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. 2010 Nov;162(2):280–288. doi: 10.1111/j.1365-2249.2010.04252.x

In vitro exposure of human fibroblasts to local anaesthetics impairs cell growth

C Fedder *,†,1, B Beck-Schimmer †,‡,1, J Aguirre §, M Hasler †,‡, B Roth-Z'graggen †, M Urner †,‡, S Kalberer †, A Schlicker †,‡, G Votta-Velis , J M Bonvini ‡, K Graetz *, A Borgeat §
PMCID: PMC2996595  PMID: 20819090

Abstract

Lidocaine, bupivacaine or ropivacaine are used routinely to manage perioperative pain. Sparse data exist evaluating the effects of local anaesthetics (LA) on fibroblasts, which are involved actively in wound healing. Therefore, we investigated the effects of the three LA to assess the survival, viability and proliferation rate of fibroblasts. Human fibroblasts were exposed to 0·3 mg/ml and 0·6 mg/ml of each LA for 2 days, followed by incubation with normal medium for another 1, 4 or 7 days (group 1). Alternatively, cells were incubated permanently with LA for 3, 6 or 9 days (group 2). Live cell count was assessed using trypan blue staining. Viability was measured by the tetrazolium bromide assay. Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine assay. Production of reactive oxygen species (ROS) was determined, measuring the oxidation of non-fluorescent-2,7′-dichlorofluorescin. Treatment of cells with the three LA showed a concentration-dependent decrease of live cells, mitochondrial activity and proliferation rate. Group arrangement played a significant role for cell count and proliferation, while exposure time influenced viability. Among the analysed LA, bupivacaine showed the most severe cytotoxic effects. Increased production of ROS correlated with decreased viability of fibroblasts in lidocaine- and bupivacaine-exposed cells, but not upon stimulation with ropivacaine. This study shows a concentration-dependent cytotoxic effect of lidocaine, bupivacaine and ropivacaine on fibroblasts in vitro, with more pronounced effects after continuous incubation. A possible mechanism of cell impairment could be triggered by production of ROS upon stimulation with lidocaine and bupivacaine.

Keywords: apoptosis, cytotoxicity, fibroblasts, local anaesthetics, reactive oxygen species

Introduction

Pain control with local anaesthetics is a major issue in perioperative medicine. Local anaesthetics (LA) are injected topically (such as intra-articular application) or applied through a perineural or wound catheter for pain management [1–7]. Clinically used concentrations of LA vary from 2 mg/ml to 10 mg/ml, depending upon the chosen type and duration of analgesia.

Lidocaine, bupivacaine and ropivacaine are all amide-type local anaesthetics. Recent publications have suggested potential adverse effects of these three LA on articular chondrocytes in vitro[8–10]. Moreover, studies have also shown toxic effects of local anaesthetics on tissues which are involved in postoperative recovery and wound healing, challenging the safe continuous application of local anaesthetics in clinical practice [11,12].

Wound healing after surgery is a natural process of regenerating tissue. A set of complex biochemical events takes place in a closely orchestrated cascade to repair tissue. These events overlap in time and may be categorized artificially into three steps: inflammatory, proliferative and remodelling phase. Fibroblasts play a crucial role in the proliferative phase. They migrate from normal tissue into the wound area from its margins, where they grow and form a new, provisional extracellular matrix by excreting collagen and fibronectin.

Due to the crucial role of fibroblasts in the wound healing process, we investigated the effects of different concentrations of local anaesthetics on viability and proliferation of fibroblasts. Based on previous results in an inflammatory model of acute lung injury [13], we hypothesized that local anaesthetics do not have an adverse effect on fibroblasts.

Material and methods

Fibroblasts

In this study, human osteosarcoma cells (LGC Standard GmbH, Wesel, Germany), osteoblast-like cell types with the morphology of human fibroblasts, were used. According to a study from Jukkola et al. in 1993, these cells have the characteristics of proliferative wound fibroblasts [14]. Cells were cultured in α-modified Eagle's medium (MEM; LGC Standard GmbH) with 10% fetal bovine serum (FBS; LGC Standard GmbH) and 10 000 U/l penicillin/streptomycin (LGC Standard GmbH) at 37°C and 5% CO2.

Lidocaine (Lidocain CO2 2% Sintetica®) was purchased from Sintetica AG, Mendrisio, Switzerland, bupivacaine (Bucain®) from DeltaSelect GmbH, Munich, Germany and ropivacaine (Naropin®) from AstraZeneca, Wedel, Germany.

Experimental groups

Serial dilutions were chosen with lidocaine, bupivacaine and ropivacaine resulting in concentrations of 0·3 mg/ml and 0·6 mg/ml, representing comparable tissue concentrations measured in clinical practice [15].

In group 1, cells were exposed to the LA for 2 days followed by another incubation time of 1, 4 or 7 days with normal medium without LA. In group 2, cells were exposed permanently to local anaesthetics for 3, 6 or 9 days. The LA-containing medium was changed every second day to provide stable and constant drug concentrations.

Control cells were incubated with medium only for the according period of time. All changes of medium performed in the treated group were performed similarly in control cells.

Cell count

On days 3, 6 and 9, living cells were counted manually in the Neubauer chamber, using trypan blue [16,17].

Cell viability

The tetrazolium bromide (MTT) assay is a well-known and recognized method to measure cell viability in vitro[18]. The method is based on the reduction of yellow tetrazoliumsalt 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide into purple formazan crystals by mitochondrial dehydrogenases. Dehydrogenases are active only in living cells. Conversion of MTT is therefore related directly to cell viability.

Cell proliferation

Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine (BrdU) assay (Roche, Basel, Switzerland). The test analyses the proliferation of cells by utilizing BrdU as an analogue of the DNA nucleotide thymidine, which is incorporated into the synthesized DNA of actively dividing cells. The amount of BrdU incorporation is reflected in the intensity of absorbance of the final reaction, measured with an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm (reference wavelength 620 nm) [19,20].

Fluorometric assays for determination of caspase-3 activity

Caspase-3 activity was determined by measuring proteolytic cleavage of the fluorogenic caspase-3 substrate Ac-Asp-Glu-Val-Asp-AMC (Calbiochem, Laeufelfingen, Switzerland). Cells were incubated for 1 h at 37°C with 2·5 µM substrate. The cleaved reporter group fluorescence was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm.

Determination of reactive oxygen species (ROS)

To quantify possible ROS generation by fibroblasts, experiments were performed measuring the oxidation of non-fluorescent 2,7′-dichlorofluorescin (DCFH) (Sigma, Buchs, Switzerland) substrate to highly fluorescent DCF by ROS. As the experimental setup could be performed only for a short exposure, LA were incubated for 5 h with fibroblasts. Cells were loaded with DCFH during a 60-min incubation in Hanks' balanced salt solution (HBSS; Sigma, Buchs, Switzerland) supplemented with 30 mM glucose (D-(+)-glucose (Sigma), pH 7·4, and 50 µM DCFH-DA (Sigma) at room temperature in the dark. Cells were washed three times with HBSS to remove any extracellular probe from the extracellular environment. Thereafter, cells were exposed to various concentrations of local anaesthetic in HBSS. The amount of generated DCF was measured using a fluorescence Synergy HT (Bio-TEK, Winooski, VT, USA). The excitation filter was set at 485 nm and the emission filter was set at 530 nm. At the same time, cell viability and activity of caspase-3 were determined.

Statistical analysis

Values were expressed as mean ± standard deviation (s.d.). Results are presented as a percentage of control. Cell count and ELISA data regarding viability, proliferation rate and caspase-3 activity were analysed using three-way analysis of variance (anova). Pearson's product–moment correlation coefficients were computed between ELISA results regarding production of ROS and cell viability. OriginPro 8G (OriginLab, Northampton, MA, USA) and spss (SPSS, Inc., Chicago, IL, USA) were used for statistical analyses. A probability of P < 0·05 was considered statistically significant.

Results

Cell count

In group 1, no negative effect of lidocaine and ropivacaine regarding cell survival was observed for the 0·3 mg/ml concentration (Fig. 1a). In the presence of bupivacaine, cell death ranged between 20% and 40%. With the 0·6 mg/ml concentration, cell survival in the lidocaine and ropivacaine group was similar with 50–90%, while a prominent effect on cell death rate was observed for bupivacaine, with 30% survival after 3 days, 5% after 6 days and no survival after 9 days of incubation (Fig. 1b).

Fig. 1.

Fig. 1

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(a) Change of cell count after 2 days' incubation with lidocaine, bupivacaine and ropivacaine with a concentration of 0·3 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± standard deviation (s.d.). (b) Change of cell count after 2 days' incubation with lidocaine, bupivacaine and ropivacaine with a concentration of 0·6 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± s.d. (c) Change of cell count after incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·3 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d. (d) Change of cell count after an incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·6 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d.

In group 2, with a permanent incubation of fibroblasts with LA at a concentration of 0·3 mg/ml, 20–30% dead cells were found with lidocaine and ropivacaine after an incubation between 3 and 9 days. Cell death was more evident in the bupivacaine group, showing a time-dependent decrease of survival (Fig. 1c). For the 0·6 mg/ml concentration, cell survival decreased over time with no major differences between the three LA (Fig. 1d).

Concentration of lidocaine, bupivacaine and ropivacaine has a significant effect on cell death (for lidocaine P < 0·001, bupivacaine P < 0·001 and ropivacaine P = 0·001). Group arrangement also influences cell survival significantly: P = 0·001 for lidocaine, P = 0·029 for bupivacaine and P = 0·01 for ropivacaine.

Cell viability

Cell viability determined in fibroblasts from group 1 showed a similar pattern to trypan blue assays: only minor impairment over time was observed for the three LA with the 0·3 mg/ml concentration (Fig. 2a). While viability was not diminished after incubation with lidocaine and ropivacaine at a 0·6 mg/ml concentration, MTT decreased time-dependently after incubation with bupivacaine (Fig. 2b).

Fig. 2.

Fig. 2

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(a) Change of viability after an incubation of 2 days with lidocaine, bupivacaine and ropivacaine with a concentration of 0·3 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± standard deviation (s.d.). (b) Change of viability after an incubation of 2 days with lidocaine, bupivacaine and ropivacaine with a concentration of 0·6 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± s.d. (c) Change of viability after an incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·3 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d. (d) Change of viability after an incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·6 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d.

In group 2, MTT did not change upon incubation with lidocaine and ropivacaine with the lower concentration. However, no cells survived after 9 days of bupivacaine exposure (Fig. 2c). With the higher concentration, fibroblasts experienced serious impairment of viability with increasing exposure time. The most pronounced effect was observed in the bupivacaine group (Fig. 2d).

Correlation analysis revealed a time- and concentration-dependent effect on cell viability for all three LA with the following values: lidocaine time P = 0·019, concentration P < 0·001; bupivacaine time P = 0·05, concentration P < 0·001; ropivacaine time P = 0·004, concentration P < 0·001. An effect based on the type of stimulation (group 1 or 2) was not observed.

Proliferation rate

Thymidine incorporation over time upon incubation with each of the three LA was not changed after exposure to a low concentration of LA (Fig. 3a). With the 0·6 mg/ml concentration, again the proliferation rate was decreased only in the bupivacaine group (Fig. 3b).

Fig. 3.

Fig. 3

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(a) Change of proliferation rate after 2 days' incubation with lidocaine, bupivacaine and ropivacaine with a concentration of 0·3 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± standard deviation (s.d.). (b) Change of proliferation rate after 2 days' incubation with lidocaine, bupivacaine and ropivacaine with a concentration of 0·6 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± s.d. (c) Change of proliferation rate after an incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·3 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d. (d) Change of proliferation rate after an incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·6 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d.

In group 2, with continued incubation with the low LA concentration, the proliferation rate decreased to 80% in the lidocaine and ropivacaine groups (Fig. 3c). This effect was more pronounced with the 0·6 mg/l concentration. Bupivacaine had a more pronounced effect on thymidine incorporation with both concentrations compared to the two other LA (Fig. 3d).

LA concentration had a statistically significant impact on proliferation rate (lidocaine: P < 0·001, bupivacaine: P < 0·001, ropivacaine P = 0·001), as did the group constellation (lidocaine: P < 0·001, bupivacaine: P = 0·009, ropivacaine P = 0·001).

Apoptosis rate

Fibroblast apoptosis was determined upon exposure to lidocaine, bupivacaine and ropivacaine. In group 1, apoptosis rate was diminished for all three LA in a similar manner for both concentrations (Fig. 4a and b).

Fig. 4.

Fig. 4

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(a) Change of apoptosis rate after 2 days' incubation with lidocaine, bupivacaine and ropivacaine with a concentration of 0·3 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± standard deviation (s.d.). (b) Change of apoptosis rate after 2 days' incubation with lidocaine, bupivacaine and ropivacaine with a concentration of 0·6 mg/ml (group 1), assessed after 3, 6 and 9 days. Values are mean ± s.d. (c) Change of apoptosis rate after an incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·3 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d. (d) Change of apoptosis rate after incubation with lidocaine, bupivacaine and ropivacaine for 3, 6 and 9 days (group 2) with a concentration of 0·6 mg/ml, assessed after 3, 6 and 9 days. Values are mean ± s.d.

With permanent incubation with LA, the apoptosis rate decreased in a time- and concentration-dependent fashion for lidocaine. An increase in the apoptosis rate was observed at 3 days of incubation with the 0·3 mg/ml (bupivacaine, ropivacaine) and 0·6 mg/ml (ropivacaine) concentrations (Fig. 4c and d).

Calculations regarding the influence of incubation time, concentration of LA and group arrangement on apoptosis reached statistical significance only for lidocaine, with P < 0·001 for concentration and P = 0·006 for group.

Production of ROS

The possibility of LA-induced expression of ROS was assessed. This could lead to an increased cell death rate. Production of ROS after a short incubation (5 h) with lidocaine or bupivacaine was enhanced with increasing concentrations from 0·3 mg/ml and 0·6 mg/ml to 1·3 mg/ml (Fig. 5a). For ropivacaine, no ROS production was observed. After incubation with lidocaine and bupivacaine a decrease in viability was measured, while viability of fibroblasts was not impaired in the presence of ropivacaine (Fig. 5b). Viability of fibroblasts correlated negatively with production of ROS (Fig. 6a and b). The highest correlation was observed in the bupivacaine group (Pearson's correlation −0·74) (Table 1), while the correlation value for lidocaine was −0·53. As no ROS were generated in the presence of ropivacaine, the correlation coefficient was not relevant for this LA. Caspase-3 activity did not increase upon short-term incubation with any of the three LA tested (data not shown).

Fig. 5.

Fig. 5

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(a) Production of reactive oxygen species. Fibroblasts were incubated for 5 h with lidocaine, bupivacaine and ropivacaine in concentrations of 0·3 mg/ml, 0·6 mg/ml and 1·25 mg/ml. Reactive oxygen species were determined. Values are mean ± standard deviation (s.d.). (b) Determination of viability (control = 100%). Fibroblasts were incubated for 5 h with lidocaine, bupivacaine and ropivacaine in concentrations of 0·3 mg/ml, 0·6 mg/ml and 1·25 mg/ml. Cell viability was determined. Values are mean ± s.d.

Fig. 6.

Fig. 6

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Correlation analysis. Production of reactive oxygen species was assessed and correlated with cell viability after incubation with lidocaine (a) and bupivacaine (b).

Table 1.

Correlation coefficients: production of reactive oxygen species versus cell viability.

Local anaesthetic Pearson's correlation coefficient P-value
Lidocaine −0·52854 < 0·001*
Bupivacaine −0·74138 < 0·001*
Ropivacaine −0·53613 < 0·001*
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*

Significant by confidence level 0·95.

Discussion

This in vitro study shows a cytotoxic effect of lidocaine, bupivacaine and ropivacaine on fibroblasts. In group 1, with exposure to local anaesthetics for 2 days followed by incubation with normal medium, cells were only slightly impaired upon stimulation with lidocaine and ropivacaine. However, bupivacaine had a significant concentration-dependent impact. In group 2, where fibroblasts were exposed permanently to LA, cells were impaired time- and concentration-dependently with all LA. The most negative effect was observed after exposure to bupivacaine. We assume that single injections do not impair the tissue.

Compared to previous investigations, the present study is original because: (i) three different local anaesthetics were tested, (ii) experiments were performed in cell culture of human fibroblasts, (iii) different concentrations of LA were evaluated, (iv) different incubation periods were assessed and (v) a possible mechanism of cytotoxicity was tested. This broad and carefully designed approach allows detailed conclusions to be drawn concerning wound healing in the presence of LA.

Previous experiments have demonstrated a possible impairment of the proliferation rate of cells such as type II pneumocytes or endothelial cells [21,22]. These data reflected the impact of local anaesthetics in very low concentrations, as found in the respiratory or vascular compartments. Our study, however, focused upon concentrations observed after injection into a wound area. A retrospective analysis of shoulder arthroscopy with intra-articular bolus injection of 0·25% bupivacaine with adrenaline described chondrolysis as a devastating complication [23]. A direct causative correlation between chondrolysis and application of local anaesthetics could not be shown, but the authors advised strongly against the use of large doses of intra-articular placement of local anaesthetics [23]. The use of topical corneal anaesthetics for pain relief after corneal abrasion also seems to have limited use [24]; Bisla and Tanelian examined epithelial regeneration in the presence of lidocaine 100–1000 µg/ml, and observed a dose-dependent impairment of epithelial wound healing with concentrations higher than 250 µg/ml, which would also demonstrate the negative effect of LA [24]. All these studies demonstrated potential harm using local anaesthetics, but did not specify the exact impairment mechanism.

Results from our study confirm other experimental findings, demonstrating that bupivacaine has a higher toxicity potential compared to lidocaine and ropivacaine [25,26]. In addition, it corroborates the results from Sturrock and Nunn, demonstrating compromised cell survival in hamster lung fibroblasts with an effective dose (ED50) of 0·06% for bupivacaine compared to 0·09% for lidocaine [27].

The present study suggests that the observed cell death is not due mainly to increased apoptosis rate, as activity of caspase-3 was correlated significantly with the amount of living cells. An exception was observed for the short stimulation period of 3 days for bupivacaine and ropivacaine. Caspase-independent mechanisms of cell death have been described in LA-induced cytotoxicity due to a change in intracellular Ca2+ homeostasis [28–33]. It is postulated in myocytes that LA induce Ca2+ release from the sarcoplasmic reticulum by interaction with ryanodine receptors [34,35]. Other studies have suggested an inhibition of Ca2+ reuptake into the sarcoplasmic reticulum, possibly regulated by Ca2+ ATPase activity [34,36]. Besides dysregulated intracellular Ca2+, the involvement of ROS production is another possible mechanism of LA-induced cell death [37,38]. As described for cocaine, LA and/or its oxidative metabolites might trigger ROS release, which has a toxic effect on hepatocytes [37,38]. Other authors claimed a correlation between the dysregulation of mitochondrial Ca2+ and ROS production, therefore reflecting a possible combination of the two proposed insult pathways [39]. A trigger such as LA or, as described by Brookes et al., ischaemia/reperfusion, might lead to a mitochondrial Ca2+ overload, mitochondrial dysfunction and ROS production which exacerbates mitochondrial damage [39].

However, cytotoxic effects of LA have been described in several studies without elucidation of the underlying mechanism [10,40,41]. Park et al. have shown increased ROS concentration correlating with cell death of Schwann cells after incubation with bupivacaine [42]. The authors thereby suggested a ROS-triggered caspase-3-activated apoptosis in neuronal cells. These conclusions were supported by results from Perez-Castro, which showed caspase-3/-7 activation in human neuroblastoma cells after 10 min incubation with lidocaine, ropivacaine and bupivacaine [43]. In our study, ROS concentrations increased upon exposure to lidocaine and bupivacaine, while at the same time caspase-3 activity was not influenced. Therefore, the ROS-induced apoptosis pathway is unlikely in our model with lidocaine and bupivacaine. Regarding ropivacaine cytotoxicity, the mechanism of ropivacaine-induced cell impairment still remains unclear and needs further evaluation.

If the cytotoxic effect is related to Na+, channel blocking is somewhat questionable. LA are well known to interact not only with Na+-, but also with K+- and Ca2+ channels [44]. In addition, they interfere with Ca uptake and release from the endoplasmic reticulum [45]. Data also indicate that LA modify N-methyl-D-aspartate (NMDA) receptor function [46]. All these, and probably many more unknown interactions, lead to a variety of properties of LA, such as myotoxicity [45], anti-inflammatory [13], anti-microbial [47] and anti-cancerogenic effects [48], which cannot be attributed to their well-known action on Na+ channels.

These in vitro data could lead to the assumption that certain local anaesthetics might have similar effects in vivo, especially by using continuous perineural application of local anaesthetic or wound instillation leading to tissue LA concentrations over several days: a factor which, according to our results, seems to be crucial for cytotoxicity. However, it should be borne in mind that, using a cell line, the in vitro model is a limitation of this study. Despite the toxic effects observed with these concentrations, further clinical studies are needed to support the present findings in vivo. Furthermore, perineural catheters for regional anaesthesia and pain therapy are used worldwide. Prospective studies with large numbers of patients did not report significant clinical neurotoxic-related complications [49–51]. However, wound healing was not assessed in detail. Whether or not neuronal cytotoxicity of LA and cytotoxicity of LA on fibroblasts is comparable remains questionable. Neuronal cells do not proliferate, while fibroblasts are highly active during the wound healing phase. Therefore, no direct conclusions can be drawn from these prospective analyses. Additionally, the average duration of the catheter was shorter in these studies: 56 h and 3·0–4·7 days, respectively [49,51].

The real clinical impact of this study warrants further investigation. However, it seems advisable to limit continuous application of LA for no more than 72–92 h, to use the lowest effective concentration and to choose the least cytotoxic LA. The application of these techniques in patients with reduced tissue healing (e.g. diabetics, smokers) needs to be investigated carefully.

Acknowledgments

This study was supported by Jubilaeumsstiftung der Schweizerischen Lebensversicherungs- und Rentenanstalt, Switzerland.

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