Spine Research Group, Competence Centre for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, Zurich, Switzerland.
Spine J. 2011 Jan;11(1):46-53. doi: 10.1016/j.spinee.2010.11.001.
Bupivacaine is commonly used as an adjunct during provocative discography and is administered intradiscally in patients with discogenic back pain. Recent studies demonstrated that bupivacaine is cytotoxic for articular chondrocytes in vitro at clinically used concentrations (0.25%-0.5%).
To analyze a concentration-dependent effect of bupivacaine on cell viability and gene expression of human intervertebral disc (IVD) cells in an in vitro model.
In vitro cell culture study.
Disc cells were isolated from human disc biopsies from 11 patients undergoing surgery because of degenerative disc disease or disc herniation.
Cell viability and gene expression after exposure to bupivacaine.
Human IVD cells were treated with different concentrations of bupivacaine for 2 (n=5) or 18 hours (n=5) and analyzed for cell viability and proliferation (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay). Additionally, cells were prestimulated with interleukin-1 beta (IL-1β) (5 ng/mL) to increase the levels of proinflammatory cytokines and matrix-degrading enzymes and thereafter treated with 0.75 mmol bupivacaine (as determined in the cell viability test) for 2 (n=5) or 18 hours (n=5). Prestimulated cells with or without bupivacaine treatment were analyzed for gene expression of IL-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), matrix metalloproteinase-3 (MMP3), MMP9, MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) using real-time reverse transcription-polymerase chain reaction. Statistical analysis was performed by using the Mann-Whitney U test with a significance level of p<.05.
After 18 hours, bupivacaine exhibited either a cytotoxic or a proliferative effect on human IVD cells, depending on the concentration. Similar but lower effects could be observed already after 2 hours. With a concentration of 0.75 mmol (proliferative effect), bupivacaine significantly decreased messenger RNA levels of TNF-α, COX-2, MMP13, and ADAMTS4 after 18 hours. In contrast, expression of IL-6, IL-8, and MMP9 did not differ; expression of IL-1β and MMP3 was stimulated with 0.75 mmol. After 2 hours, we observed a reduction in the expression of COX-2, MMP3, MMP13, and ADAMTS4, without any effect regarding IL-1β.
Application of bupivacaine in clinically relevant concentrations was toxic for IVD cells in vitro. A low concentration stimulated cell proliferation and reduced gene expression of certain matrix-degrading enzymes and proinflammatory cytokines. If these results can be corroborated in tissue explant models or animal studies, caution regarding provocative discography with bupivacaine is prompted.
布比卡因常用于激发性椎间盘造影术的辅助药物，并在椎间盘源性腰痛患者中直接注射到椎间盘内。最近的研究表明，在临床使用浓度（0.25%-0.5%）下，布比卡因对关节软骨细胞具有细胞毒性。
分析布比卡因在体外模型中对人椎间盘（IVD）细胞活力和基因表达的浓度依赖性影响。
体外细胞培养研究。
从 11 名因退行性椎间盘疾病或椎间盘突出症而行手术的患者的椎间盘活检中分离椎间盘细胞。
暴露于布比卡因后的细胞活力和增殖（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物测定法）。此外，用白细胞介素-1β（IL-1β）（5ng/mL）预先刺激细胞（以增加促炎细胞因子和基质降解酶的水平），然后用 0.75mmol 布比卡因（如细胞活力试验中确定）处理 2 小时（n=5）或 18 小时（n=5）。用布比卡因处理或不处理的预刺激细胞进行实时逆转录-聚合酶链反应分析白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、肿瘤坏死因子-α（TNF-α）、环氧化酶-2（COX-2）、基质金属蛋白酶-3（MMP3）、基质金属蛋白酶-9（MMP9）、基质金属蛋白酶-13（MMP13）和整合素金属蛋白酶与血栓反应蛋白 4（ADAMTS4）的基因表达。使用 Mann-Whitney U 检验进行统计分析，显著性水平为 p<.05。
18 小时后，布比卡因对人 IVD 细胞表现出细胞毒性或增殖作用，这取决于浓度。在 2 小时后已经可以观察到类似但更低的作用。在 0.75mmol 的浓度下（增殖作用），布比卡因在 18 小时后显著降低 TNF-α、COX-2、MMP13 和 ADAMTS4 的信使 RNA 水平。相比之下，IL-6、IL-8 和 MMP9 的表达没有差异；IL-1β 和 MMP3 的表达被 0.75mmol 刺激。在 2 小时后，我们观察到 COX-2、MMP3、MMP13 和 ADAMTS4 的表达减少，而 IL-1β 没有任何影响。
在临床相关浓度下应用布比卡因对体外 IVD 细胞具有毒性。低浓度刺激细胞增殖，并降低某些基质降解酶和促炎细胞因子的基因表达。如果这些结果可以在组织外植体模型或动物研究中得到证实，那么在使用布比卡因进行激发性椎间盘造影术时需要谨慎。
