Impact of Local vs. Systemic Senescent Bone Cell Accumulation on Healthy Osteocyte Mechanosensation

PROJECT SUMMARY / ABSTRACT The goals of this proposal are to: (1) acquire the experimental skills and career training necessary to expand my research program into cellular aging/senescence and molecular biology, (2) investigate how the accumulation of senescent cells (SnCs) and their senescence-associated secretory phenotypes (SASPs) impact osteocyte mechanobiology in a 3D bone-mimicking microenvironment, and (3) generate foundational data to support future NIH R01 applications. Our previous studies have shown that SnC accumulation with age at various sites has anti-resorptive and anabolic effects, ultimately leading to bone loss. However, the mechanisms by which local and systemic SnCs disrupt osteocyte mechanosensation during aging remain unclear. This proposal addresses three crucial knowledge gaps: (Aim 1) How do local vs. systemic SnCs and SASP influence cytoskeletal stiffness and membrane viscoelasticity in healthy osteocytes? (Aim 2) How do age-related changes in cytoskeletal stiffness impact osteocyte responses to mechanical stimuli? (Aim 3) What are the reciprocal effects of senescence between osteocytes and bone marrow mesenchymal stem cells (BMSCs)? My preliminary data show that senescent osteocytes exhibit time-dependent increases in cytoskeletal stiffness, with local interactions between senescent and healthy osteocytes having a greater impact than systemic SASP exposure. Based on these data, my central hypothesis is that senescent osteocytes impair healthy osteocyte mechanosensation through cytoskeletal stiffening, with local and systemic interactions differentially impacting healthy osteocytes. To test this, I will develop novel 2D and 3D co-culture models using our visible light-induced 3D bioprinting technique and biocompatible hydrogel-based bio-inks to mimic the bone microenvironment. These models will allow investigation into how SnCs within the bone microenvironment, both locally and systemically, affect osteocyte mechanobiology and mechanosensation. Micromechanical properties of osteocytes and their surrounding matrix will be measured using optical fiber-based interferometry nanoindentation coupled with live- cell immunofluorescence staining. These biophysical measurements will be correlated with gene and protein expression analyses to mechanistically determine the impact of SnCs in the bone microenvironment on osteocyte mechanobiology. The successful completion of these aims will provide critical mechanistic insights into how bone mechanobiology is altered by aging and cellular senescence, supporting the development of therapeutic strategies to rejuvenate or eliminate SnCs to restore osteocyte function. The proposed training plan integrates advanced techniques in cellular senescence and biomolecular assays, with opportunities to extend into transgenic mouse models, which will support my future research projects and R01 applications.