Reduction of Environmental Temperature Mitigates Local Anesthetic Cytotoxicity in Bovine Articular Chondrocytes

The purpose of this study was to assess whether reducing environmental temperature will lead to increased chondrocyte viability following injury from a single-dose of local anesthetic treatment. Bovine articular chondrocytes from weight bearing portions of femoral condyles were harvested and cultured. 96-well plates were seeded with 15,000 chondrocytes per well. Chondrocytes were treated with one of the following conditions: ITS Media, 1x PBS, 2% lidocaine, 0.5% bupivacaine, or 0.5% ropivacaine. Each plate was then incubated at 37°C, 23°C, or 4°C for one hour and then returned to media at 37°C. Chondrocyte viability was assessed 24 hours after treatment. Chondrocyte viability is presented as a ratio of the fluorescence of the treatment group over the average of the media group at that temperature (ratio ± SEM). At 37°C, lidocaine (0.35 ± 0.04) and bupivacaine (0.30 ± 0.05) treated chondrocytes show low cell viability when compared to the media (1.00 ± 0.03) control group (p < 0.001). Lidocaine treated chondrocytes were significantly more viable at 23°C (0.84 ± 0.08) and 4°C (0.86±0.085) than at 37°C (p < 0.001). Bupivacaine treated chondrocytes were significantly more viable at 4°C (0.660 ± 0.073) than at 37°C or 23°C (0.330 ± 0.069) (p < 0.001 and p = 0.002 respectively). Reducing the temperature from 37°C to 23°C during treatment with lidocaine increases chondrocyte viability following injury. Chondrocytes treated with bupivacaine can be rescued by reducing the temperature to 4°C. Key points Confirm that local anesthetics, specifically bupivacaine and lidocaine, are toxic to chondrocytes in monolayer Chondrocyte viability significantly improved for chondrocytes treated with bupivacaine when the environment was cooled to 23°C. Chondrocyte viability significantly improved for chondrocytes treated with bupivacaine or lidocaine when the environment was cooled to 4°C It is the recommendation of the authors that physicians should be wary of the risks of injecting local anesthetics into the intra-articular space. Active cooling of the joint could potentially protect the articular cartilage from insult following treatment with local anesthetics. Key words: Clinical hypothermia, local anesthetics, articular cartilage, osteoarthritis, chondrolysis Introduction Osteoarthritis is characterized by articular cartilage degeneration, pain, and loss of joint motion. Annually, approximately 30 million Americans suffer from osteoarthritis, costing more than $100 billion a year in treatment (Brown et al., 2006). Local anesthetic injections are effective for treatment and diagnosis of joint pain, and are commonly used in the clinic, both pre- and post-operatively. To use of these drugs outside of local administration or in nerve blocks are not indicated by the FDA. That is, use of local anesthetics intra-articularly is an off label use that now carries a warning for potential risk of chondrolysis by the FDA (Beyzadeoglu et al., 2012). Clinicians have observed cases of chondrolysis following treatment with local anesthetics to manage postoperative pain (Anakwenze et al., 2010; Noyes et al., 2012; Piper et al., 2011). In-vitro studies have confirmed that these intra-articular local anesthetic injections, in particular bupivacaine and lidocaine, are cytotoxic to chondrocytes (Braun et al., 2011; Chu et al., 200; Dragoo et al., 2010; 2012; Lo et al., 2009b; Miyazaki et al., 2011; Piper and Kim, 2008). These studies examined various cell sources, formulations, combinations with other drugs, and dose responses to better characterize the toxicity of local anesthetics to chondrocytes (Braun et al., 2011; Chu et al., 2006; Dragoo et al., 2010, 2012; Lo et al., 2009b; Miyazaki et al., 2011; Piper and Kim, 2008). However, they do not adequately propose ways of mitigating the adverse effects caused by intra-articular local anesthetic injections. Although the exact mechanism has not yet been described, it has been proposed that local anesthetics lead to cell death by disrupting mitochondrial function in chondrocytes (Grishko et al., 2010). Therefore, one possible way of mitigating the cytotoxic effects of local anesthetics would be to reduce cellular metabolism. Moderate hypothermia has been used clinically for neuronal or myocardial protection following acute spinal trauma, surgical ischemia, and cardiogenic shock (Dietrich et al., 2011; Schmidt-Schweda et al., 2013; Zierer et al., 2012). In-vitro cell culture experiments and in-vivo small animal models have confirmed the benefit of therapeutic hypothermia to improving histological and functional outcomes in these injury models (Herlambang et al., 2011; Li et al., 2012; Lo et al., 2009a). Kocaoglu et al. demonstrated that cartilage explants submerged in low temperature saline reduced cellular metabolism (Kocaoglu et al., 2011). Active cooling to the knee joint has shown a reduction in intra-articular temperatures to 20-25°C after 1 hour (Warren et al., 2004). The purpose of this study was to determine whether reducing the environmental temperature during treatment would reduce local anesthetic cytotoxicity in bovine articular chondrocytes. The intent of our study is to model active cooling of the joint following intra-articular injections with local anesthetics, such as those performed in clinic or post-operatively, and measures the viability of chondrocytes following such a procedure. We hypothesize that reduction of environmental temperature to 23°C and 4°C, inducing hypothermia, during treatment with local anesthetics would decrease chondrocyte death when compared to chondrocytes treated under normal physiological temperatures (37°C).