Local anesthetics are frequently delivered into joints to provide postoperative analgesia. The intra-articular delivery of local anesthetics after arthroscopy through single injections or through continuously acting pain pumps and indwelling catheters has been shown to substantially minimize postoperative pain and maintain joint motion1-4. Such use of local anesthetics is a common practice worldwide. In one survey of all orthopaedic units associated with the Swedish Arthroscopic Society, all but one of thirty-seven practices used intra-articular local anesthesia for postoperative pain relief following knee arthroscopy5.
Despite their efficacy in pain control, local anesthetics may also have detrimental effects. Numerous studies have linked their use to the development of chondrolysis, a complication characterized by the rapid destruction of articular cartilage6-8. Notably, shoulder arthroscopy patients diagnosed with rapid onset of glenohumeral chondrolysis routinely have a history of intra-articular infusion of bupivacaine or lidocaine8. Consistent with these clinical findings, numerous in vivo and in vitro studies have shown that common local anesthetics such as bupivacaine, ropivacaine, and lidocaine can be toxic to chondrocytes. Such toxicity depends on the specific anesthetic, duration of exposure, and dose administered9-13. For example, 0.5% ropivacaine induced less cell death after a thirty-minute exposure in vitro compared with 0.5% bupivacaine in both isolated human articular chondrocytes and intact articular cartilage13.
Although the chondrotoxicity of local anesthetics has been well studied, the effects of these drugs on reparative cells within a joint are unknown. Many orthopaedic procedures, including tendon repair, ligament reconstruction, and surgical marrow stimulation procedures such as microfracture, depend on patient healing responses. Mesenchymal stem cells likely play a key role in these processes. Under appropriate conditions, these cells can form bone, fat, tendon, and cartilage14,15. These regenerative cells are found in multiple tissues, including bone marrow, adipose, and synovium14. In addition to the healing resulting from the action of endogenous mesenchymal stem cells, surgical administration of exogenous mesenchymal stem cells may also enhance tissue regeneration16.
We hypothesized that the toxicity of local anesthetics on chondrocytes would also extend to mesenchymal stem cells. If so, local anesthetics could both directly injure tissues within a joint and decrease the ability of tissues to heal. In this in vitro study, we evaluated the effects of commonly used doses of lidocaine, ropivacaine, and bupivacaine on the viability of mesenchymal stem cells from three healthy donors. Our goal was to identify treatments that minimized toxicity.
Mesenchymal Stem Cell Monolayer Culture
Commercially available bone-marrow-derived human mesenchymal stem cells (Lonza, Walkersville, Maryland) from a twenty-year-old man (Lot 8F354), a thirty-year-old man (Lot 0F4452), and a twenty-two-year-old woman (Lot 0F3825) were plated in monolayer culture in high-glucose DMEM (Dulbecco’s modification of Eagle’s medium) (Mediatech, Manassas, Virginia) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and 1% antibiotic/antimycotic solution (Hyclone) on tissue-culture-treated polystyrene at a density of 5000 cells/cm2.
The cells were grown at 37°C in a humidified 5% CO2 incubator until 90% confluent, were trypsinized, and were expanded until the fifth or sixth passage. They were then seeded onto ninety-six-well plates at a density of 2500 cells/well (8000 cells/cm2) and cultured for forty-eight hours, leading to 90% confluence prior to experimental treatment.
Treatment Groups
Mesenchymal stem cell cultures were subdivided into six replicate wells per condition and exposed to one of the following drug dilutions: (1) 2% lidocaine, pH 6 (APP Pharmaceuticals, Schaumburg, Illinois), (2) 1% lidocaine, pH 6, (3) 0.5% bupivacaine, pH 6 (Hospira, Lake Forest, Illinois), (4) 0.25% bupivacaine, pH 6, (5) 0.5% ropivacaine, pH 5 (APP Pharmaceuticals), and (6) 0.2% ropivacaine, pH 5. The higher concentration of each anesthetic was used as provided by the manufacturer. The supplied solutions were preservative-free, with sodium chloride (to adjust osmolarity) and sodium hydroxide or hydrochloric acid (to adjust pH) added by the manufacturer. Control groups were treated with 0.9% saline solution (APP Pharmaceuticals), which was also used to prepare the lower concentration of lidocaine and of bupivacaine from the higher concentration. The 0.2% ropivacaine solution was used as provided by the manufacturer or diluted from 0.5% ropivacaine with 0.9% saline solution.
The cell culture media was aspirated, and 200 μL of the appropriate treatment solution was added to each well. Cells were incubated in the treatment solution for sixty minutes at 37°C in a humidified 5% CO2 incubator, washed in 1× phosphate-buffered saline (PBS) solution, and returned to the incubator in fresh culture media. Mesenchymal stem cell viability was measured twenty-four hours later.
Assessment of Viability
Live Cell Counts
The cell culture media was gently aspirated from each well, and viable mesenchymal stem cells were stained with a LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies, Grand Island, New York). Briefly, 100 μL of a 1:2000 dilution of calcein AM (acetomethoxy) in PBS was added to each well. Cells were incubated for forty-five minutes at room temperature and then visualized with use of an Axiovert 200M fluorescent microscope (Carl Zeiss Microscopy, Thornwood, New York) with a fluorescein filter. Digital photographs of the center of each well were taken at 5× magnification. The cell viability in each field was quantified by superimposing a grid onto the digital image in Photoshop (Adobe Systems, San Jose, California), and calcein-stained live cells, which exhibit green fluorescence, were counted. The number of dead cells was also quantified in a subset of the experiments by staining with ethidium homodimer-1 and visualization with use of a rhodamine filter. Ethidium-stained dead cells and calcein-stained live cells were counted, and the percentage of live cells for each treatment was calculated.
ATP (Adenosine Triphosphate) Content
The ATP content in treated samples was measured with use of the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wisconsin) according to the manufacturer’s instructions. Briefly, the CellTiter-Glo buffer and the CellTiter-Glo substrate were combined to form the CellTiter-Glo reagent. The reagent was added to the culture media in each well in a 1:1 ratio, mixed well, and incubated for ten minutes at room temperature. Luminescence was then measured with use of a Wallac 1420 VICTOR2 Multilabel Counter (PerkinElmer, Waltham, Massachusetts).
Statistical Analysis
Live cell counts from the three independent trials, calculated as a percentage of the control, were analyzed together with use of mixed-model analysis of variance (ANOVA), with the specified drug concentration as the fixed factor and trial as a random factor. Live cell count results are reported for each drug concentration as the least-squares mean (the calculated best-fit value across all trials) and the standard error. ATP content results are reported for each of the three individual donors as the mean luminescence (the CellTiter-Glo output) and the standard error. Differences in ATP content among treatment groups were assessed for each donor with use of ANOVA. Results for the three donors were combined with use of mixed-model ANOVA, with the specified drug concentration as the fixed factor and donor as a random factor. ATP content results are reported as the least-squares mean luminescence and the standard error. Because multiple comparisons were made, the significance of differences among treatments was calculated with use of the Tukey honestly significant difference test; a p value of <0.05 was considered significant. Statistical analysis was performed with use of JMP (version 9.0; SAS Institute, Cary, North Carolina).
Source of Funding
This work was performed with funding from the Department of Orthopaedic Surgery of the University of California San Francisco and with resources of the Veterans Affairs Medical Center, San Francisco, California. There was no external funding source.
Comparison of Cell Viability Measured with the Live Cell Count, Percentage of Live Cells, and ATP Content
In a pilot experiment, mesenchymal stem cells from one of the donors (the twenty-year-old male) were exposed to each of the six anesthetic conditions, and viability was determined with each of three methods: (1) live cell count, (2) percentage of live cells, and (3) ATP content. Six replicates were performed for each of the three evaluation methods for each condition. Linear regression yielded an r2 value of 0.84 between the live cell count and the percentage of live cells and an r2 value of 0.79 between the live cell count and the ATP content. Given the strength of these correlations, we evaluated viability with use of the live cell count and the ATP content for this donor and with use of only the ATP content for the other two donors.
Live Cell Counts
The number of live cells remaining after treatment with local anesthetic was determined with use of mesenchymal stem cells from the twenty-year-old male donor. Treatment with 0.25% and 0.5% bupivacaine in three independent trials reduced the live cell count to 94% and 79%, respectively, of the value in the control group. Treatment with 0.2% and 0.5% ropivacaine reduced the live cell count to 83% and 84%, respectively, of the value in the control group. No difference among these four groups was significant. Treatment with both concentrations of lidocaine, however, resulted in a significant reduction in the live cell count compared with all other groups. Treatment with 1% lidocaine reduced the live cell count to 49% of the value in the control group (p < 0.0001), and 2% lidocaine reduced it to 32% (p < 0.0001) (Figs. 1 and 2).
ATP Content
As an alternative, independent measure of cell viability, cellular ATP content after local anesthetic treatment was determined with use of mesenchymal stem cells from each of the three donors (the twenty-year-old man, the twenty-two-year-old woman, and the thirty-year-old man). The results for each donor as well as the results of a model combining information from all three donors are shown in Figure 3. In all four cases, 2% lidocaine treatment significantly reduced viability compared with treatment with either ropivacaine or bupivacaine. In the combined model, the ATP content in the 2% lidocaine group was only 49% of that in the 0.2% ropivacaine group. For the twenty-year-old donor, the ATP content in the 1% and 2% lidocaine groups was only 56% and 39%, respectively, of that in the 0.2% ropivacaine group (p < 0.05). However, the difference between the 1% lidocaine group and the other treatment groups was not significant for the other two donors or for the combined model. No significant differences were found between the ropivacaine and bupivacaine groups.
The chondrotoxicity of local anesthetics including bupivacaine, ropivacaine, and lidocaine in both human and animal models has been widely reported. However, to our knowledge, the effects of these drugs on human mesenchymal stem cells have not been previously addressed. Given the likely role of these cells in tissue healing following orthopaedic procedures, the toxicity of local anesthetics on these cells may be clinically important14. The present study indicated that bupivacaine and ropivacaine at the commonly used 0.5% concentration were significantly less toxic than a concentration of lidocaine with equivalent anesthetic potency. These in vitro results suggest that intra-articular administration of lidocaine may have detrimental effects on mesenchymal stem cell populations.
The relative toxicities of bupivacaine, ropivacaine, and lidocaine on mesenchymal stem cells differed from their toxicities on human chondrocytes. Previous in vitro and in vivo studies have shown that bupivacaine is more toxic to cartilage cells than ropivacaine and lidocaine11,13,17. Specifically, Piper and Kim directly compared the toxicity of 0.5% ropivacaine with that of 0.5% bupivacaine and found that although ropivacaine did induce cell death in human chondrocytes, it was significantly less chondrotoxic than bupivacaine13. The observed cytotoxicity differences between chondrocyte and mesenchymal stem cell populations suggest a differential effect of local anesthetics on different cell types.
The inherent differences in physical and chemical properties among these drugs likely contribute to the differences in their relative toxicities on different cell types. Bupivacaine and ropivacaine are highly lipophilic molecules, whereas lidocaine is only slightly lipophilic18,19. Treatment of human leukemia cells with these anesthetics induces different patterns of cell death. Bupivacaine and ropivacaine predominantly cause necrosis, whereas lidocaine induces DNA fragmentation and apoptosis (programmed cell death)19. Studies of human T-cell lymphoma and neuroblastoma cell lines showed that lidocaine treatment leads to apoptosis but ropivacaine does not. These differences may be due to differential activation of caspases, which are proteolytic enzymes that play key roles in apoptosis20,21. The three anesthetics may also differentially affect inflammation. Bupivacaine induces nitric oxide synthase-2 activity in rat glial cells and astrocytes but ropivacaine does not22. This pathway is normally activated as part of an inflammatory response, suggesting that bupivacaine toxicity may be attributable in part to the production of nitric oxide during ongoing inflammation. In contrast, ropivacaine has anti-inflammatory properties13.
The relative potencies of lidocaine, bupivacaine, and ropivacaine were accounted for in our comparisons among the drugs. Bupivacaine and ropivacaine are highly potent anesthetics with long-lasting effects, whereas lidocaine is a moderately potent anesthetic with shorter effects23. Specifically, bupivacaine and ropivacaine are approximately four times more potent anesthetics than lidocaine24. Therefore, the effects of 0.5% bupivacaine and 0.5% ropivacaine on pain should be similar to that of 2% lidocaine, but with a longer duration of action and less toxicity on mesenchymal stem cells. Although the CellTiter-Glo assay for ATP content was slightly less sensitive to differences among conditions than the live cell count, similar trends were found with both methods of analysis.
One limitation of the present study is the fact that the experiments were performed in vitro. Further experiments are needed to assess the in vivo effects of these drugs on mesenchymal stem cell viability. However, the experimental conditions in the present study (growth of cells in tissue culture to yield a monolayer in the fifth or sixth passage) are commonly used in studies of mesenchymal stem cells25,26, and such cells are defined in part by their ability to adhere, proliferate, and differentiate into mesenchymal tissues under such in vitro conditions27. Another limitation is the fact that viability measurements were only performed after sixty minutes of anesthetic treatment. This duration of exposure was chosen on the basis of a previous study of the pharmacokinetics of bupivacaine after intra-articular injection into the knee28. The absorption of this anesthetic from the joint into the bloodstream is rapid, with the peak blood concentration being reached within the first hour after injection. We are not aware of similar data for lidocaine or ropivacaine. We did not test for time dependence or for potential early-acting toxicity in the drug treatments. The effect of local anesthetic treatment on the differentiation potential of mesenchymal stem cells is also of interest in future experiments. Although cells treated with bupivacaine and ropivacaine in the present study appeared to be metabolically and morphologically normal, the drugs may subtly affect differentiation patterns. To study these effects, mesenchymal stem cells treated with varying classes and doses of local anesthetic can be assayed for the ability to form bone, cartilage, and fat.
In conclusion, in contrast to the toxicity observed in human and bovine chondrocytes, commonly used concentrations of ropivacaine and bupivacaine appeared to have limited toxicity on human mesenchymal stem cells. In contrast, lidocaine significantly decreased mesenchymal stem cell viability. Although these experiments are limited by their in vitro nature, these data suggest that ropivacaine and bupivacaine may represent better options with respect to mesenchymal stem cell health. Taking into further account the chondrotoxicity of bupivacaine and the chondrolysis associated with postoperative bupivacaine infusions, ropivacaine may be the safest of these three choices for intra-articular analgesia.
Disclosure: None of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of any aspect of this work. None of the authors, or their institution(s), have had any financial relationship, in the thirty-six months prior to submission of this work, with any entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. Also, no author has had any other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.