Molecular biological analysis of bone metabolism by compressive mechanical stress in human synovial cells of the temporomandibular joint

1. 目的：研究压缩机械应力对颞下颌关节（TMJ）疾病滑膜细胞破骨形成的影响，以阐明滑膜细胞破骨形成的机制。研究设计：从大鼠膝关节分离滑膜细胞，并使用常规方法持续压缩。采用逆转录聚合酶链反应、免疫印迹和免疫荧光染色等方法分析滑膜细胞中受体激活物核因子κ b配体（RANKL） mRNA和蛋白的表达。将小鼠骨髓细胞与滑膜细胞共同培养7 d，检测破骨细胞。结果：RANKL mRNA和蛋白在滑膜细胞中的表达随压缩力的增加而增加。将小鼠骨髓细胞与连续压缩的滑膜细胞一起培养，形成抗酒石酸酸性磷酸酶阳性的多核细胞。骨保护素完全抑制滑膜细胞压缩培养诱导的破骨细胞形成。结论：我们的研究结果表明，RANKL在压缩滑膜细胞中的表达促进了破骨细胞的形成，而持续的压缩力可能导致tmj的破骨细胞骨破坏。目的：虽然生化研究已经检测了颞下颌关节（TMJ）疾病（TMDs）患者的滑液（SF），但骨破坏和重塑的分子机制细节仍不清楚。在这项研究中，我们从TMD患者的SF中诱导并表征了破骨细胞样细胞，并研究了这些细胞在TMD发病过程中的作用。方法：我们从TMD患者抽吸手术后收集SF细胞，培养破骨细胞样细胞，并检测其特征，包括破骨细胞标志物和骨吸收活性。此外，我们通过连续传代培养从TMD患者的SF中获得成纤维细胞。使用这些成纤维细胞，我们使用免疫细胞化学染色检测成纤维细胞标记物，并分析核因子- κ b配体受体激活因子（RANKL） mRNA水平。采用酶联免疫吸附法（ELISA）检测血清中RANKL的可溶性形式（sRANKL）。结果：通过加入重组人（rh）巨噬细胞集落刺激因子（M-CSF）和1,25 -二羟基维生素D3 [1,25 (OH) 2D3]或前列腺素E2 (PGE2)， TMD患者SF细胞可诱导破骨细胞样细胞。这些多核巨细胞呈抗酒石酸酸性磷酸酶（TRAP）阳性，并具有吸收骨的能力。TMD患者SF成纤维细胞标记物阳性，RANKL mRNA表达上调。患者组SF中sRANKL的检出率明显高于对照组。结论：TMD患者关节浸润的SF细胞在其以进行性骨破坏或骨重塑为特征的发病机制中起重要作用。少

1. OBJECTIVES: We investigated the effects of compressive mechanical stress on osteoclastogenesis of synovial cells to clarify the mechanism of osteoclast formation by those cells in temporomandibular joint (TMJ) disorders. STUDY DESIGN: Synovial cells were isolated from rat knee joints and continuously compressed using a conventional method. The expression of receptor activator nuclear factor kappaB ligand (RANKL) mRNA and protein in synovial cells was analyzed by reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence staining. Mouse bone marrow cells were cultured with synovial cells for 7 days to detect osteoclasts. RESULTS: The expressions of RANKL mRNA and protein in synovial cells were increased with compressive force. When mouse bone marrow cells were cultured with continuously compressed synovial cells, tartrate-resistant acid phosphatase-positive multinucleated cells were formed. Osteoprotegerin completely inhibited osteoclast formation induced … More by culturing with compressed synovial cells. CONCLUSION: Our results indicated that the expression of RANKL in compressed synovial cells enhanced osteoclast formation, whereas continuous compressive force may induce osteoclastic bone destruction in the TMJ.2. OBJECTIVE: Although biochemical studies have examined the synovial fluid (SF) of patients with temporomandibular joint (TMJ) disorders (TMDs), the details of the molecular mechanism of bone destruction and remodeling remain unknown. In this study, we induced and characterized osteoclast-like cells from the SF of patients with TMD and investigated the participation of these cells in the pathogenesis of TMD. METHODS: We collected SF cells from patients with TMD after a pumping procedure, cultured osteoclast-like cells, and examined their characteristics, including osteoclast markers and bone resorption activities. In addition, we obtained fibroblastic cells from the SF of TMD patients by continuous sub-culturing. Using these fibroblastic cells, we examined fibroblast markers using immunocytochemical staining and analyzed the receptor activator of nuclear-factor-kappaB ligand (RANKL) mRNA levels. Detection of soluble form of RANKL (sRANKL) in the SF was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Osteoclast-like cells were induced from the SF cells of patients with TMD by adding recombinant human (rh) macrophage colony stimulating factor (M-CSF) and either 1, 25-dihydroxy vitamin D3 [1, 25 (OH) 2D3] or prostaglandin E2 (PGE2). These multinucleated giant cells were positive for tartrate-resistant acid phosphatase (TRAP) and had the ability to absorb bone. The fibroblastic cells from the SF of TMD patients were positive for fibroblast markers and RANKL mRNA was up-regulated. Detection of sRANKL in SF of patient group was significantly higher than control group. CONCLUSION: The results suggest that the joint-infiltrating SF cells from TMD patients play important roles in the pathogenesis of these disorders, which is characterized by progressive bone destruction or remodeling. Less