Visualization and characterization of Ca2+-dependent actomyosin contractions in ex vivo osteocytes

Osteocytes exhibit many unique characteristics which have been thought to play a role in their mechanosensing capabilities, including longevity, a unique stellate morphology, and their existence as a vast, interconnected 3D network within the bone matrix. As such, it is important to maintain these features when investigating osteocyte responses to mechanical load. Thus, we have developed an ex vivo model of osteocyte mechanotransduction in which we visualize and capture real-time, short term responses of osteocytes to mechanical loading in explanted mice tibiae. This model utilizes a custom mechanical loading device to apply physiological mechanical stimulation to the tibiae while the cells are imaged with confocal microscopy. We currently use tibiae dissected from transgenic mice expressing a fluorescent F-actin tag stained with Ca²⁺ indicator dye to visualize actin network dynamics and intracellular Ca²⁺ events in osteocytes, respectively. We are characterizing the frequency of Ca²⁺ transients and actin dynamics as well as the sensitivity of these events to varying mechanical load. The goal of these studies is to draw connections between the two intracellular events, and investigate their potential as a mechanotransduction mechanism in osteocytes in response to whole-bone mechanical loading.