The adaptation of bone to mechanical load is facilitated by osteocytes, the primary mechanosensory cells of bone. Across many cell types, membrane dynamics are understood to actively regulate mechanosensitivity by modulating the spatial organization and turnover of mechanosensitive proteins at the cell surface. However, the mechanisms regulating osteocyte membrane dynamics and their contribution to mechanotransduction remain poorly understood. This dissertation addresses this gap by developing and applying a novel intravital imaging platform to visualize and manipulate osteocyte membrane trafficking in living bone.

We first establish fluorescent core–shell silica nanoparticles (C’Dots) as an exogenous probe for imaging osteocytes in an intravital murine metatarsal model. These nanoparticles enable real-time visualization of cellular uptake and clearance dynamics, including integrin-targeted interactions, revealing previously unobserved aspects of osteocyte membrane behavior in vivo.

Building on this platform, we next investigate the role of endocytic pathways in regulating osteocyte membrane dynamics. Pharmacological modulation of endocytosis, including cholesterol depletion and dynamin inhibition, demonstrates that nanoparticle trafficking is sensitive to both membrane composition and pathway-specific perturbations, with distinct effects observed across nanoparticle functionalizations and between sexes.

Finally, we examine the relationship between membrane dynamics and mechanosensitivity. Cholesterol depletion reduces membrane trafficking activity while enhancing load-induced calcium signaling, indicating that membrane organization directly influences osteocyte mechanotransduction.

Together, this work develops a new platform and framework for studying osteocyte biology in vivo and establishes membrane dynamics as a regulator of osteocyte mechanosensitivity. These findings provide foundational insight into bone mechanobiology and identify membrane trafficking and composition as potential targets for modulating skeletal adaptation and disease.