A study of fluid flow induced intracellular calcium changes in osteoblast-like cells

This research studied the intracellular calcium concentration, $\rm \lbrack Ca\sp{2+}\rbrack \sb{i},$ response of primary osteoblast-like cells (PROB) to a laminar fluid flow with and without the addition of newborn bovine serum. The focus was to determine if the presence of serum enhanced the cells\u27 ability to transduce physical stimuli in the form of shear stress and streaming potential into a $\rm \lbrack Ca\sp{2+}\rbrack \sb{i}$ signal. Additionally, this work examined the effect of shear stress loading rate and long term cell culture passaging on the observed $\rm \lbrack Ca\sp{2+}\rbrack \sb{i}$ response. A parallel plate flow system and fluorescence microscopy using fura-2 AM dye were used to stimulate the cells with shear stresses ranging from 0 to 35 dynes/cm$\sp2$ in physiologic saline supplemented with 0%, 2% or 10% serum, and to concurrently record the fluorescent images of individual cell $\rm \lbrack Ca\sp{2+}\rbrack \sb{i}$ changes. The results showed that the PROB $\rm \lbrack Ca\sp{2+}\rbrack \sb{i}$ response to serum-supplemented flow was amplified relative to the serum-free flow response primarily due to a complementary ligand-receptor stimulation of the cells by serum-born agonists via convective mass transport. The transduction pathway in each case occurred via the inositol phospholipid cascade for a mobilization of calcium from the endoplasmic reticulum. The streaming potential exhibited no statistically significant effect in the $\rm\lbrack Ca\sp{2+}\rbrack \sb{i}$ response to serum-supplemented flow. Varying the continuous shear stress loading rate from 0.5 to 4.2 dynes/cm$\sp2$/sec in serum-free flow did not significantly alter the mean peak $\rm\lbrack Ca\sp{2+}\rbrack \sb{i}$ response to 25 dynes/cm$\sp2$ maximum shear stress. However, the incremental loading of the same shear stress with an effective loading rate of 0.26 dynes/cm$\sp2$/sec did significantly reduce the response suggesting a time-dependent property of the transduction activation. Finally, the long term cell culture passaging of the PROB showed a dramatic reduction in the mean peak $\rm \lbrack Ca\sp{2+}\rbrack \sb{i}$ response to serum-free flow but a consistent response to the serum-supplemented flow. This finding suggests an adaptive deterioration of the mechano-sensor due to prolonged cell culturing