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

人颞下颌关节滑膜细胞压缩机械应力对骨代谢的分子生物学分析

基本信息

批准号：
18592196
负责人：
TAKANO Hiroshi
金额：
$ 1.9万
依托单位：
Akita University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18592196/
关键词：
dentistry cell tissue mechanical stress

项目摘要

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

1.目的：我们研究了机械压力对滑膜细胞破骨细胞生成的影响，以阐明颞下颌关节（TMJ）疾病中滑膜细胞形成破骨细胞的机制。研究设计：从大鼠膝关节分离滑膜细胞，并使用常规方法连续压缩。通过逆转录-聚合酶链反应、免疫印迹和免疫荧光染色分析滑膜细胞中受体激活因子核因子κ B配体（RANKL）mRNA和蛋白的表达。将小鼠骨髓细胞与滑膜细胞一起培养7天以检测破骨细胞。结果：关节滑膜细胞RANKL mRNA和蛋白表达随压力的增加而增加。当小鼠骨髓细胞与连续挤压的滑膜细胞一起培养时，形成了抗酒石酸盐酸性磷酸酶阳性的多核细胞。骨保护素完全抑制诱导的破骨细胞形成 ...更多信息通过与压缩的滑膜细胞一起培养。结论：我们的研究结果表明，RANKL在受压滑膜细胞中的表达促进了破骨细胞的形成，而持续的压力可能会导致TMJ中破骨细胞的骨破坏。目的：虽然生化研究已经检查了颞下颌关节（TMJ）疾病（TMD）患者的滑液（SF），骨破坏和重建的分子机制的细节仍然未知。在这项研究中，我们诱导和特点的破骨细胞样细胞从SF的TMD患者，并调查这些细胞参与TMD的发病机制。方法：我们收集了TMD患者的SF细胞，培养破骨细胞样细胞，并检测其特性，包括破骨细胞标志物和骨吸收活性。此外，我们还从TMD患者的SF中通过连续传代培养获得成纤维细胞。使用这些成纤维细胞，我们使用免疫细胞化学染色检查成纤维细胞标志物，并分析核因子-κ B配体受体激活因子（RANKL）mRNA水平。通过酶联免疫吸附试验（ELISA）检测SF中可溶性RANKL（sRANKL）。研究结果：用重组人巨噬细胞集落刺激因子（M-CSF）和1，25-二羟基维生素D_3 [1，25（OH）_2D_3]或前列腺素E_2（PGE_2）从TMD患者的SF细胞中诱导出破骨细胞样细胞。这些多核巨细胞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的成纤维细胞成纤维细胞标志物阳性，RANKL mRNA表达上调。患者组SF中sRANKL的检测明显高于对照组。结论：结果表明，关节浸润SF细胞从TMD患者在这些疾病的发病机制中发挥重要作用，这是一个渐进的骨破坏或重建的特点。少