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

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 degrees C, 37 degrees C, 40 degrees C, 42 degrees C, 45 degrees C, 47 degrees C, and 50 degrees C for 15, 30, and 60 minutes or with phosphate-buffered saline solution at 37 degrees C, 45 degrees C, and 50 degrees C for 30 and 60 minutes followed by 0.25% bupivacaine at 20 degrees 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 degrees C to 42 degrees C did not cause chondrocyte death. Temperatures at or above 45 degrees C caused significant chondrocyte death, particularly at 50 degrees C for 60 minutes, compared with 37 degrees C at 60 minutes (P < .01). When the chondrocytes were incubated at 50 degrees 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 degrees C caused significant chondrocyte death. Bupivacaine treatment in the presence of 45 degrees C and 50 degrees 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.