Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold.: An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis
Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold.: An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis
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DOI：
10.1016/j.joca.2006.01.008
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发表时间：
2006-07-01
影响因子：
7
通讯作者：
Freyria, A. -M.
中科院分区：
文献类型：
--
作者：
Cortial, D.;Gouttenoire, J.;Freyria, A. -M.
目的：为了确定最好的协议，用于制备的组织工程化软骨，以调查潜在的抗关节炎和/或抗骨关节炎的药物effects of drugs.Methods：小牛关节软骨细胞，接种在胶原海绵中生长培养长达1个月。在第14天，培养物接受白细胞介素（IL）-1 β（范围为0.1至20 ng/ml）1至3天。采用实时聚合酶链反应（PCR）分析细胞外基质蛋白、胶原结合整合素、基质金属蛋白酶（MMPs）、聚集蛋白聚糖酶、TIMPs、IL-1 Ra和I κ-B α的基因表达。使用酶谱法和荧光肽底物分析培养基中的金属蛋白酶活性。我们选择用接种有107个软骨细胞的胶原海绵培养15或17天，显示最小的细胞增殖、最大的硫酸化糖胺聚糖（sGAG）沉积和COL 2A 1的高表达，聚集蛋白聚糖和α 10整联蛋白亚基的表达以及COLA 2和α 11整联蛋白亚基的低表达。在1 ng/ml IL-1 β存在下，我们在第15天观察到MMP-1上调450倍，MMP-13上调60倍，ADAMTS-4和MMP-3上调54倍，ADAMTS-5和IL-1 Ra上调10倍。仅在第17天观察到COL 2A 1和聚集蛋白聚糖下调2.5倍。在蛋白质水平的总MMP-1和MMP-13的剂量依赖性增加，注意到小于15%的活性form.Conclusions：这种体外模型的软骨细胞培养在三维（3D）似乎很适合调查这些细胞的反应，炎症细胞因子和评估潜在的抗炎作用的药物。(C)2006年国际骨关节炎研究学会。由爱思唯尔有限公司出版。保留所有权利。
Objective: To determine the best protocol for the preparation of a tissue-engineered cartilage to investigate the potential anti-arthritic and/or anti-osteoarthritic effects of drugs.Methods: Calf articular chondrocytes, seeded in collagen sponges were grown in culture for up to 1 month. At day 14 cultures received interleukin (IL)-1 beta (ranging from 0.1 to 20 ng/ml) for 1 to 3 days. Analyses of gene expression for extracellular matrix proteins, collagen-binding integrins, matrix metalloproteinases (MMPs), aggrecanases, TIMPs, IL-1Ra and I kappa-B alpha were carried out using real-time polymerase chain reaction (PCR). Metalloproteinase activities were analysed in the culture medium using both zymography and fluorogenic peptide substrates.Results: We selected a culture for 15 or 17 days with collagen sponges seeded with 107 chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha 10 integrin sub-unit and low expression of COLA2 and the alpha 11 integrin sub-unit. In the presence of 1 ng/ml IL-1 beta, we observed at day 15 up-regulations of 450-fold for MMP-1, 60-fold for MMP-13, 54-fold for ADAMTS-4 and MMP-3 and 10-fold for ADAMTS-5 and IL-1Ra. Down-regulations of 2.5-fold for COL2A1 and aggrecan were observed only at day 17. At the protein level a dose-dependent increase of total MMP-1 and MMP-13 was noted with less than 15% in the active form.Conclusions: This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.