Summary
Although it is widely accepted that osteocytes regulate bone homeostasis by sensing, integrating and
transducing mechanical and hormonal signals, characterization of dynamic signaling within the osteocyte
network has been challenging due to its location embedded within the bone matrix. Osteocytes reside within a
mineralized lacunar-canalicular (MLC) structure allowing sensing of mechanical forces and transduction this
signal through gap-junctions and secreted exchange of soluble biochemical signals. The MLC structure
modulates access of essential nutrients between vasculature and entombed osteocytes in a spatially gradient
manner. New understanding on osteocyte signaling will be necessary to develop new therapeutics for treating
diseases that involve osteocyte dysfunction. To that end, the goal of this work is to develop a new in vitro model
that will not only mimic the in vivo like MLC structure, but also facilitate the study of signaling dynamics within an
osteocyte network upon targeted mechanical stimulation or cell damage. The hypothesis that, “the nutrient
gradient that osteocyte encounter is a function of the mineralized lacunar-canalicular (MLC) structure, which in
turn regulates their signal propagation dynamics”, will be tested using three specific aims. Aim 1 will use a Hybrid
Laser Printing (HLP) platform to develop a microfluidic chip that mimics the MLC structure with associated
gradient nutrient transport properties. Aim 2 will identify experimental conditions to generate osteocyte network
within MLC chips using the mouse MLO-Y4 osteocyte cell line. Aim 3 will characterize propagation characteristics
of calcium signaling (amplitude, range, velocity, refractory period, spike-synchrony) within osteocyte networks
upon targeted mechanical stimulation, cell-damage, ablation of cell-cell connections, or in the presence of
signaling inhibitors. In summary, individual and combined effects of (i) MLC structure-induced gradient nutrient
access (ii) mineralized matrix, (iii) environmental hypoxia, and (iv) single cell manipulation, on calcium signaling
dynamics will provide new insights into osteocyte mechanotransduction. In the long term, this model can be
extended to patient-specific cells to screen therapeutics that target skeletal pathologies associated with
osteocyte malfunctions.