Sato Rei, Maruyama Kentaro, Nemoto Eiji, Sakisaka Yukihiko, Suzuki Shigeki, Li Jiajun, Numazaki Kento, Tada Hiroyuki, Yamada Satoru
Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai, Japan.
Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Sendai, Japan.
Front Physiol. 2022 Feb 14;13:825596. doi: 10.3389/fphys.2022.825596. eCollection 2022.
Cementum resorption, unlike bone resorption, is clinically known to occur only with limited pathological stimuli, such as trauma, orthodontic forces, and large apical periodontitis; however, the molecular mechanisms that control osteoclast formation on the cementum surface remain unclear. In this study, we focused on extracellular vesicles (EVs) secreted by cementoblasts and analyzed their effects on osteoclast differentiation. EVs were extracted from the conditioned medium (CM) of the mouse cementoblast cell line OCCM-30. Transmission electron microscopy (TEM) analysis confirmed the presence of EVs with a diameter of approximately 50-200 nm. The effect of the EVs on osteoclast differentiation was examined using the mouse osteoclast progenitor cell line RAW 264.7 with recombinant receptor activator of nuclear factor (NF)-κB ligand (rRANKL) stimulation. EVs enhanced the formation of tartrate-resistant acid phosphatase (TRAP) activity-positive cells upon rRANKL stimulation. EVs also enhanced the induction of osteoclast-associated gene and protein expression in this condition, as determined by real-time PCR and Western blotting, respectively. On the other hand, no enhancing effect of EVs was observed without rRANKL stimulation. A Western blot analysis revealed no expression of receptor activator of NF-κB ligand (RANKL) in EVs themselves. The effect on rRANKL-induced osteoclast differentiation was examined using the CM of cementoblasts in terms of TRAP activity-positive cell formation and osteoclast-associated gene expression. The conditioned medium partly inhibited rRANKL-induced osteoclast differentiation and almost completely suppressed its enhancing effect by EVs. These results indicate that cementoblasts secreted EVs, which enhanced RANKL-induced osteoclast differentiation, and simultaneously produced soluble factors that neutralized this enhancing effect of EVs, implicating this balance in the regulation of cementum absorption. A more detailed understanding of this crosstalk between cementoblasts and osteoclasts will contribute to the development of new therapies for pathological root resorption.
与骨吸收不同，临床已知牙骨质吸收仅在有限的病理刺激下发生，如创伤、正畸力和严重根尖周炎；然而，控制牙骨质表面破骨细胞形成的分子机制仍不清楚。在本研究中，我们聚焦于成牙骨质细胞分泌的细胞外囊泡（EVs），并分析了它们对破骨细胞分化的影响。从小鼠成牙骨质细胞系OCCM-30的条件培养基（CM）中提取EVs。透射电子显微镜（TEM）分析证实存在直径约为50-200nm的EVs。使用小鼠破骨细胞祖细胞系RAW 264.7并经重组核因子（NF）-κB配体（rRANKL）刺激，检测EVs对破骨细胞分化的影响。在rRANKL刺激下，EVs增强了抗酒石酸酸性磷酸酶（TRAP）活性阳性细胞的形成。在这种情况下，通过实时PCR和蛋白质印迹法分别测定，EVs还增强了破骨细胞相关基因和蛋白质表达的诱导。另一方面，在没有rRANKL刺激的情况下，未观察到EVs的增强作用。蛋白质印迹分析显示EVs本身不表达NF-κB配体受体激活剂（RANKL）。从TRAP活性阳性细胞形成和破骨细胞相关基因表达方面，使用成牙骨质细胞的条件培养基检测对rRANKL诱导的破骨细胞分化的影响。条件培养基部分抑制了rRANKL诱导的破骨细胞分化，并几乎完全抑制了EVs对其的增强作用。这些结果表明，成牙骨质细胞分泌的EVs增强了RANKL诱导的破骨细胞分化，同时产生了中和EVs这种增强作用的可溶性因子，这意味着这种平衡参与了牙骨质吸收的调节。对成牙骨质细胞与破骨细胞之间这种相互作用的更详细了解将有助于开发病理性牙根吸收的新疗法。
