Calcium response in osteocytic networks under steady and oscillatory fluid flow

The fluid flow in the lacunar-canalicular system of bone is an essential mechanical stimulation on the osteocyte networks. Due to the complexity of human physical activities, the fluid shear stress on osteocyte bodies and processes consists of both steady and oscillatory components. In this study, we investigated and compared the intracellular calcium ([Ca2+](i)) responses of osteocytic networks under steady and oscillatory fluid flows. An in vitro osteocytic network was built with MLO-Y4 osteocyte-like cells using micro-patterning techniques to simulate the in vivo orderly organization of osteocyte networks. Sinusoidal oscillating fluid flow or unidirectional steady flow was applied on the cell surface with 2 Pa peak shear stress. It was found that the osteocytic networks were significantly more responsive to steady flow than to oscillatory flow. The osteocytes can release more calcium peaks with higher magnitudes at a faster speed under steady flow stimulation. The [Ca2+](i) signaling transients under the steady and oscillatory flows have significantly different spatiotemporal characters, but a similar responsive percentage of cells. Further signaling pathway studies using inhibitors showed that endoplasmic reticulum (ER) calcium store, extracellular calcium source, ATP, PGE(2) and NO related pathways play similar roles in the [Ca2+](i) signaling of osteocytes under either steady or oscillating flow. The spatiotemporal characteristics of [Ca2+](i) transients under oscillating fluid flow are affected more profoundly by pharmacological treatments than under the steady flow. Our findings support the hypothesis that the [Ca2+](i) responses of osteocytic networks are significantly dependent on the profiles of fluid flow