Real-time visualization and modulation of endocytic dynamics in osteocytes in vivo

Endocytosis is a critical cellular process involved in many physiological functions, including mechanotransduction. Recent advancements in intravital imaging have led to in vivo analysis of endocytosis, and these tools can now be translated to study the highly mechanosensitive bone tissue. Here, we present a live-cell study of endocytosis in osteocytes, mechanosensory cells embedded in mouse bone. Using intravital multiphoton microscopy and fluorescently labeled nanoparticles, we visualized real-time uptake and trafficking within osteocytes in vivo. We applied pharmacologic inhibitors to assess both general and receptor-specific modes of endocytosis. Our results demonstrate rapid and dynamic nanoparticle internalization by osteocytes, with distinct differences in uptake kinetics and subcellular distribution depending on nanoparticle surface functionalization. Notably, we observed sex-specific differences in dynamin-dependent endocytic activity. These results offer the first in vivo derived insights into how osteocytes take up materials and provide new evidence for chemically altering receptor-mediated endocytosis in live bone tissue.