The role of mechanical forces in bone formation: Evaluation of long-term responses by osteoblasts to low fluid shear stress in vitro

Bone strength is maintained through the continuous process of bone remodelling within which bone cells work together. Mechanical loading is an important regulator of this process with bone cells being particularly sensitive to fluid shear stress (FSS). The aim of this research was to investigate long-term responses by osteoblasts to low FSS using both monolayer and 3D in vitro models. The osteoblast cell lines IDG-SW3 and MLO-A5 were cultured for 28 days or 21 days respectively in 6-well plates and were exposed to low FSS generated with a see-saw rocker (<0.05 Pa) or an orbital shaker (<0.9 Pa). There were no differences in metabolic activity, cell proliferation, alkaline phosphatase (ALP) activity, mineralisation, collagen deposition and osteocytogenic differentiation between static and dynamic groups. Despite not reacting to FSS, IDG-SW3 responded to biochemical stimulation. Deposition of collagen and ALP activation were inhibited when ascorbic acid (50μg/ml) was omitted (p<0.001). Mineralisation was positively related to the concentration of β-glycerophosphate (β-GP) which also regulated osteocytogenesis. Treatment with 1 mM strontium ranelate reduced ALP activity and mineralisation (p<0.001), but enhanced expression of the osteocyte marker Dmp1-GFP (p<0.001). To investigate whether IDG-SW3 were more responsive to low FSS in a 3D environment, cells were embedded in collagen (2 mg/ml). Modification of a transwell insert enabled long-term cell culture under hydrostatic pressure-driven low fluid flow. After 21 days, cells had contracted the collagen by 50% compared to cell-free controls (p<0.001). However, no significant differences in gel contraction, cell distribution and mineralisation were detected between static and flow-stimulated groups, further confirming the results from the monolayer experiments.