Intraarticular injections of local anesthetics, alone or in combination with corticosteroids or epinephrine, are approved and regularly used in perioperative settings for diagnostic and therapeutic purposes.1 However, both in vitro and in vivo studies have indicated cytotoxic effects of local anesthetic drugs on articular chondrocytes.2–6
As chondrocyte loss has been implicated in the development of osteoarthritis,7 the extent of cytotoxicity of local anesthetics on chondrocytes is crucial to determine. However, the basic mechanisms of chondrotoxicity are not well elucidated. Recent reports of postarthroscopic chondrolysis of the knee8 and glenohumeral joint9 have received considerable attention. In most of the patients, postoperative pain pumps generated a prolonged exposure to concentrated local anesthetics, which may have the potential for chondrotoxicity. The effects of a single bolus injection, e.g., 20 mL of 0.25% bupivacaine,10 remain unclear.
The purpose of this study was to assess the chondrotoxic effects of mepivacaine, ropivacaine, and bupivacaine. We hypothesized that specific cytotoxic potencies directly correlate with analgesic potencies, and that cytotoxic effects in intact cartilage are different than in osteoarthritic tissue.
Cell Isolation and Culture Conditions
Ethical standards of the studies were approved by the IRB. The requirement for written informed consent was waived by the IRB. Two dimensional experiments were performed with articular cartilage harvested from 4 patients (age range 42–62 years) undergoing total joint arthroplasty. The samples were dissected into small pieces (0.2–0.5 mm3), washed in Dulbecco phosphate buffered saline (Cambrex, Oberhaching, Germany) and digested in Roswell Park Memorial Institute 1640 medium (Gibco Invitrogen, Karlsruhe, Germany) containing penicillin/streptomycin (1%; Gibco Invitrogen), collagenase P (16.6%; Boehringer, Mannheim, Germany), DNase IIB (16.6%; Sigma, Taufkirchen, Germany), and hyaluronidase (16.6%; Sigma). Digestion was performed at 37°C for 17 hours. Cells were filtered, centrifuged (2000g, 5 minutes), and plated in 75 cm2 culture flasks with Roswell Park Memorial Institute 1640 medium (Gibco Invitrogen) supplemented with heat-inactivated fetal calf serum (10%; PAN, PAN Biotech, Aidenbach, Germany), penicillin/streptomycin (1%), glutamine (1%; Gibco Invitrogen), and HEPES (1%; Gibco Invitrogen) at a 37°C humidified 5% CO2 atmosphere. Chondrocytes from the patients were grown independently and split once (first passage cells). Cells from each patient were subjected to all experiments and transferred to 24-well plates (100,000 cells per well) 48 hours before experimental treatment.
We investigated the effects of pipecoloxylidide local anesthetics mepivacaine, ropivacaine, and bupivacaine in clinically relevant concentrations. Based on their chemical structure, lipophilicity, and thus, analgesic potency increase from mepivacaine to ropivacaine to bupivacaine. Postulating a potency ratio of 1:0.67:0.25, bupivacaine 0.5% is equipotent to ropivacaine 0.75% and mepivacaine 2%.11–15 Thus, possible correlation between specific cytotoxic effects and analgesic potencies were assessed.
Analysis of Chondrocyte Viability, Apoptosis, and Necrosis Using Flow Cytometry
First passage cells were used to prevent dedifferentiation. Chondrocytes were exposed for 1 hour to 1 mL of local anesthetic solutions with the following concentrations and total amounts: 0.03125% (total amount: 0.3125 mg), 0.0625% (0.625 mg), 0.125% (1.25 mg), 0.25% (2.5 mg), and 0.5% (5 mg) of bupivacaine (Sigma); 0.03125% (0.3125 mg), 0.0625% (0.625 mg), 0.125% (1.25 mg), 0.25% (2.5 mg), 0.5% (5 mg), and 0.75% (7.5 mg) of ropivacaine (Fagron, Barsbuettel, Germany); 0.03125% (0.3125 mg), 0.0625% (0.625 mg), 0.125% (1.25 mg), 0.25% (2.5 mg), 0.5% (5 mg), 1% (10 mg), and 2% (20 mg) of mepivacaine (Sigma). Different concentrations of local anesthetics were applied to different cells. All drugs were preservative-free and dissolved in buffered saline solution (pH 7.0). Control cells were exposed to buffered saline for 1 hour. Treatment solutions containing nonadherent chondrocytes were removed and centrifuged. Cell pellets were washed in buffered saline and returned to the corresponding wells with culture medium. After 24 and 96 hours, cell viability was determined using flow cytometry. Viability rates were analyzed 24 hours, 96 hours, and 7 days after a 1-hour exposure to saline solutions with pH of 7.4, 7.0, 6.0, 5.0, and 4.0 to examine the influence of pH on chondrocytes.
The Annexin-V-Fluos Staining Kit (Roche, Mannheim, Germany) was used to identify apoptotic, necrotic, and viable cells after local anesthetic treatment. Annexin V shows high specificity for phosphatidylserine exposed on the external surface of apoptotic cells.16,17 As necrotic cells also express phosphatidylserine due to the loss of membrane integrity, annexin-V staining is not able to distinguish between apoptotic and necrotic cells. Propidium iodide solely stains the DNA of leaky necrotic cells and is used for differentiation.
After exposure to local anesthetics, chondrocytes were trypsinized and collected by centrifugation. Cell culture media from each well was added to the resulting cell suspension from the same well not to lose nonadherent cells. Cells were washed in Dulbecco phosphate buffered saline and incubated with fluorescein-labeled annexin V and propidium iodide staining solution for 15 minutes at 15°C to 20°C. Quantitative analysis of 10,000 cells per treatment group was performed on a flow cytometry machine (FACSCalibur, BD Biosciences, Baltimore, MD) and the percentage of viable, apoptotic, and necrotic cells was calculated with the analysis software FloJo (Tree Star, Ashland, OR).
Analysis of Chondrocyte Viability with Live–Dead Staining
Chondrocytes of 4 patients were treated with 1 mL of bupivacaine 0.5% (5 mg), ropivacaine 0.75% (7.5 mg), mepivacaine 2% (20 mg), or buffered saline for 1 hour. Chondrocytes were also exposed to saline solutions of pH 7.0 and 5.0 for 1 hour. Nonadherent cells in the removed treatment solutions were washed and returned. Cell viability was analyzed with a calcein AM (Sigma) and ethidium homodimer-1 (Sigma) staining 1 hour, 24 hours, 4, and 7 days after exposure. Intracellular esterase activity in living cells converts the membrane-permeable calcein AM to the green-fluorescent calcein. Ethidium homodimer-1 enters cells with damaged membranes and produces a red fluorescence on binding to nucleic acids in dead cells. Cells were stained with 4-µM calcein AM and 2-µM ethidium homodimer-1 for 2 hours. Cells were imaged by fluorescence microscopy and scored for cell viability.
Analysis of Cell Morphology and Caspase Activity
Apoptosis is mediated by a cascade of aspartate-specific cysteine proteases or caspases.18–20 The CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen, Carlsbad, CA) was applied to distinguish viable and apoptotic cells. After activation of caspase-3 or caspase-7 in apoptotic cells, the membrane-permeable substrate is cleaved and enabled to bind to DNA to exhibit a green fluorescence signal. Chondrocytes of 4 patients were exposed to 1 mL of bupivacaine 0.5% (5 mg), ropivacaine 0.75% (7.5 mg), mepivacaine 2% (20 mg), or normal saline for 1 hour. Nonadherent cells in the removed treatment solutions were returned, and chondrocytes were cultured with medium for 24 or 48 hours. CellEvent Caspase-3/7 Green Detection Reagent was added at a final concentration of 1 µM and incubated for 1 hour at 37°C. Cells were imaged by fluorescence microscopy.
Analysis of Chondrocyte Viability in Human Articular Cartilage
Osteochondral cores were harvested from the femoral articular cartilage of 2 patients undergoing total joint arthroplasty. Five cores were taken from intact and 5 from osteoarthritic parts of each donor. Intact cores were characterized by a macroscopically smooth and glistening articular surface and a histologically undamaged cartilage structure. Osteoarthritic cores exhibited superficial fibrillation with small erosions and revealed intermittent hypercellularity and vertical clefts into the transitional zone in histologic imaging. Intact and osteoarthritic cores were submerged in 5 mL of bupivacaine 0.5%, ropivacaine 0.75%, mepivacaine 2%, or saline solution for 1 hour. They were subsequently washed and cultured with chondrocyte growth medium for 24 or 96 hours. Cartilage cores were stained with 4-µM calcein AM and 2-µM ethidium homodimer-1 for 24 hours. Cores were fixed in 4% phosphate buffered paraformaldehyde (Roth, Karlsruhe, Germany) for 1 hour, infiltrated with increasing concentrations (10%–30%) of sucrose and finally embedded in Tissue-Tec (Sakura, Zoeterwoude, the Netherlands). Disks were cryosectioned at 18 µm using a HM 500 OM cryotome (Microm, Berlin, Germany). Sections were imaged by fluorescence microscopy to analyze chondrocyte viability.
Mean values and standard deviation (mean ± SD) were calculated for the outcome variables in viability assessments using flow cytometry and live–dead staining. Cells of 4 patients were used in all experimental groups (n = 4). Analysis of variance (ANOVA) with Dunnett T post hoc testing was used to assess chondrotoxicity of varying concentrations of 1 anesthetic drug against control. ANOVA with Tukey Honestly Significant Difference post hoc testing were used for comparison of equipotent concentrations of local anesthetics using SPSS for Windows 19.0 (SPSS Inc., Chicago, IL). Statistical significance was assumed as P < 0.01. The results of caspase activity detection and viability assessments in articular cartilage are given in a qualitative form.
There was no difference in chondrocyte viability after a 1-hour exposure to saline solutions with pH of 7.4, 7.0, 6.0, 5.0, and 4.0 through 1 week after treatment (for all observation time points: P > 0.01; ANOVA; Fig. 1).
Bupivacaine, ropivacaine, and mepivacaine showed time-dependent and concentration-dependent cytotoxic effects on human chondrocytes (Fig. 2). Twenty-four and 96 hours after treatment, bupivacaine exposure resulted in a decreasing number of viable cells (P = 0.0004 and P < 0.0001; ANOVA) and increased the fraction of apoptotic (P < 0.0001 and P < 0.0001; ANOVA) and necrotic cells (P = 0.0001 and P < 0.0001; ANOVA). Pairwise comparisons with control demonstrated that bupivacaine 0.5% caused a decline in viability to 63% ± 8% after 24 hours (P < 0.0001; Dunnett) and 26% ± 9% after 96 hours (P < 0.0001; Dunnett). The fraction of apoptotic cells increased to 13% ± 4% after 24 hours (P < 0.0001; Dunnett) and 38% ± 6% after 96 hours (P < 0.0001; Dunnett). After 24 hours, 25% ± 7% of chondrocytes were necrotic (P < 0.0001; Dunnett) and 36% ± 7% after 96 hours (P < 0.0001; Dunnett). Viability assessments after 24 and 96 hours were not different in chondrocytes treated with up to 0.25% bupivacaine as compared with the control group.
Chondrocyte viability was not significantly reduced 24 hours after treatment with ropivacaine (P > 0.01; ANOVA). Ninety-six hours later, a decrease in viability (P = 0.0067; ANOVA) and an increase in necrosis (P = 0.0015; ANOVA) were detected compared with control. After exposure to ropivacaine 0.75%, viability was 76% ± 18% (P = 0.0084; Dunnett). The fraction of necrotic cells was increased to 13% ± 7% (P = 0.0020; Dunnett). Concentrations <0.75% did not reduce viability.
Compared with control, mepivacaine reduced viable cells (P < 0.0001; ANOVA) and enlarged the number of apoptotic (P < 0.0001; ANOVA) and necrotic chondrocytes (P < 0.0001; ANOVA) after 24 hours. Viability decreased to 63% ± 7% (P< 0.0001; Dunnett) after exposure to mepivacaine 2% with 9% ± 3% (P = 0.0001; Dunnett) of apoptotic and 29% ± 6% (P < 0.0001; Dunnett) of necrotic chondrocytes. Mepivacaine 1% reduced viability to 69% ± 10% (P= 0.0006; Dunnett) with a necrotic fraction of 22% ± 9% (P= 0.0050; Dunnett). Lower concentration of mepivacaine did not influence the distribution of viable, apoptotic, and necrotic cells. Viability assessments showed an increase of apoptotic cells in the mepivacaine 2% treatment group after 96 hours (P = 0.0038; Dunnett).
Equipotent concentrations of local anesthetics caused differences in chondrocyte viability (P = 0.0045; ANOVA) and necrosis (P = 0.0064; ANOVA) after 24 hours. Viability rates were higher after ropivacaine 0.75% treatment compared with bupivacaine 0.5% (P = 0.0087; Tukey) and mepivacaine 2% (P = 0.0078; Tukey). Ropivacaine caused less necrosis than mepivacaine (P = 0.0060; Tukey). Viability assessment between the bupivacaine and mepivacaine groups did not differ statistically. Comparing all treatment groups 96 hours later, differences were detected in viability (P = 0.0003; ANOVA), apoptosis (P = 0.0006; ANOVA), and necrosis (P= 0.0006; ANOVA). Bupivacaine 0.5% reduced chondrocyte viability compared with ropivacaine 0.75% (P = 0.0011; Tukey) and mepivacaine 2% (P = 0.0004; Tukey). The rate of apoptotic and necrotic cells was higher than in the ropivacaine (P = 0.0021 and P = 0.0009; Tukey) and mepivacaine group (P = 0.0027 and P = 0.0009; Tukey). Viability assessment between the ropivacaine and mepivacaine groups did not show significant differences.
While the decrease in viability was primarily caused by necrosis after 24 hours, the number of apoptotic cells increased after 96 hours in all groups.
Chondrocyte viability was assessed in monolayer cultures using a live–dead staining and compared with saline control (Figs. 3 and 4). After exposure to local anesthetics, chondrocytes lost adherence to the culture disk and cell morphology changed from a longish and branched fibroblastic appearance to a rounded form. The number of dead cells and cell detritus increased as a time-dependent and substance-dependent process, where chondrotoxicity increased from ropivacaine to mepivacaine to bupivacaine in an ascending order (Fig. 3). Chondrotoxicity of bupivacaine was delayed by several hours compared with the ropivacaine and mepivacaine groups. Cell proliferation was observed in surviving cells after 4 and 7 days (data not shown).
Compared with control, chondrocyte viability was decreased by local anesthetics after 1 hour and 24 hours (P < 0.0001 and P < 0.0001; ANOVA). After exposure to bupivacaine 0.5%, viability rates were reduced to 78% ± 9% after 1 hour (P = 0.0183; Dunnett) and to 16% after 24 hours (P < 0.0001; Dunnett). Ropivacaine 0.75% reduced viable cells to 80% ± 7% (P = 0.0475; Dunnett) after 1 hour and to 80% ± 10% (P = 0.0095; Dunnett) after 24 hours. Decreases of viability to 36% ± 6% (P < 0.0001; Dunnett) after 1 hour and to 30% ± 11% (P < 0.0001; Dunnett) after 24 hours were found in the mepivacaine group (Fig. 4).
Equipotent concentrations of anesthetic substances showed differences in cytotoxicity after 1 hour (P < 0.0001; ANOVA) and 24 hours (P = 0.0007; ANOVA). Death rates were increased 1 hour after mepivacaine exposure compared with bupivacaine (P < 0.0001; Tukey) and ropivacaine treatment (P < 0.0001; Tukey). After 24 hours, chondrocyte viability in the ropivacaine group was higher than after bupivacaine (P = 0.0006; Tukey) and mepivacaine exposure (P = 0.0059; Tukey).
Caspase activity was determined to distinguish whether apoptosis or necrosis was responsible for chondrotoxicity of local anesthetics (Fig. 5). Cells exposed to bupivacaine 0.5% exhibited extensive chondrocyte apoptosis after 24 hours (Fig. 5, A and E). Cells were nonadherent, rounded without cytoplasmatic branched projections, and nuclei fluorescenced green. Cell fragments increased during the observation period of 48 hours (data not shown). In contrast, viable chondrocytes after saline solution treatment stayed adherent to the culture disk and appeared longish with branched projections forming cell–cell contacts. No fluorescent nuclei or cell fragments could be detected (Fig. 5, D and H). As compared with the bupivacaine group, the fraction of apoptotic cells was distinctly lower after treatment with ropivacaine 0.75% (Fig. 5, B and F). Chondrocytes treated with mepivacaine 2% expressed apoptotic features more frequently than in the ropivacaine, but less often than in the bupivacaine group (Fig. 5, C and G).
Correlating the effects between the experiments for the same patient, similar courses of the dose–response curves were found for all samples. Local anesthetic chondrotoxicity did not differ between individuals in this study. Observing the effects of local anesthetic treatment as a composite of results of the separate experiments, chondrotoxicity was dose-dependent for all treatment drugs and increased substance specifically from ropivacaine to mepivacaine to bupivacaine. Time dependency showed in a postponed onset of cytotoxicity after bupivacaine treatment. Immediate cell death was mainly due to necrosis followed by apoptosis afterward.
Osteochondral cores from macroscopically intact and osteoarthritic human cartilage were analyzed for chondrotoxicity of local anesthetic treatment (Figs. 6 and 7). As compared with saline control (Fig. 6D), intact and osteoarthritic cartilage exposed to bupivacaine 0.5% for 1 hour exhibited extensive chondrocyte necrosis after 24 hours. Cell damage was worse in osteoarthritic compared with intact cartilage (Fig. 6, A and E). The number of observable viable cells in intact osteochondral cores after treatment with ropivacaine 0.75% was similar to control (Fig. 6B). Ropivacaine treatment of osteoarthritic cartilage resulted in an increased chondrocyte death rate in the top 25 to 50 µm (Fig. 6F). Exposure to mepivacaine 2% did not increase chondrocyte death rate in intact osteochondral cores (Fig. 6C). In contrast, the same concentration of mepivacaine distinctly decreased chondrocyte viability in osteoarthritic cartilage. Analysis showed predominantly dead cells within the superficial 100 to 200 µm after 24 hours (Fig. 6G).
Four days after treatment, cores of intact cartilage were macroscopically and histologically comparable with probes analyzed after 24 hours. No differences regarding death rates or cellular density were visible (data not shown). In contrast, osteoarthritic cartilage revealed signs of increased cell death (Fig. 7, E–H). Cartilage treated with ropivacaine and mepivacaine showed occasional necrotic cells along the surface layer as well as hypocellularity and empty lacunae (Fig. 7, F and G). Bupivacaine exposure resulted in substantial chondrocyte necrosis at the surface and subsurface with the near absence of viable cells on fluorescence imaging. Cell-free zones were readily apparent as evidenced by empty lacunae (Fig. 7E).
The important findings of this study are that chondrotoxic effects increased in a concentration-dependent and drug-dependent manner from ropivacaine to mepivacaine to bupivacaine in cell cultures. Contrary to our hypothesis, chondrotoxicity did not directly correlate with anesthetic potency. Cellular death rates were higher in osteoarthritic compared with intact cartilage after local anesthetic treatment. Immediate cell death was mainly due to necrosis followed by apoptosis.
Chondrocyte death is associated with cartilage degradation and osteoarthritis with a close correlation between the frequency of chondrocyte apoptosis and severity of osteoarthritic changes.21,22 Because articular cartilage does not contain tissue macrophages, necrotic and apoptotic remnants cannot be removed and remain to cause further tissue damage.20,22 Therefore, any cytotoxic procedures and drugs possibly promoting the onset and deterioration of osteoarthritis should be avoided.
Several investigations report deleterious chondrotoxic effects of local anesthetics in clinical and experimental settings, especially after long-term exposition. Ropivacaine was found to be less chondrotoxic than bupivacaine in human chondrocytes;23,24 mepivacaine was less toxic than bupivacaine in equine chondrocytes.25 Immediate cell death was mainly due to necrosis whereas the number of apoptotic cells increased after several days.3,5,24 Reported data of chondrocyte cultures2,3 or animal models2–4,26 may not exactly be transferable to human tissue.
In our experimental approach, we tried to further improve the methodologic approach of previous examinations. Because chondrocyte apoptosis and necrosis are time-dependent, viability needs to be assessed at different time points; otherwise, toxic effects may be missed.6,23,25
Cellular adherence to culture disks can be impaired by local anesthetic exposure. Chondrocytes could be discarded during the replacement of treatment solution.5,6,23 Therefore, we washed and returned nonadherent cells.
Flow cytometry is commonly used for chondrocyte viability assessments.2,3,5,24,25 Cell preparation can cause membrane damage. Cell detritus detected during flow cytometry likely indicated the loss of damaged cells. Viability could thus be overestimated. To address this limitation, we analyzed viability rates and apoptosis markers with cell-staining methods. Our results indicated a reduced proportion of viable cells compared with flow cytometry.
Buffering of bupivacaine solutions increased chondrocyte death,6 indicating a higher chondrotoxicity of the free base. The use of the soluble form and the exclusion of chondrocidal effects of an acidic pH may prohibit possible interferences in this work.
Pharmacokinetics and clearance rates of intraarticular anesthetics are not fully understood. Bupivacaine absorption in large joints occurs within the first hour.27,28 Clearance may be accelerated perioperatively by lavage fluid, effusion, and inflammatory reactions. Therefore, local anesthetic treatment for 1 hour should reflect a clinically relevant exposure time.
Although cytotoxicity of local anesthetics in chondrocyte cultures is unchallenged, in vivo effects remain controversial. Bupivacaine caused substantial chondrocyte necrosis despite a normal histologic appearance in rats.4 Subsurface pathologic changes are not detectable macroscopically, indicating that toxic effects of local anesthetics would not be evident on clinical inspection and would take several years to develop. Fewer chondrocytes to repair and maintain the matrix would reduce tolerance to mechanical loading and injury.29
Our data suggest more severe chondrocyte damage in osteoarthritic compared with intact cartilage, which may be of clinical relevance because local anesthetics are mainly used for pain management in osteoarthritic, injured, or surgically manipulated joints. Chondrotoxicity of bupivacaine increased in bovine cartilage with the superficial layer removed.3 Gradual destruction of extracellular matrix in osteoarthritic cartilage30 improves diffusion capacities of intraarticular local anesthetics.31 Osteoarthritis, surgical procedures, or articular injuries cause oxidative stress compromising chondrocyte cell functions,32 additionally increasing vulnerability to local anesthetics. Cell cultures in this study consisted of a mixed population of more or less damaged chondrocytes, likely reflecting the situation in osteoarthritic cartilage.
In conclusion, our findings show that in a time-dependent and concentration-dependent manner, bupivacaine, ropivacaine, and mepivacaine are more chondrotoxic in degenerated compared with intact human cartilage. Chondrotoxicity substance specifically increases from ropivacaine to mepivacaine to bupivacaine, without clearly correlating with analgesic potency. Necrotic and apoptotic pathways contribute to chondrocyte destruction. The clinical impact of these findings still remains unclear. Additional studies involving patients having received intraarticular local anesthetic administration would be useful to further assess the clinical relevance of local anesthetic chondrotoxicity.
Name: Anita Breu, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Anita Breu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Katharina Rosenmeier.
Contribution: This author helped conduct the study.
Attestation: Katharina Rosenmeier has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Richard Kujat, PhD.
Contribution: This author helped conduct the study.
Attestation: Richard Kujat has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Peter Angele, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Peter Angele has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Wolfgang Zink, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Wolfgang Zink has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Terese T. Horlocker, MD.
We thank Daniela Drenkard and Regina Lindner for excellent technical assistance
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© 2013 International Anesthesia Research Society
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