Chang-Meen Sung1, Young-Sool Hah2, Jin-Su Kim3, Jeoung-Bin Nam4, Ra Jeong Kim5, Sang-Jin Lee6, Hyung Bin Park7. 1. Department of Orthopaedic Surgery, School of Medicine, Gyeongsang National University, Jinju, Korea. 2. Biomedical Research Institute, Gyeongsang National University Hospital, Jinju, Korea. 3. Surgery of Foot & Ankle, Eulji Medical Center, Eulji College of Medicine, Eulji University, Seoul, Korea. 4. Musculoskeletal Research Center, Gyeongsang National University Hospital, Jinju, Korea. 5. Department of Convergence Medical Science, Gyeongsang National University, Jinju, Korea. 6. Department of Orthopaedic Surgery, Barun Hospital, Jinju, Korea. 7. Department of Orthopaedic Surgery, School of Medicine, Gyeongsang National University, Jinju, Korea hbinpark@gnu.ac.kr.
Abstract
BACKGROUND: Concern has recently arisen over the safety of local anesthetics used on human tissues. HYPOTHESIS: Aminoamide local anesthetics have cytotoxic effects on human rotator cuff tenofibroblasts. STUDY DESIGN: Controlled laboratory study. METHODS: Cultured human rotator cuff tenofibroblasts were divided into control, phosphate buffered saline (PBS), and local anesthetic study groups; the PBS study group was further subdivided by pH level (pH 7.4, 6.0, and 4.4). The 6 local anesthetic subgroups (0.2% and 0.75% ropivacaine, 0.25% and 0.5% bupivacaine, and 1% and 2% lidocaine) were also studied at 10% dilutions of their original concentrations. Exposure times were 5, 10, 20, 40, or 60 minutes for the higher concentrations and 2, 6, 12, 24, 48, or 72 hours for the lower concentrations. Cell viability was evaluated through live, apoptotic, and necrotic cell rates using the annexin V-propidium iodide double-staining method. Intracellular reactive oxygen species (ROS) and the activity of mitogen-activated protein kinases (MAPKs) and caspase-3/7 were investigated. RESULTS: The control and PBS groups showed no significant differences in cell viability (P > .999). In the local anesthetic study groups, cell viability decreased significantly with increases in anesthetic concentrations (P < .001) and exposure times (P < .001), with the exception of the lidocaine subgroups, where this effect was masked by the very high cytotoxicity of even low concentrations. Among the studied local anesthetic subgroups, 0.2% ropivacaine was the least toxic. The levels of intracellular ROS of each local anesthetic subgroup also increased significantly (P < .05). The studied local anesthetics showed increases in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 as well as in levels of caspase-3/7 activity (P < .001). CONCLUSION: The cytotoxicity of the anesthetics studied to tenofibroblasts is dependent on exposure time and concentration. Of the evaluated anesthetics, ropivacaine is the least toxic in the clinically used concentration. The studied anesthetics induce tenofibroblast cell death, mediated by the increased production of ROS, by the increased activation of ERK1/2, JNK, and p38 and by the activation of caspase-3/7. CLINICAL RELEVANCE: This study identified the cytotoxic mechanisms of aminoamide local anesthetics acting on rotator cuff tenofibroblasts. The greatest margin of safety was found in lower anesthetic concentrations in general and more specifically in the use of ropivacaine.
BACKGROUND: Concern has recently arisen over the safety of local anesthetics used on human tissues. HYPOTHESIS: Aminoamide local anesthetics have cytotoxic effects on human rotator cuff tenofibroblasts. STUDY DESIGN: Controlled laboratory study. METHODS: Cultured human rotator cuff tenofibroblasts were divided into control, phosphate buffered saline (PBS), and local anesthetic study groups; the PBS study group was further subdivided by pH level (pH 7.4, 6.0, and 4.4). The 6 local anesthetic subgroups (0.2% and 0.75% ropivacaine, 0.25% and 0.5% bupivacaine, and 1% and 2% lidocaine) were also studied at 10% dilutions of their original concentrations. Exposure times were 5, 10, 20, 40, or 60 minutes for the higher concentrations and 2, 6, 12, 24, 48, or 72 hours for the lower concentrations. Cell viability was evaluated through live, apoptotic, and necrotic cell rates using the annexin V-propidium iodide double-staining method. Intracellular reactive oxygen species (ROS) and the activity of mitogen-activated protein kinases (MAPKs) and caspase-3/7 were investigated. RESULTS: The control and PBS groups showed no significant differences in cell viability (P > .999). In the local anesthetic study groups, cell viability decreased significantly with increases in anesthetic concentrations (P < .001) and exposure times (P < .001), with the exception of the lidocaine subgroups, where this effect was masked by the very high cytotoxicity of even low concentrations. Among the studied local anesthetic subgroups, 0.2% ropivacaine was the least toxic. The levels of intracellular ROS of each local anesthetic subgroup also increased significantly (P < .05). The studied local anesthetics showed increases in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 as well as in levels of caspase-3/7 activity (P < .001). CONCLUSION: The cytotoxicity of the anesthetics studied to tenofibroblasts is dependent on exposure time and concentration. Of the evaluated anesthetics, ropivacaine is the least toxic in the clinically used concentration. The studied anesthetics induce tenofibroblast cell death, mediated by the increased production of ROS, by the increased activation of ERK1/2, JNK, and p38 and by the activation of caspase-3/7. CLINICAL RELEVANCE: This study identified the cytotoxic mechanisms of aminoamide local anesthetics acting on rotator cuff tenofibroblasts. The greatest margin of safety was found in lower anesthetic concentrations in general and more specifically in the use of ropivacaine.
Authors: Clayton W Nuelle; Cristi R Cook; Aaron M Stoker; James L Cook; Seth L Sherman Journal: J Orthop Translat Date: 2016-09-28 Impact factor: 5.191