Liu Cong-Jin, Yang Xiao, Shi Jing-Wen, Shi Chang-Yang, Wu Xin-Tong, Lu Fei-Xiang, Fan Yu-Bo, Sun Lian-Wen
Key Laboratory of Biomechanics and Mechanobiology (Beihang University), Ministry of Education; Key Laboratory of Innovation and Transformation of Advanced Medical Devices, Ministry of Industry and Information Technology; National Medical Innovation Platform for Industry-Education Integration in Advanced Medical Devices (Interdiscipline of Medicine and Engineering); School of Biological Science and Medical Engineering, Beihang University, Beijing, 100191, China.
Calcif Tissue Int. 2025 Aug 2;116(1):103. doi: 10.1007/s00223-025-01411-8.
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 Ca 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.
非酶糖基化（NEG）产物是导致各种类型骨质疏松症的关键因素，因为它们会影响骨基质的物理特性以及成骨细胞和破骨细胞的功能。然而，它们对骨细胞机械敏感性的影响仍知之甚少。骨细胞嵌入腔隙-小管系统（LCS）中，其突起与小管基质有丰富的连接以放大其膜应变。因此，我们主要研究了骨基质中NEG交联对骨基质与骨细胞突起之间连接的影响，以及骨细胞对机械刺激的反应。为了制备不同程度的非酶糖基化交联骨基质，我们使用两种浓度的D-核糖（0.1M和0.4M）孵育脱钙牛骨切片。然后将类骨细胞（MLO-Y4）接种到这些骨切片上，检测细胞形态、细胞突起的机械性能、F-肌动蛋白细胞骨架、机械传感元件（整合素αVβ3和基底膜聚糖）的表达，以探索机械转导结构的变化。随后，在对细胞突起施加机械刺激后，检测骨细胞的细胞内钙反应。最后，检测骨钙素（OCN）和核因子κB受体激活剂配体（RANKL）/骨保护素（OPG），以探索骨细胞功能的变化。结果表明，与对照组相比，0.1M组和0.4M组的戊糖素（PEN）含量显著增加，两组骨硬度也显著增加；0.1M组整合素αVβ3的表达以及对局部机械刺激的细胞内钙反应较高；0.4M组F-肌动蛋白强度、整合素αVβ3和基底膜聚糖强度较低。与对照组相比，0.1M组和0.4M组的OCN表达显著降低。这些结果揭示可能存在一个糖基化阈值来影响骨细胞的机械敏感性：低水平的NEG交联可以通过这些特殊的机械传感元件促进骨细胞突起与骨基质的连接，以及骨细胞对局部机械刺激的反应，尽管高水平的NEG交联破坏了这种功能连接。然而，这两种水平的NEG交联都对骨细胞调节骨重塑的能力有负面影响。本研究为糖基化驱动的骨脆性机制及对抗年龄相关性和其他形式骨质疏松症的治疗策略提供了新的见解。
