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.
Although it is largely accepted that osteocyte signaling plays a key role in a variety of bone diseases, evaluation of signaling propagation within the osteocyte network has been challenging due to their location within a calcified bone matrix. To that end, this work will develop a new model that not only replicates the in vivo like mineralized lacunar-canalicular structure and associated gradient of nutrient transport but also allows real-time study of signal propagation within an osteocyte network upon targeted mechanical stimulation.
