Intravital imaging of osteocyteα_vβ₃integrin dynamics with locally injectable fluorescent nanoparticles

AbstractOsteocytes are the resident mechanosensory cells in bone. They are responsible for skeletal homeostasis and adaptation to mechanical cues. Integrin proteins play an prominent role in osteocyte mechanotransduction, however the details are not well stratifiedin vivo. Intravital imaging with multiphoton microscopy presents an opportunity to study molecular level mechanobiological eventsin vivo, and could be used to study integrin dynamics in osteocytes. However, fluorescent imaging limitations with respect to excessive optical scattering and low signal to noise ratio caused by mineralized bone matrix make such investigations non-trivial. Here we demonstrate that ultra-small and bright fluorescent core-shell silica nanoparticles (<7nm diameter), known as Cornell Prime Dots (C’Dots), are well-suited for thein vivobone microenvironment and can improve intravital imaging capabilities. We report validation studies for C’Dots as a novel, locally injectedin vivoosteocyte imaging tool for both non-specific cellular uptake and for targeting integrins. The pharmacokinetics of C’Dots reveal distinct sex differences in nanoparticle cycling and clearance in osteocytes, which represents a novel topic of study in bone biology. Integrin-targeted C’Dots were used to study osteocyte integrin dynamics. To the best of our knowledge, we report here the first evidence of osteocyte integrin endocytosis and recyclingin vivo. Our results provide novel insights in osteocyte biology and will open up new lines of investigation that were previously unavailablein vivo.