title: Bupivacaine--the deadly friend of intervertebral disc cells?
abstract: BACKGROUND
  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%).
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  In vitro cell culture study.
  
  
  METHODS
  Disc cells were isolated from human disc biopsies from 11 patients undergoing surgery because of degenerative disc disease or disc herniation.
  
  
  METHODS
  Cell viability and gene expression after exposure to bupivacaine.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Spine Research Group, Competence Centre for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, Zurich, Switzerland.
authors.forename: Lilian
authors.initials: L
authors.lastname: Quero
authors.forename: Marina
authors.initials: M
authors.lastname: Klawitter
authors.forename: Andreas G
authors.initials: AG
authors.lastname: Nerlich
authors.forename: Massimo
authors.initials: M
authors.lastname: Leonardi
authors.forename: Norbert
authors.initials: N
authors.lastname: Boos
authors.forename: Karin
authors.initials: K
authors.lastname: Wuertz
mesh_terms: D002045:Bupivacaine / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018709:Statistics, Nonparametric
chemical_list: D016207:Cytokines; D020782:Matrix Metalloproteinases; D002045:Bupivacaine