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Cytotoxic responses of human chondrocytes to bupivacaine, levobupivacaine, and ropivacaine

Pudkrong Kaewpichit
Pudkrong Kaewpichit
, 
Somrat Charuluxananan
Somrat Charuluxananan
, 
Monpichar Srisa-Art
Monpichar Srisa-Art
, 
Sarocha Sisawat
Sarocha Sisawat
, 
Vitavat Aksornkitti
Vitavat Aksornkitti
, 
Chalermsri Chayudsatid
Chalermsri Chayudsatid
 and 
Amornpun Sereemaspun
Amornpun Sereemaspun
   | Sep 25, 2019
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Article Category: Brief communication (original)
Published Online: Sep 25, 2019
Page range: 169 - 178
DOI: https://doi.org/10.1515/abm-2019-0017
Keywords
© 2018 Pudkrong Kaewpichit et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Figure 1

Effect of local anesthetics on viability of CHON-001 cells after 15-, 30-, 60-, and 120-min exposure as determined by FITC-conjugated annexin V and PI staining and cytometry using a Beckman Coulter Epics XL flow cytometer. The sets of bars represent the mean percentages of chondrocyte viability after treatment with local anesthetic or controls; from left to right, PBS vehicle control (black bars), 0.4 mM H2O2 positive control (dark-gray bars), 0.25% ropivacaine (medium-gray bars), 0.5% ropivacaine (medium-gray bars), 0.25% levobupivacaine (light-gray bars), 0.5% levobupivacaine (light-gray bars), 0.25% bupivacaine (white bars), 0.5% bupivacaine (white bars). Low (0.25%) and high (0.5%) concentrations of bupivacaine and 0.5% levobupivacaine treated groups significantly decreased cell viability at 60 min, compared with treatment by the PBS vehicle control. **P < 0.001, ***P < 0.001; error bars represent standard deviation; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI, propidium iodide
Effect of local anesthetics on viability of CHON-001 cells after 15-, 30-, 60-, and 120-min exposure as determined by FITC-conjugated annexin V and PI staining and cytometry using a Beckman Coulter Epics XL flow cytometer. The sets of bars represent the mean percentages of chondrocyte viability after treatment with local anesthetic or controls; from left to right, PBS vehicle control (black bars), 0.4 mM H2O2 positive control (dark-gray bars), 0.25% ropivacaine (medium-gray bars), 0.5% ropivacaine (medium-gray bars), 0.25% levobupivacaine (light-gray bars), 0.5% levobupivacaine (light-gray bars), 0.25% bupivacaine (white bars), 0.5% bupivacaine (white bars). Low (0.25%) and high (0.5%) concentrations of bupivacaine and 0.5% levobupivacaine treated groups significantly decreased cell viability at 60 min, compared with treatment by the PBS vehicle control. **P < 0.001, ***P < 0.001; error bars represent standard deviation; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI, propidium iodide

Figure 2A–D

Pattern of CHON-001 cell death after 120 min exposure to local anesthetics as analyzed using a Beckman Coulter Epics XL flow cytometer after staining with annexin V-conjugated FITC and PI. Compared with chondrocytes treated with PBS vehicle as a control (A), the numbers of necrotic chondrocytes increased after treatment with 0.25% bupivacaine (C), 0.5% bupivacaine (D), 0.4 mM H2O2 positive control (B) FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI, propidium iodide
Pattern of CHON-001 cell death after 120 min exposure to local anesthetics as analyzed using a Beckman Coulter Epics XL flow cytometer after staining with annexin V-conjugated FITC and PI. Compared with chondrocytes treated with PBS vehicle as a control (A), the numbers of necrotic chondrocytes increased after treatment with 0.25% bupivacaine (C), 0.5% bupivacaine (D), 0.4 mM H2O2 positive control (B) FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI, propidium iodide

Figure 2E–H

Pattern of CHON-001 cell death after 120 min exposure to local anesthetics as analyzed using a Beckman Coulter Epics XL flow cytometer after staining with annexin V-conjugated FITC and PI. The numbers of necrotic chondrocytes increased after treatment with 0.25% levobupivacaine (E), 0.5% levobupivacaine (F), while chondrocytes treated with ropivacaine (G, H) showed no substantial difference in cell viability compared with those treated with PBS vehicle as a control (A). FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI, propidium iodide
Pattern of CHON-001 cell death after 120 min exposure to local anesthetics as analyzed using a Beckman Coulter Epics XL flow cytometer after staining with annexin V-conjugated FITC and PI. The numbers of necrotic chondrocytes increased after treatment with 0.25% levobupivacaine (E), 0.5% levobupivacaine (F), while chondrocytes treated with ropivacaine (G, H) showed no substantial difference in cell viability compared with those treated with PBS vehicle as a control (A). FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI, propidium iodide

Figure 3

Production of dermatan sulfate by bupivacaine-treated CHON-001 cells as determined by a Beckman Coulter P/ACE MDQ capillary electrophoresis system equipped with a photodiode array detector operating at 195 nm (AU). (A) Dermatan sulfate production by chondrocytes treated with 0.125% bupivacaine for 24 h was greater than production by chondrocytes treated with 0.25% bupivacaine for 24 h. Labeled peaks correspond to dermatan sulfate showing migration time (min) and relative area under the peak (arbitrary units). Treatments for 24 h as indicated. (B) Bars show the relative increase in dermatan sulfate production by the chondrocytes after the various treatments including both concentrations of bupivacaine (single measurements). AU, arbitrary units; DMEM, Dulbecco’s modified Eagle’s medium
Production of dermatan sulfate by bupivacaine-treated CHON-001 cells as determined by a Beckman Coulter P/ACE MDQ capillary electrophoresis system equipped with a photodiode array detector operating at 195 nm (AU). (A) Dermatan sulfate production by chondrocytes treated with 0.125% bupivacaine for 24 h was greater than production by chondrocytes treated with 0.25% bupivacaine for 24 h. Labeled peaks correspond to dermatan sulfate showing migration time (min) and relative area under the peak (arbitrary units). Treatments for 24 h as indicated. (B) Bars show the relative increase in dermatan sulfate production by the chondrocytes after the various treatments including both concentrations of bupivacaine (single measurements). AU, arbitrary units; DMEM, Dulbecco’s modified Eagle’s medium

Figure 4

ROS production by CHON-001 cells after exposure to local anesthetics for 0–60 min as determined by 2′,7′-dichlorodihydrofluorescein diacetate reagent. The chondrocytes were treated with PBS vehicle as a control (black bars), bupivacaine (white bars) 0.25% (A) and 0.5% (B), ropivacaine (dark-gray bars) 0.25% (C) and 0.5% (D), levobupivacaine (light-gray bars) 0.25% (E) and 0.5% (F). The local anesthetics immediately decreased ROS production compared with PBS. *P < 0.05, **P < 0.01, and ***P < 0.001; error bars represent standard deviation m, minutes; PBS, phosphate-buffered saline; ROS, reactive oxygen species
ROS production by CHON-001 cells after exposure to local anesthetics for 0–60 min as determined by 2′,7′-dichlorodihydrofluorescein diacetate reagent. The chondrocytes were treated with PBS vehicle as a control (black bars), bupivacaine (white bars) 0.25% (A) and 0.5% (B), ropivacaine (dark-gray bars) 0.25% (C) and 0.5% (D), levobupivacaine (light-gray bars) 0.25% (E) and 0.5% (F). The local anesthetics immediately decreased ROS production compared with PBS. *P < 0.05, **P < 0.01, and ***P < 0.001; error bars represent standard deviation m, minutes; PBS, phosphate-buffered saline; ROS, reactive oxygen species

Figure 5

NO production by CHON-001 cells after exposure to local anesthetics for 120 min as measured by using a colorimetric Nitrite/Nitrate Assay kit. The bars represent the mean NO production after treatment with PBS vehicle as a control (black bars), bupivacaine (white bars, 0.25% left, 0.5% right), levobupivacaine (light-gray bars, 0.25% left, 0.5% right), ropivacaine (medium-gray bars, 0.25% left, 0.5% right), and 0.4 mM H2O2 (dark-gray bars, far right). *P < 0.05, **P < 0.01, and ***P < 0.001; error bars represent standard deviation; m, minutes; NO, nitric oxide
NO production by CHON-001 cells after exposure to local anesthetics for 120 min as measured by using a colorimetric Nitrite/Nitrate Assay kit. The bars represent the mean NO production after treatment with PBS vehicle as a control (black bars), bupivacaine (white bars, 0.25% left, 0.5% right), levobupivacaine (light-gray bars, 0.25% left, 0.5% right), ropivacaine (medium-gray bars, 0.25% left, 0.5% right), and 0.4 mM H2O2 (dark-gray bars, far right). *P < 0.05, **P < 0.01, and ***P < 0.001; error bars represent standard deviation; m, minutes; NO, nitric oxide
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