Pulsating fluid flow affects pre-osteoblast behavior and osteogenic differentiation through production of soluble factors

Bone mass increases after error-loading, even in the absence of osteocytes. Loaded osteoblasts may produce a combination of growth factors affecting adjacent osteoblast differentiation. We hypothesized that osteoblasts respond to a single load in the short-term (minutes) by changing F-actin stress fiber distribution, in the intermediate-term (hours) by signaling molecule production, and in the long-term (days) by differentiation. Furthermore, growth factors produced during and after mechanical loading by pulsating fluid flow (PFF) will affect osteogenic differentiation. MC3T3-E1 pre-osteoblasts were either/not stimulated by 60 min PFF (amplitude, 1.0 Pa; frequency, 1 Hz; peak shear stress rate, 6.5 Pa/s) followed by 0-6 h, or 21/28 days of post-incubation without PFF. Computational analysis revealed that PFF immediately changed distribution and magnitude of fluid dynamics over an adherent pre-osteoblast inside a parallel-plate flow chamber (immediate impact). Within 60 min, PFF increased nitric oxide production (5.3-fold), altered actin distribution, but did not affect cell pseudopodia length and cell orientation (initial downstream impact). PFF transiently stimulated Fgf2, Runx2, Ocn, Dmp1, and Col1⍺1 gene expression between 0 and 6 h after PFF cessation. PFF did not affect alkaline phosphatase nor collagen production after 21 days, but altered mineralization after 28 days. In conclusion, a single bout of PFF with indirect associated release of biochemical factors, stimulates osteoblast differentiation in the long-term, which may explain enhanced bone formation resulting from mechanical stimuli.