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Abstract
This work presents the results of research investigating the response of primary cultured bone cells (PROBs) to fluid flow. The ultimate goal of this research was to elucidate the biophysical transduction mechanism by which the extracellular stimulus of fluid flow is transduced to an intracellular signal, which may in turn affect cell function. This research was undertaken using a parallel-plate flow chamber and fura-2 fluorescence microscopy which permitted intracellular calcium ( (Ca$\sp{2+}$) $\sb{\rm i}$) to be measured cell-by-cell in preconfluent bone cells capable of expressing the osteoblast phenotype when mature. Fluid flow rates corresponding to 0-70 dynes/cm$\sp2$ of fluid-induced chamber wall shear stress were examined using a serum-free physiologic saline perfusate, a modified Hanks' Buffered Saline (HBS). Several major findings of this work are enumerated below: (1) A Heterogeneous response with respect to peak amplitude and latency to peak response was observed for the culture with an overriding dose-dependent relationship between the mean peak amplitude of response and shear stress magnitude. Furthermore, a dose-dependence was observed between the number of responsive cells (responding $>$50% over basal levels) and shear stress magnitude. Heterogeneity in the sense that not all cells could be re-stimulated by repeated exposure to flow was also observed. The cell response appears to be independent of whether cells are clustered together or isolated. (2) In contrast to PROBs, neither the clonal murine MC3T3-E1 nor rat osteosarcoma ROS 17/2.8 osteogenic cell lines demonstrated a cell (Ca$\sp{2+}$) $\sb{\rm i}$ response to HBS fluid flow. Clonal bovine pulmonary artery endothelial cells (BPAECs) were examined as a biological control. Consistent with reported findings, the BPAECs failed to respond in the normal HBS perfusate and responded robustly in HBS plus serum. (3) There was no significant contribution of the concomitant streaming potential to the mean peak (Ca$\sp{2+}$) $\sb{\rm i}$ response. Similarly, no role by the concomitant hydrostatic pressure was observed. (4) The inositol phospholipid biochemical pathway, inositol trisphosphate (IP$\sb3$)-induced calcium release from internal stores, was demonstrated with antagonist and blocker studies to be important in the observed (Ca$\sp{2+}$) $\sb{\rm i}$ response to flow. In addition, a concomitant need for extracellular calcium or influx through mechanosensitive channels was also observed. These results suggest that IP$\sb3$ and calcium may act as coagonists mediating the flow response.
