Teixeira Graciosa Q, Boldt Antje, Nagl Ines, Pereira Catarina Leite, Benz Karin, Wilke Hans-Joachim, Ignatius Anita, Barbosa Mário A, Gonçalves Raquel M, Neidlinger-Wilke Cornelia
1 Institute of Orthopaedic Research and Biomechanics, Center for Musculoskeletal Research, University of Ulm , Ulm, Germany .
2 Instituto de Investigação e Inovação em Saúde, Universidade do Porto , Porto, Portugal .
Tissue Eng Part C Methods. 2016 Jan;22(1):8-19. doi: 10.1089/ten.tec.2015.0195. Epub 2015 Nov 13.
Resolution of intervertebral disc (IVD) degeneration-associated inflammation is a prerequisite for tissue regeneration and could possibly be achieved by strategies ranging from pharmacological to cell-based therapies. In this study, a proinflammatory disc organ culture model was established. Bovine caudal disc punches were needle punctured and additionally stimulated with lipopolysaccharide (10 μg/mL) or interleukin-1β (IL-1β, 10-100 ng/mL) for 48 h. Two intradiscal therapeutic approaches were tested: (i) a nonsteroidal anti-inflammatory drug, diclofenac (Df) and (ii) human mesenchymal stem/stromal cells (MSCs) embedded in an albumin/hyaluronan hydrogel. IL-1β-treated disc organ cultures showed a statistically significant upregulation of proinflammatory markers (IL-6, IL-8, prostaglandin E2 [PGE2]) and metalloproteases (MMP1, MMP3) expression, while extracellular matrix (ECM) proteins (collagen II, aggrecan) were significantly downregulated. The injection of the anti-inflammatory drug, Df, was able to reduce the levels of proinflammatory cytokines and MMPs and surprisingly increase ECM protein levels. These results point the intradiscal application of anti-inflammatory drugs as promising therapeutics for disc degeneration. In parallel, the immunomodulatory role of MSCs on this model was also evaluated. Although a slight downregulation of IL-6 and IL-8 expression could be found, the variability among the five donors tested was high, suggesting that the beneficial effect of these cells on disc degeneration needs to be further evaluated. The proinflammatory/degenerative IVD organ culture model established can be considered a suitable approach for testing novel therapeutic drugs, thus reducing the number of animals in in vivo experimentation. Moreover, this model can be used to address the cellular and molecular mechanisms that regulate inflammation in the IVD and their implications in tissue degeneration.
椎间盘（IVD）退变相关炎症的消退是组织再生的前提条件，并且可能通过从药物治疗到基于细胞的治疗等一系列策略来实现。在本研究中，建立了一种促炎性椎间盘器官培养模型。对牛尾椎间盘组织块进行针刺，并额外用脂多糖（10μg/mL）或白细胞介素-1β（IL-1β，10 - 100 ng/mL）刺激48小时。测试了两种椎间盘内治疗方法：（i）一种非甾体抗炎药双氯芬酸（Df）和（ii）包埋于白蛋白/透明质酸水凝胶中的人间充质干/基质细胞（MSCs）。经IL-1β处理的椎间盘器官培养物显示促炎标志物（IL-6、IL-8、前列腺素E2 [PGE2]）和金属蛋白酶（MMP1、MMP3）表达有统计学意义的上调，而细胞外基质（ECM）蛋白（胶原蛋白II、聚集蛋白聚糖）则显著下调。注射抗炎药Df能够降低促炎细胞因子和MMPs的水平，并且出人意料地提高ECM蛋白水平。这些结果表明椎间盘内应用抗炎药有望成为治疗椎间盘退变的方法。同时，还评估了MSCs在该模型上的免疫调节作用。尽管可以发现IL-6和IL-8表达有轻微下调，但在所测试的五个供体之间变异性很高，这表明这些细胞对椎间盘退变的有益作用需要进一步评估。所建立的促炎性/退行性IVD器官培养模型可被视为测试新型治疗药物的合适方法，从而减少体内实验中的动物数量。此外，该模型可用于研究调节IVD炎症的细胞和分子机制及其在组织退变中的意义。
