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. 2012 Mar;28(3):397-404.
doi: 10.1016/j.arthro.2011.08.308. Epub 2011 Dec 14.

Supraphysiologic Temperature Enhances Cytotoxic Effects of Bupivacaine on Bovine Articular Chondrocytes in an in Vitro Study

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Supraphysiologic Temperature Enhances Cytotoxic Effects of Bupivacaine on Bovine Articular Chondrocytes in an in Vitro Study

R Nelson Mead et al. Arthroscopy. .
Free PMC article

Abstract

Purpose: The purpose of this study was to determine the effects of temperature or 0.25% bupivacaine treatment in combination with supraphysiologic temperatures on chondrocyte viability.

Methods: Bovine articular chondrocytes in suspension culture were treated with phosphate-buffered saline solution at 20°C, 37°C, 40°C, 42°C, 45°C, 47°C, and 50°C for 15, 30, and 60 minutes or with phosphate-buffered saline solution at 37°C, 45°C, and 50°C for 30 and 60 minutes followed by 0.25% bupivacaine at 20°C for 60 minutes. Chondrocyte viability was analyzed by flow cytometry with the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR). Annexin V and ethidium double staining determined whether apoptosis or necrosis occurred.

Results: Temperatures from 20°C to 42°C did not cause chondrocyte death. Temperatures at or above 45°C caused significant chondrocyte death, particularly at 50°C for 60 minutes, compared with 37°C at 60 minutes (P < .01). When the chondrocytes were incubated at 50°C, subsequent exposure to bupivacaine significantly increased chondrocyte death compared with the saline solution-treated control group (P < .001). There were additive cytotoxic effects when bupivacaine was combined with supraphysiologic temperatures. It was also found that bupivacaine at supraphysiologic temperatures caused necrosis of articular chondrocytes.

Conclusions: Temperatures at or above 45°C caused significant chondrocyte death. Bupivacaine treatment in the presence of 45°C and 50°C temperatures significantly increased necrosis of bovine articular chondrocytes in this in vitro study.

Clinical relevance: Immediate intra-articular injection of bupivacaine after heat-generating procedures may cause damage to the cartilage because of the additive cytotoxic effects of bupivacaine and elevated temperature.

Figures

Figure 1
Figure 1
Effects of temperature on viability of bovine articular chondrocytes. A to G. Scatter plots of flow cytometry analysis in the cells treated for 30 min. The abscissa shows calcein fluorescence and the ordinate shows ethidium fluorescence. Quadrant 1 (Q1) represents the dead cells; quadrant 2 (Q2) represents the newly dead cells; quadrant 3 (Q3) represents live cells without calcein staining; and quadrant 4 (Q4) represents the live cells. H. Quantitative analysis. The bars represent mean percentages of the dead cells after 15-, 30- and 60- minute treatments at the indicated temperatures. Error bars represent the standard deviations. Asterisks indicate significant differences between the indicated groups and the corresponding control groups at 37° C.
Figure 2
Figure 2
Effects of bupivacaine and temperature on viability of bovine articular chondrocytes. A to F. Scatter plots of flow cytometry analysis in the cells treated for 30 min. The abscissa shows calcein fluorescence and the ordinate shows ethidium fluorescence. Quadrant 1 (Q1) represents the dead cells; quadrant 2 (Q2) represents the newly dead cells; quadrant 3 (Q3) represents live cells without calcein staining; and quadrant 4 (Q4) represents the live cells. G. Quantitative analysis. The bars represent mean percentages of the dead cells after 30 and 60 minutes treatments at the indicated temperatures. Error bars represent the standard deviations. Asterisks indicate significant differences between the indicated groups and the corresponding saline control groups at the same temperature.
Figure 3
Figure 3
Bupivacaine causes necrosis of bovine articular chondrocytes. A to C. Scatter plots of flow cytometry analysis in the cells treated for 30 min. The abscissa shows Annexin-V FITC fluorescence and the ordinate shows ethidium fluorescence. Quadrants 1 (Q1) and 2 (Q2) represent cells that have undergone necrosis and stained positive with ethidium due to cell membrane breakdown. Quadrant 3 (Q3) represents live cells without any staining. Quadrant 4 (Q4) represents cells that have undergone apoptosis and stained positive with Annexin-V FITC but not ethidium. D. Quantitative analysis. The solid bars represent the mean percentages of apoptotic cells and the open bars represent the mean percentages of necrotic cells. The error bars are the standard deviations. Asterisk indicates significant difference between the necrotic cells and the apoptotic cells at the same temperature.

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