Jacobs:Research:Mechanotransduction in Bone via Oscillatory Fluid Flow

Objective
Bone cells occupy fluid filled voids (lacunae) in the mineralized matrix and interconnected by small tubes (canaliculi). As the bone matrix is cyclically loaded, fluid flows in the lacunar-canalicular network from regions of high matrix strain to low matrix strain and back in an oscillatory fashion. Although, it has been demonstrated that bone cells respond to steady and pulsatile fluid flow with a transient elevation in intracellular calcium concentration, increased release of paracrine factors, and increased gene transcription, our preliminary data indicate that these responses are fundamentally different from those observed for oscillating flow. To date no experimental system has been designed to study responsiveness to physiologic oscillating fluid flow as a function of frequency and flow rate. To this end, we have developed a functioning oscillatory fluid flow exposure apparatus. This has allowed us to observe a frequency dependent intracellular calcium response to physiologic levels of oscillating fluid flow.

Our central hypothesis is that oscillatory fluid flow in vivo provides an important mechanism of mechanotransduction, and furthermore shear stress level, frequency, time course, low level steady flow, and cell morphology modulate the cellular response.

Research Plan
This project is divided into four specific aims:


 * Determine the range of frequencies and shear stress magnitudes that are stimulatory to bone cells. The response will be quantified in terms of intracellular calcium concentration ([Ca2+]i), prostaglandin E2 (PGE2) release, and mRNA level for the bone specific protein osteopontin. Thus, our outcome measures include immediate, intermediate, and late time points. The testing matrix will include oscillating flow at 0.0Hz (steady), 0.5Hz, 1Hz, 2Hz, 5Hz, 10Hz, and 20Hz at stress magnitudes of 5, 10, 20, and 40 dynes/cm2.


 * The [Ca2+]i, PGE2, and osteopontin mRNA responses to physiologic oscillating fluid flow applied various times after an initial exposure will be quantified. We predict that the cells will exhibit a decreased sensitivity after exposure to oscillating flow from which they gradually recover.


 * The [Ca2+]i, PGE2, and osteopontin mRNA responses to physiologic oscillating fluid flow in combination with low level steady flow will be determined in hFOB cells. We predict that, although insufficient to elicit a response by itself, low level steady flow will work synergistically with oscillating flow to increase responsiveness to oscillating flow and regulate bone cell metabolism.


 * The geometric dimensions of individual cells will be correlated with [Ca2+]i responsiveness to oscillating fluid flow both within and perpendicular to the substrate plane. The 2D shape of the cells in the digital images acquired in SA1 will be analyzed to determine aspect ratio and orientation with flow and correlated with [Ca2+]i responsiveness. [Ca2+]i responsiveness to oscillating fluid flow will also be determined in a plane perpendicular to the substrate and correlated with cell height.