLO-Y4 cells are an osteocyte—like cell line that expresses negligible levels of fgf23. To date, no study has yet investigated the effect of a 3-dimensional culture system upon MLO-Y4 cells. The overall study objective was therefore to examine the effects of 3D culture upon MLO-Y4 expression of fgf23 using a collagen-glycosaminoglycan (GAG) 3D scaffold. In addition the effect of mechanical cues, scaffold stiffness and fluid flow shear stress, in directing cell behaviour was also studied.