Integrated analysis of human osteocyte lacunar ultrastructure and bone proteome profiling reveals coupling between immune and osteogenic signaling pathways.

Osteocytes reside within lacunae where they sense mechanical loading and regulate bone formation and resorption. Although lacunae define the osteocyte population, their number and volume are themselves regulated, and evidence regarding lacunar structural properties remains inconsistent. The relationship between lacunar ultrastructure and the bone proteome has not previously been explored. We therefore integrated high-resolution ultrastructural and proteomic analyses of femoral bone from osteoporotic patients undergoing hip arthroplasty after fracture (n = 15) and individuals undergoing surgery for osteoarthritis with normal or near-normal bone mineral density (n = 42). Lacunar morphology was assessed using high-resolution micro-computed tomography, and proteomic profiling was performed by liquid chromatography-tandem mass spectrometry, quantifying approximately three thousand proteins. Lacunar density and total lacunar volume normalized to bone volume were inversely correlated with total hip bone mineral density T-score (r = -0.462 and r = -0.471), indicating increased lacunar number and total lacunar volume in osteoporotic bone. In both sexes, lacunar density correlated strongly with total lacunar volume per bone volume (males: r = 0.69, p = 1.2 × 10-6; females: r = 0.70, p = 1.1 × 10-3), demonstrating tight coupling between lacunar number and volume. Exploratory pathway enrichment analysis revealed enrichment of pathways related to ROBO receptor signaling, translational regulation, and complement-mediated inflammatory processes. These findings link osteocyte lacunar ultrastructure to the bone proteome and identify osteocytes as central regulators integrating immune signaling, mechanosensing, and bone turnover.