Impacts of Nonenzymatic Glycation Crosslinks in Bone Matrix on the Mechanosensitivity of Osteocyte

The products of nonenzymatic glycation (NEG) are a key contributor to various types of osteoporosis, as they affect both the physical properties of bone matrix and the function of osteoblasts and osteoclasts. However, their impact on the mechanosensitivity of osteocytes remains poorly understood. Osteocytes are embedded in lacunar-canalicular system (LCS), and their processes have abundant connections with canalicular matrix to amplify their membrane strain. Thus, we mainly studied the effects of NEG crosslinks in bone matrix on the connections between bone matrix and osteocyte processes, as well as the responses of osteocyte to the mechanical stimulation. To develop the nonenzymatic glycation crosslinked bone matrix in different degrees, we used two concentrations of D-ribose (0.1M and 0.4M) to incubate the decalcified bovine bone slices. Then the osteocyte-like cells (MLO-Y4) were seeded onto these bone slices, and the cell morphology, the mechanical properties of cell processes, the F-actin cytoskeleton, the expression of mechanical sensing elements (integrin αVβ3 and perlecan), were detected to explore the changes in mechanotransduction structure. Followed by, the intracellular Ca²⁺ responses of osteocytes were detected after applying mechanical stimulation to the cell processes. Finally, osteocalcin (OCN) and receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) were detected explore the changes in osteocyte function. Results showed that the PEN content increased significantly in 0.1M group and 0.4M group compared to CON group, the hardness of bone also significantly increased in two groups; the expression of integrin αVβ3, as well as the intracellular calcium responses to local mechanical stimulation were higher in 0.1M group; the F-actin intensity, integrin αVβ3 and PLN intensity were lower in 0.4M group. The OCN expression decreased significantly in 0.1M group and 0.4M group compared to CON group. These revealed a glycation threshold may exist to influence osteocyte mechanosensitivity: the low levels of NEG crosslinks could promote the connections of osteocyte processes and bone matrix via these special mechanical sensing elements, as well as the responses of osteocyte to the local mechanical stimulation, although the high levels of NEG crosslinks disrupted this functional connectivity. However, both of these two levels of NEG crosslinks had negative effects on the ability of osteocytes regulating the bone remodeling. This study offered novel insights into the mechanism of glycation-driven bone fragility and therapeutic strategies to counteract age-related and other forms of osteoporosis.