Buprenorphine has a concentration-dependent cytotoxic effect on equine chondrocytes in vitro

Gabriel Castro-Cuellar Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Jeannette Cremer Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Chin-Chi Liu Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Patricia Queiroz-Williams Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Chiara Hampton Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN

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Britta Sigrid Leise Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Abstract

OBJECTIVE

To investigate the cytotoxic effects of 2 different concentrations of buprenorphine and compare them with bupivacaine and morphine on healthy equine chondrocytes in vitro.

SAMPLE

Primary cultured equine articular chondrocytes from 3 healthy adult horses.

PROCEDURES

Chondrocytes were exposed for 0 and 2 hours to the following treatments: media (CON; negative control); bupivacaine at 2.2 mg/mL (BUPI; positive control); morphine at 2.85 mg/mL (MOR); buprenorphine at 0.12 mg/mL (HBUPRE); or buprenorphine at 0.05 mg/mL (LBUPRE). Chondrocyte viability was assessed using live/dead staining, water-soluble tetrazolium salt-8 (WST-8) cytotoxic assay, LDH assay, and flow cytometry. All continuous variables were evaluated with a mixed ANOVA with treatment, time, and their interactions as the fixed effects and each horse as the random effect.

RESULTS

Buprenorphine showed a concentration-dependent chondrotoxic effect. The viability of chondrocytes was significantly decreased with exposure to HBUPRE and BUPI compared to CON, MOR, and LBUPRE.

CLINICAL RELEVANCE

Negligible chondrotoxic effects were observed in healthy cultured equine chondrocytes exposed to 0.05 mg/mL of buprenorphine, whereas higher concentrations (0.12 mg/mL) showed a marked cytotoxic effect. Based on these results, low concentrations of buprenorphine appear to be safe for intra-articular administration. Further evaluation of this dose in vivo is needed before recommending its clinical use.

Abstract

OBJECTIVE

To investigate the cytotoxic effects of 2 different concentrations of buprenorphine and compare them with bupivacaine and morphine on healthy equine chondrocytes in vitro.

SAMPLE

Primary cultured equine articular chondrocytes from 3 healthy adult horses.

PROCEDURES

Chondrocytes were exposed for 0 and 2 hours to the following treatments: media (CON; negative control); bupivacaine at 2.2 mg/mL (BUPI; positive control); morphine at 2.85 mg/mL (MOR); buprenorphine at 0.12 mg/mL (HBUPRE); or buprenorphine at 0.05 mg/mL (LBUPRE). Chondrocyte viability was assessed using live/dead staining, water-soluble tetrazolium salt-8 (WST-8) cytotoxic assay, LDH assay, and flow cytometry. All continuous variables were evaluated with a mixed ANOVA with treatment, time, and their interactions as the fixed effects and each horse as the random effect.

RESULTS

Buprenorphine showed a concentration-dependent chondrotoxic effect. The viability of chondrocytes was significantly decreased with exposure to HBUPRE and BUPI compared to CON, MOR, and LBUPRE.

CLINICAL RELEVANCE

Negligible chondrotoxic effects were observed in healthy cultured equine chondrocytes exposed to 0.05 mg/mL of buprenorphine, whereas higher concentrations (0.12 mg/mL) showed a marked cytotoxic effect. Based on these results, low concentrations of buprenorphine appear to be safe for intra-articular administration. Further evaluation of this dose in vivo is needed before recommending its clinical use.

Successful pain relief after diagnostic or therapeutic arthroscopic procedures has been achieved using intra-articular (IA) administration of various pain medications in horses.1,2 Intra-articular drugs should have analgesic effects and be devoid of any detrimental effects on cartilage. Local anesthetics appear to lack the latter attribute, as chondrotoxic effects following IA administration have been reported in different species, including dogs, cattle, and horses.35

Selected opioids, specifically morphine, can be used as an alternative to local anesthetics for IA pain management. Intra-articular morphine exhibits both anti-inflammatory and analgesic effects and has been used for more than 20 years to treat joint pain in humans and horses.2,6 Opioid receptors are present in equine synovial membranes,7 and upregulation of μ-opioid receptors in inflamed joints has been linked to analgesic efficacy,8 further supporting IA use of morphine for pain management.

Buprenorphine is a partial µ-opioid receptor agonist that is approximately 25 times more potent than morphine and with a higher receptor affinity.9 Buprenorphine has been used systemically for the treatment of pre- and postoperative pain in several species including humans, swine, mice, dogs, cats, and horses.1016 In humans, IA buprenorphine after arthrocentesis of the temporomandibular joint provided pain relief and increased joint mobility when compared to saline.17 Other studies18,19 in people report that IA buprenorphine provided superior analgesia when compared with IA morphine after arthroscopic knee surgery.

Before recommending IA use of buprenorphine in horses for pain management, it is of paramount importance to assess its toxicity to cartilage, in particular to chondrocytes, if any exists. To the authors’ knowledge, chondrotoxic effects of buprenorphine are currently unknown. Therefore, the aim of this study was to evaluate the chondrotoxic effects of buprenorphine and compare them with bupivacaine and morphine on healthy cultured equine chondrocytes in vitro. The null hypothesis was that buprenorphine is devoid of any toxic effects on cultured equine chondrocytes independent of the concentration administered or duration of exposure.

Materials and Methods

Chondrocyte culture

Equine articular chondrocytes used in this study were isolated and propagated as previously reported by Nixon et al.20 Briefly, equine articular cartilage was aseptically collected from both femorotibial, left scapulohumeral, and left metacarpophalangeal joints of 3 separate horses euthanized for reasons unrelated to this study. After collection, cartilage was placed in Hank’s balanced salt solution containing penicillin and streptomycin (P/S) (Gibco). Subsequently, cartilage from each horse was minced under sterile conditions and rinsed with phosphate-buffered saline (PBS; GE Healthcare Life Sciences) solution. Minced cartilage was then enzymatically digested in collagenase type II (Invitrogen). Cartilage was incubated at 37 °C for 12 hours in digestion media, after which cell suspension was filtered through a sterile cell strainer (100 µM) (Fisher Scientific). The filtered fluid with cells was then centrifuged at 260 X g for 10 minutes at 20 °C to pellet the cells, and the supernatant was removed. The cell pellet was resuspended in Dulbecco’s modified Eagle’s medium (DMEM)-high glucose/F12 (DMEM/F12; Gibco) and washed once by centrifugation at 260 X g for 10 minutes at 20 °C. The supernatant was then removed, and the cell pellet was resuspended in DMEM/F12 medium with 10% fetal calf serum, 50 μg/mL ascorbic acid (Millipore Sigma), 100 U/mL P/S (Gibco), and 2.5 μg/mL amphotericin B (Gibco). Chondrocytes were counted and plated at a density of 3 to 4 X 105 cells/cm2 and were incubated at 37 °C in 5% CO2 and a humidified atmosphere until cells formed adequate monolayers for propagation. Cells from passages 1 through 3 from each of the 3 horses were used for the experiments.

Treatment groups

Chondrocytes were exposed to the following treatments: (1) media only (CON; DMEM/F12; Gibco) as a negative control; (2) bupivacaine (BUPI; preservative-free bupivacaine HCl; Hospira Inc) diluted in DMEM/F12 (2.2 mg/mL final concentration) as a positive control; (3) morphine (MOR; preservative-free morphine sulfate; Hospira Inc) diluted in DMEM/F12 (2.85 mg/mL final concentration); (4) high-dose buprenorphine (HBUPRE; buprenorphine HCl; Par Pharmaceutical) diluted in DMEM/F12 (0.12 mg/mL final concentration); or (5) low-dose buprenorphine (LBUPRE; buprenorphine HCl; Par Pharmaceutical) diluted in DMEM/F12 (0.05 mg/mL final concentration). Positive and negative controls were included to compare the viability of chondrocytes among treatments. Previous studies21,22 reported deleterious effects on equine chondrocytes exposed to concentrations of bupivacaine at or above 2.2 mg/mL, while no alterations in cell viability were reported with morphine use in vitro at doses of 2.85 mg/mL. Equine chondrocytes were exposed to each of the 5 treatments for 0 hours (T0) and 2 hours (T2). Chondrocyte viability was evaluated using live/dead fluorescence staining, water-soluble tetrazolium salt-8 (WST-8) assay to detect mitochondrial activity, measurement of lactate dehydrogenase (LDH) concentration, and flow cytometry to differentiate apoptosis from necrosis.

Cell viability tests

Live/dead fluorescence staining of chondrocytes

Equine chondrocytes were cultured in 24-well plates. After confluency was achieved, DMEM/F-12 media was removed from each well, and treatments (described above) were added in triplicate. Plates were incubated for 0 hours (T0; treatments applied and immediately stained) and 2 hours (T2; treatments applied and incubated at 37 °C for 2 hours; followed by staining). After treatment incubation at the respective times, chondrocytes were immediately stained with Hoechst 33342 (Molecular Probes), which stains both live and dead cells, and propidium iodide (PI; Molecular Probes), which stains dead cells. Treatments were not removed before the addition of the stains to prevent inadvertent removal of dead cells that may have lifted from the plate. Five-hundred microliters of Hoechst 33342 (diluted 1:1,000 with PBS) and 400 μL PI (diluted 1:500 with PBS) were added to each well. After the addition of both Hoechst 33342 and PI stains, the 24-well plates were incubated at room temperature (21 °C) in the dark for 10 minutes. Immediately following incubation, equine chondrocytes from each treatment group were observed using a ZOE Bio-Rad Fluorescent Cell Imager. Cells were initially observed under phase contrast microscopy, followed by fluorescence microscopy using blue (350/380 nm) and red (530/617 nm) spectrums at 10X magnification. Two to 4 images that were representative of the entire chondrocyte population were captured from each well using the microscope’s camera and saved on a USB. Ideal settings for gain, exposure, LED intensity, and contrast were set initially for the phase, blue, and red image collection. These settings were then used for the collection of all images in the study to eliminate bias when subjectively evaluating between treatment groups. The phase and merged (blue and red combined) images were subjectively assessed for cell morphology and positive PI staining (demonstrated as purple in the merged images).

Mitochondrial activity determined by WST-8 assay

Chondrocytes were seeded at 3 X 104 cells/cm2 in 96-well plates and incubated at 37 °C until confluency was achieved. For the experiment, DMEM/F12 media were removed, and each treatment as described above was added to its respective well and incubated for the designated times. All treatments were evaluated in quadruplicate. A no-cell well (media ± treatment only) was included in each group as a background control, and the WST-8 assay (Dojindo Molecular Technologies Inc) was performed per the manufacturer’s instructions. Immediately after treatments were added to the T0 group and 2 hours after treatments were added to the T2 group, 10 µL of the WST-8 reagent was also added to each well and incubated at 37 °C for 2 hours. After the 2-hour incubation, the optical density for each well was determined at 450 nm using a spectrophotometer (BioTek Synergy; Agilent Technologies Inc).

The optical density (OD) for each well was determined at 450 nm using a spectrophotometer. The no-cell well OD (background) was subtracted from the OD for all wells in each respective treatment group to eliminate variation in OD due to treatment dilutions.

LDH concentration determination

Equine chondrocytes were cultured to confluency in 24-well plates. DMEM/F12 media were removed from each well, and the respective treatments were added in duplicate. No-cell stimulation (media ± treatment) and killed cells (2 μL of 10% triton-X per 100 μL media) were included in the LDH assay as a negative and positive control, respectively. Immediately after treatments were added to the T0 group and 2 hours after treatments were added to the T2 group, 5 μL of supernatant was removed from each well and added to 95 μL of LDH storage buffer. After collection, the supernatant was immediately frozen at −80 °C until further analysis.

Lactate dehydrogenase analysis was performed using the LDH-Glo Cytotox Assay (Promega) per the manufacturer’s instructions. Briefly, 50 μL of supernatant in LDH storage buffer from each sample was added to their respective wells on a 96-well plate followed by the addition of 50 μL the LDH detection reagent. Serial dilutions of LDH standard were also included on the plate. All samples and standards were tested in duplicate. The plates were incubated at room temperature (21 °C) for 60 minutes. Following incubation, luminescence was detected for each sample using a plate reader (BioTek Synergy; Agilent Technologies Inc), and concentrations were determined from the standard curve.

Flow cytometry

Confluent cultured chondrocytes grown to confluency were trypsinized, centrifuged to obtain a cell pellet, and washed once with PBS. Chondrocytes were aliquoted into polystyrene tubes at a concentration of 1 X 105 cells/tube with media ± treatment (as described above). Tubes for T2 were incubated at 37 °C in 5% CO2 for 2 hours. After incubation (T0 and T2), tubes were centrifuged at 1,000 rpm for 5 minutes to pellet the cells. The cells were washed by resuspension of them in 1 mL of cold PBS followed by centrifugation at 1,000 rpm for 5 minutes. The cells in each tube were then resuspended per manufacturer’s instructions (Molecular Probes), with 100 µL of 1X annexin-binding buffer followed by the addition of 5 µL/tube of fluorescein isothiocyanate annexin V stain (annexin V-FITC) and 1 µL/tube of PI stain. The tubes were incubated for 15 minutes in the dark at room temperature, (21 °C), and then 400 µL of the 1X annexin-binding buffer was added to each tube and gently mixed. Tubes were kept on ice before analysis by flow cytometry (BD FACSCaliber; BD Biosciences). Annexin V-FITC and PI emissions were collected with 530/30-nm and 585/42-nm bandpass filters, respectively. Data were analyzed and are displayed as a 2-parameter dot plot of annexin-V FITC versus PI fluorescence using the Cellquest Pro software (BD Biosciences).

Statistical analysis

Data analyses were performed using JMP Pro 16.1.0 software (SAS Institute Inc). All continuous variables (optical density from the WST-8 assay and LDH concentrations) were evaluated using a mixed effect ANOVA with treatment, time, and their interactions as the fixed effects and each animal as the random effect. Flow cytometry data was also analyzed within each treatment with each quadrant as the fixed effect and each animal as the random effect. Assumptions of all ANOVA models (linearity, normality of residuals, and homoscedasticity of residuals) and influential data points were assessed by examining standardized residual and quantile plots. When a fixed effect was detected, Tukey’s post hoc test comparisons were performed with least square means for the effect. WST-8, LDH, and flow cytometry data were summarized and are presented as mean ± SD. Significance was set at P < .05.

Results

Live/dead fluorescence staining

Live/dead staining was subjectively evaluated for each treatment group under phase and fluorescence microscopy. Phase images from the BUPI and HBUPRE treatment groups began to exhibit cellular changes, in the form of rounding of some cells noted at T0. Rounding of the cells continued at T2 for the BUPI and HBUPRE groups with increased distance noted between cells (Figure 1). Subjectively, chondrocyte death was noted to be increased in the BUPI and HBUPRE groups, as an increase in PI staining was subjectively detected. The cell death was demonstrated by an increased proportion of purple cells observed in Hoescht/PI merged images at both T0 and T2. While not as pronounced as the BUPI and HBUPRE groups, mild subjective increases in dead cells were also noted in the MOR and LBUPRE groups when compared to CON at T2.

Figure 1
Figure 1

Live/dead cell determination at T2. Images obtained following stimulation of equine chondrocytes with media only (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (HBUPRE; 0.12 mg/mL), or low-dose buprenorphine (LBUPRE; 0.05 mg/mL). The top images display chondrocytes following stimulation using phase microscopy at X10 magnification (scale bar = 100 µm). The bottom images display chondrocytes following stimulation with the addition of live/dead Hoescht and propidium iodine stains, respectively. The cells were viewed at X10 magnification (scale bar = 100 µm) via fluorescence microscopy using blue and red spectrums; the images from both stains were then merged. Hoescht dye penetrates cell membranes of all cells independent of live/dead status, staining them blue. Propidium iodine dye penetrates only dead cells, staining them red. Therefore, in the merged images, dead cells appear purple. Note changes in the cell morphology under phase and increased number of purple cells under fluorescence in the BUPI and HBUPRE groups.

Citation: American Journal of Veterinary Research 84, 3; 10.2460/ajvr.22.08.0143

WST-8 assay

At T0, mitochondrial activity (as determined by mean OD) was lowest in the BUPI treatment group (0.051 ± 0.035; Figure 2). Conversely, mitochondrial activity was highest in the CON (0.235 ± 0.035), MOR (0.283 ± 0.035), and LBUPRE (0.356 ± 0.035) treatment groups. These values were significantly different (P < .0001) when compared with BUPI and HBUPRE (0.141 ± 0.035). LBUPRE mitochondrial activity was significantly higher than CON (P < .0001), and BUPI was significantly lower (P < .0001) than CON.

Figure 2
Figure 2

Chondrocyte viability determined by water-soluble tetrazolium salt-8 assay to assess mitochondrial activity. Higher optical density represents greater mitochondrial activity and greater viability. Mean ± SD optical densities (OD) from cultured equine chondrocytes are represented following exposure for 0 hours (A) or 2 hours (B) to media (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (H BUPRE; 0.12 mg/mL), or low-dose buprenorphine (L BUPRE; .05 mg/mL). Different lowercase letters indicate significant differences (P ≤ .0001) between treatment groups.

Citation: American Journal of Veterinary Research 84, 3; 10.2460/ajvr.22.08.0143

At T2, mitochondrial activity was highest in chondrocytes subjected to CON (0.250 ± 0.035), MOR (0.220 ± 0.035), or LBUPRE (0.236 ± 0.035). Chondrocytes exposed to BUPI (0.029 ± 0.035) had the lowest mitochondrial activity followed by HBUPRE (0.057 ± 0.035) (Figure 2). CON, MOR, and LBUPRE had significantly higher mitochondrial activity (P < .0001) when compared with those exposed to BUPI or HBUPRE. No difference in mitochondrial activity was found among chondrocytes exposed to CON, MOR, or LBUPRE, nor between those chondrocytes exposed to HBUPRE and BUPI. Mitochondrial activity was significantly different (P = .015) between treatment times within a treatment group (T0 vs T2) for LBUPRE only.

LDH assay

At T0, supernatant from equine chondrocytes exposed to BUPI (7.71 ± 1.94 mU/mL) or HBUPRE (7.79 ± 1.94 mU/mL) had the highest concentrations of LDH, whereas supernatant from chondrocytes exposed to CON (2.59 ± 1.94 mU/mL), MOR (2.78 ± 1.94 mU/mL), or LBUPRE (3.46 ± 1.94 mU/mL) had lower LDH concentrations.

At T2, supernatant from equine chondrocytes exposed to CON (0.33 ± 1.94 mU/mL), MOR (2.37 ± 1.94 mU/mL), or LBUPRE (3.65 ± 1.94 mU/mL) had the lowest LDH concentrations. In contrast, chondrocytes exposed to BUPI (9.16 ± 1.94 mU/mL) or HBUPRE (12.58 ± 1.94 mU/mL) had higher LDH concentrations.

Independent of exposure time, chondrocyte viability determined by LDH concentration was significantly decreased (P < .016) in the HBUPRE and BUPI groups when compared with the MOR or CON groups (Figure 3). Furthermore, independently of exposure time, chondrocyte viability was significantly lower (P = .0045) in HBUPRE group when compared with the LBUPRE group. There were no significant differences between treatment times (T0 vs. T2) within treatment groups.

Figure 3
Figure 3

Chondrocyte death demonstrated by increased concentrations of lactate dehydrogenase. Mean ± SD lactate dehydrogenase (LDH; mU/mL) concentrations of cultured equine chondrocytes following exposure for 0 (T0) and 2 (T2) hours to media (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (HBUPRE; 0.12 mg/mL), or low-dose buprenorphine (LBUPRE; 0.05 mg/mL). Different lowercase letters indicate significant differences (P ≤ .05) between treatment groups. There were no differences between the T0 or T2 times.

Citation: American Journal of Veterinary Research 84, 3; 10.2460/ajvr.22.08.0143

Flow cytometry

Flow cytometry scatter plot data were compared among treatment groups at each treatment time to differentiate cell death due to apoptosis or necrosis. At T0, there was no difference in the percentage of cell death due to apoptosis or necrosis when compared among treatments.

At T2, the percentage of cells positive for PI and annexin-V staining were significantly (P < .05) higher in BUPI (77.87 ± 10.66) compared with CON (16.55 ± 10.66; P = .015) and MOR (16.08 ± 10.66; P = .014; Figure 4). No other differences were found among the other treatment groups.

Figure 4
Figure 4

Mean ± SD percent gated chondrocytes determined by flow cytometry following exposure for 2 hours to media (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (H BUPRE; 0.12 mg/mL), or low-dose buprenorphine (L BUPRE; 0.05 mg/mL). (A) Demonstrates mean ± SD percent gated chondrocytes localized to the upper left (UL) quadrant staining positive for PI (necrosis). (B) Demonstrates mean ± SD percent gated chondrocytes localized to the UR quadrant staining positive for both PI and annexin-V. (C) Demonstrates mean ± SD percent gated chondrocytes localized to the lower left (LL) quadrant that were negative for both PI and annexin-V. (D) Demonstrates mean ± SD percent gated chondrocytes localized to the lower right (LR) quadrant staining positive for annexin-V (apoptosis). Different lowercase letters indicate significant differences (P ≤ .05) between treatment groups, which were only present in the upper right (UR) quadrant.

Citation: American Journal of Veterinary Research 84, 3; 10.2460/ajvr.22.08.0143

Discussion

Results from the present study indicate buprenorphine has a concentration-dependent cytotoxic effect of buprenorphine on equine articular chondrocytes in vitro. Healthy cultured equine chondrocytes exposed to high concentrations of buprenorphine (0.12 mg/mL) had subjectively increased chondrocyte death 2 hours after administration demonstrated by an increased number of dead cells observed via PI staining. Additionally, chondrocytes exposed to high concentrations of buprenorphine demonstrated 4 times less mitochondrial activity than chondrocytes exposed to media, morphine, or low concentrations of buprenorphine. Only bupivacaine, which has also been reported to have concentration-dependent cytotoxic effects on canine and human chondrocytes,22,23 had a similar effect on PI staining and mitochondrial activity in this study. Cytotoxicity of bupivacaine to equine chondrocytes in vitro has been reported previously at the dose used in this study;5,21 therefore, it was used as a positive control. Furthermore, supernatant from chondrocytes treated with the high buprenorphine concentration had the highest LDH concentration of all treatments at both treatment times, revealing that cell membrane integrity was disrupted and cell viability decreased. In contrast, equine chondrocytes exposed to low concentrations of buprenorphine (0.05 mg/mL) demonstrated similar viability to that of the media control and morphine-treated cells. These findings suggest that higher concentrations of buprenorphine may result in chondrocyte toxicity and should be considered a potential risk for clinical use.

When using the average synovial fluid volume of the middle carpal joint for calculation,24 the high buprenorphine concentration (0.12 mg/mL) used in this study was calculated to be equivalent to the lower end (5 µg/kg) of the systemic analgesic dose reported in equids, which ranges from 5 to 10 µg/kg.25 The low buprenorphine concentration used in this study was equivalent to a systemic subclinical dose of 2 µg/kg. Two previous studies2 evaluating the pharmacokinetics and analgesic efficacy of IA morphine in horses with lipopolysaccharide (LPS)-induced synovitis, used a systemic subclinical dose of 0.05 mg/kg morphine, which remained within the joint for at least 24 hours and decreased lameness scores after LPS administration. Furthermore, studies26 in humans have reported doses of IA buprenorphine as low as 100 µg/joint, which is equivalent to 1.4 µg/kg for an average 70 kg person. Systemic analgesic dosages of buprenorphine in humans range from 3 to 8.5 µg/kg. Even though information on the analgesic efficacy of IA buprenorphine in horses is lacking, a subclinical dose of 2 µg/kg was chosen based on reports that analgesia from IA buprenorphine in humans,18 and IA morphine in horses,2 can be accomplished using lower doses than those used systemically. While low-dose buprenorphine displayed minimal to no cytotoxic effects in vitro in this study, in vivo evaluation of this dosage is needed to determine if IA analgesic effects can be achieved before its use in clinical cases is recommended.

When used alone, the μ-opioid receptor agonist morphine was previously reported to have no significant cytotoxic effects on equine chondrocytes,21 as was found in the current study. As buprenorphine is a partial μ-opioid receptor agonist, it was expected to have a similar response as morphine. However, the current study found a concentration-dependent cytotoxic effect of buprenorphine on healthy cultured equine chondrocytes. Concentration-dependent cytotoxicity has been demonstrated in chondrocytes exposed to selected opioids and local anesthetics.27,28 For example the µ-opioid receptor agonist meperidine exhibits concentration-dependent chondrotoxicity; 5 mg/mL meperidine was found to be significantly less chondrotoxic compared to 10 mg/mL and 15 mg/mL meperidine.27 As mentioned previously, bupivacaine (2.5 mg/mL and 5 mg/mL) resulted in decreased canine chondrocyte viability based on significantly less MTT activity when compared to a negative control and low dose bupivacaine (0.6 mg/mL).22 Buprenorphine can act as a local anesthetic by inducing a concentration-dependent blockade of sodium channels when injected perineurally and neuraxially.2931 As previously mentioned, a comparable effect has also been reported with meperidine, which is also µ-opioid receptor agonist and a sodium channel blocker.29,30 Cytotoxicity from local anesthetics is thought to be related to a concentration-dependent increase in ion influx that leads to changes in oxidative metabolism within the cell. This leads to an increase in reactive oxygen species production and mitochondrial dysfunction, which is followed by disruption in transmembrane potential that ultimately results in apoptosis or necrosis of the cell.3235 Cellular morphological changes after exposure to local anesthetics have also been reported. Chondrocytes exposed to lidocaine exhibited concentration-dependent spherical protrusions on the cell surface (a phenomenon known as membrane blebbing), followed by cell shrinkage and death.36 This effect of local anesthetics on cell morphology and organization of membrane-associated cytoskeletal structures has also been reported to occur in fibroblasts and tenofibroblasts.34,37 The decreased viability observed in chondrocytes exposed to high-dose but not low-dose buprenorphine may be due to its similarities to local anesthetics (lidocaine and bupivacaine) and meperidine in its mechanism of action and concentration-dependent chondrotoxic effects. This effect could be mediated by the same cellular processes reported for local anesthetics and meperidine; however, further studies are needed to confirm this hypothesis.

Other possible causes for the cytotoxicity seen in this study could be the pH of the treatments and/or crystalline development. An acidic environment (low pH) has been proposed as a potential source for decreased viability of chondrocytes. Although bovine chondrocytes exposed to extremely acidic solutions (pH = 2.4) have demonstrated some decrease in cell viability, pH solutions ranging from 7.4 to 3.2 have been reported to have no effect on chondrocyte viability.38,39 While the pH of the treatments was not controlled or measured in this study, the pH of the drugs used has been reported. The pH of bupivacaine ranges from 5.55 to 6.65,40 the pH of preservative-free morphine reportedly varies from 2.5 to 6.5,41 and the pH of buprenorphine HCl ranges from 4 to 5;42 therefore, based on the acidic nature of all the drugs used drugs in this study and their variable effects on cell viability it is unlikely that pH alone would have resulted in the cytotoxic effects seen in this study. Mixing a local anesthetic with culture media at a different pH can cause crystal formation, and crystallization has been suggested to potentially result in micro trauma to the cells and subsequently cell death.28 For instance, the local anesthetic ropivacaine forms crystals at physiologic pH (7.37),43 whereas bupivacaine and lidocaine form crystals at a pH of 7.7 and 12.9, respectively.44 In the present study, however, no gross crystallization nor cloudiness was observed when the treatments were combined with the culture media and, therefore, were not suspected to be a cause of cell death in this study.

Flow cytometry was performed to assess whether necrosis or apoptosis was the main cause of cell death in this study. Only bupivacaine was found to have significantly greater cell death 2 hours posttreatment administration. Cell death occurred by both necrosis, evident by PI staining, and apoptosis, evident by annexin-V staining. This finding does vary from a previous report5 where necrosis was considered as the main mechanism of chondrocyte death in equine cultured chondrocytes exposed to local anesthetics. No significant difference was seen between apoptosis and necrosis within the buprenorphine treatments at any treatment time. Further research is needed to elucidate the mechanism of chondrocyte death after exposure to buprenorphine.

The main limitation of this study from a clinical point of view is the fact that this was an in vitro study. This implies that the setting in which drugs were tested does not accurately resemble the joint environment, which is dynamic, involving the interaction of different tissue types (cartilage and synovium) and the surrounding synovial fluid. The joint environment and cellular responses to the administration of medications may be expected to affect the kinetics of drugs differently from what may occur in vitro. For instance, most of these drugs are administered after arthroscopic surgery where invariably joint distention and inflammation would be present. This could result in further dilution of the drug intra-articularly and possibly hasten drug elimination from the joint. In this study, a 2-dimensional monolayer cell culture was used, which does not accurately recreate the natural microenvironment to which chondrocytes are normally exposed in vivo. In this regard, 3-dimensional (3-D) cell cultures are superior for extrapolating in vitro results to in vivo environments.45 In contrast to monolayer cultures, 3-D cultures better develop in vivo characteristics including cellular morphologies, structural organization, intercellular interactions, and gene expression patterns.46 Despite the superiority of 3-D cultures, their use is still limited due to difficulties associated with establishing cultures.45 Therefore, the results reported here and their extrapolation to an in vivo setting should be interpreted carefully.

In conclusion, the present study demonstrated a dose-dependent chondrotoxic effect exerted by buprenorphine (0.12 mg/mL) in an in vitro setting after 2 hours of exposure. However, a lower concentration (0.05 mg/mL) appears to have a negligible effect on chondrocyte viability in vitro. Further studies are necessary to determine the chondrotoxic mechanism(s) associated with higher buprenorphine concentrations and to determine if the lower concentration used in this study is clinically relevant for IA administration in the horse to treat joint pain.

Acknowledgments

The study was funded by the Louisiana State University Department of Veterinary Clinical Sciences Research Program Grant and Equine Health Studies Program. The funding sources did not have any involvement in the study design, data analysis, and interpretation or writing and publishing of the manuscript.

The authors declare that there were no conflicts of interest.

Dr. Castro-Cuellar contributed to study design, study execution, and preparation and revision of the manuscript. Dr. Leise contributed to study design, study execution, data interpretation, and preparation and revision of the manuscript. Dr. Cremer contributed to study design, study execution, and manuscript review. Dr. Liu contributed to data analysis and interpretation and manuscript review. Drs. Queiroz-Williams and Hampton contributed to study design and manuscript review. All authors gave final approval of the manuscript.

Presented in part at the Anesthesia and Pain Management section of the International Veterinary Emergency and Critical Care Symposium, virtual meeting, September 2020.

The authors thank Michael Keowen, Marilyn Dietrich, and Heather Bell for their technical help in the completion of this study and Dr. Lee Ann Fugler for help in manuscript preparation.

References

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  • Figure 1

    Live/dead cell determination at T2. Images obtained following stimulation of equine chondrocytes with media only (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (HBUPRE; 0.12 mg/mL), or low-dose buprenorphine (LBUPRE; 0.05 mg/mL). The top images display chondrocytes following stimulation using phase microscopy at X10 magnification (scale bar = 100 µm). The bottom images display chondrocytes following stimulation with the addition of live/dead Hoescht and propidium iodine stains, respectively. The cells were viewed at X10 magnification (scale bar = 100 µm) via fluorescence microscopy using blue and red spectrums; the images from both stains were then merged. Hoescht dye penetrates cell membranes of all cells independent of live/dead status, staining them blue. Propidium iodine dye penetrates only dead cells, staining them red. Therefore, in the merged images, dead cells appear purple. Note changes in the cell morphology under phase and increased number of purple cells under fluorescence in the BUPI and HBUPRE groups.

  • Figure 2

    Chondrocyte viability determined by water-soluble tetrazolium salt-8 assay to assess mitochondrial activity. Higher optical density represents greater mitochondrial activity and greater viability. Mean ± SD optical densities (OD) from cultured equine chondrocytes are represented following exposure for 0 hours (A) or 2 hours (B) to media (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (H BUPRE; 0.12 mg/mL), or low-dose buprenorphine (L BUPRE; .05 mg/mL). Different lowercase letters indicate significant differences (P ≤ .0001) between treatment groups.

  • Figure 3

    Chondrocyte death demonstrated by increased concentrations of lactate dehydrogenase. Mean ± SD lactate dehydrogenase (LDH; mU/mL) concentrations of cultured equine chondrocytes following exposure for 0 (T0) and 2 (T2) hours to media (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (HBUPRE; 0.12 mg/mL), or low-dose buprenorphine (LBUPRE; 0.05 mg/mL). Different lowercase letters indicate significant differences (P ≤ .05) between treatment groups. There were no differences between the T0 or T2 times.

  • Figure 4

    Mean ± SD percent gated chondrocytes determined by flow cytometry following exposure for 2 hours to media (CON), bupivacaine (BUPI; 2.2 mg/mL), morphine (MOR; 2.85 mg/mL), high-dose buprenorphine (H BUPRE; 0.12 mg/mL), or low-dose buprenorphine (L BUPRE; 0.05 mg/mL). (A) Demonstrates mean ± SD percent gated chondrocytes localized to the upper left (UL) quadrant staining positive for PI (necrosis). (B) Demonstrates mean ± SD percent gated chondrocytes localized to the UR quadrant staining positive for both PI and annexin-V. (C) Demonstrates mean ± SD percent gated chondrocytes localized to the lower left (LL) quadrant that were negative for both PI and annexin-V. (D) Demonstrates mean ± SD percent gated chondrocytes localized to the lower right (LR) quadrant staining positive for annexin-V (apoptosis). Different lowercase letters indicate significant differences (P ≤ .05) between treatment groups, which were only present in the upper right (UR) quadrant.

  • 1.

    Santos LC, de Moraes AN, Saito ME. Effects of intraarticular ropivacaine and morphine on lipopolysaccharide-induced synovitis in horses. Vet Anaesth Analg. 2009;36(3):280286. doi:10.1111/j.1467-2995.2009.00452.x

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 2.

    Lindegaard C, Thomsen MH, Larsen S, Andersen PH. Analgesic efficacy of intra-articular morphine in experimentally induced radiocarpal synovitis in horses. Vet Anaesth Analg. 2010;37(2):171185. doi:10.1111/j.1467-2995.2009.00521.x

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3.

    Anz A, Smith MJ, Stoker A, et al. The effect of bupivacaine and morphine in a coculture model of diarthrodial joints. Arthroscopy. 2009;25(3):225231. doi:10.1016/j.arthro.2008.12.003

    • Search Google Scholar
    • Export Citation
  • 4.

    Lo IK, Sciore P, Chung M, et al. Local anesthetics induce chondrocyte death in bovine articular cartilage disks in a dose- and duration-dependent manner. Arthroscopy. 2009;25(7):707715. doi:10.1016/j.arthro.2009.03.019

    • Search Google Scholar
    • Export Citation
  • 5.

    Park J, Sutradhar BC, Hong G, Choi SH, Kim G. Comparison of the cytotoxic effects of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes. Vet Anaesth Analg. 2011;38(2):127133. doi:10.1111/j.1467-2995.2010.00590.x

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Kalso E, Tramèr MR, Carroll D, McQuay HJ, Moore RA. Pain relief from intra-articular morphine after knee surgery a qualitative systematic review. Pain. 1997;71(2):127134. [10.1016/S0304-3959(97)03344-7]

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Sheehy JG, Hellyer PW, Sammonds GE, et al. Evaluation of opioid receptors in synovial membranes of horses. Am J Vet Res. 2001;62(9):14081412. doi:10.2460/ajvr.2001.62.1408

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8.

    van Loon JP, de Grauw JC, Brunott A, Weerts EA, van Weeren PR. Upregulation of articular synovial membrane mu-opioid-like receptors in an acute equine synovitis model. Vet J. 2013;196(1):4046. doi:10.1016/j.tvjl.2012.07.030

    • Search Google Scholar
    • Export Citation
  • 9.

    KuKanich B, W A. Opioids In: Grimm KA Lamont LA, Tranquili WJ, Greene JA, Robertson SA, ed. Veterinary Anesthesia and Analgesia: the Fifth Edition of Lumb and Jones. Wiley Blackwell; 2015:207226.

    • Search Google Scholar
    • Export Citation
  • 10.

    Rodriguez NA, Cooper DM, Risdahl JM. Antinociceptive activity of and clinical experience with buprenorphine in swine. Contemp Top Lab Anim Sci. 2001;40(3):1720.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    Roughan JV, Flecknell PA. Buprenorphine a reappraisal of its antinociceptive effects and therapeutic use in alleviating post-operative pain in animals. Lab Anim. 2002;36(3):322343. doi:10.1258/002367702320162423

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12.

    Christoph T, Kogel B, Schiene K, Méen M, De Vry J, Friderichs E. Broad analgesic profile of buprenorphine in rodent models of acute and chronic pain. Eur J Pharmacol. 2005;507(1–3):8798. doi:10.1016/j.ejphar.2004.11.052

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13.

    Steagall PV, Taylor PM, Rodrigues LC, Ferreira TH, Minto BW, Aguiar AJ. Analgesia for cats after ovariohysterectomy with either buprenorphine or carprofen alone or in combination. Vet Rec. 2009;164(12):359363. doi:10.1136/vr.164.12.359

    • Search Google Scholar
    • Export Citation
  • 14.

    Love EJ, Taylor PM, Whay HR, Murrell J. Postcastration analgesia in ponies using buprenorphine hydrochloride. Vet Rec. 2013;172(24):635. doi:10.1136/vr.101440

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Rigotti C, De Vries A, Taylor PM. Buprenorphine provides better anaesthetic conditions than butorphanol for field castration in ponies: results of a randomised clinical trial. Vet Rec. 2014;175(24):623. doi:10.1136/vr.102729

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Jonan AB, Kaye AD, Urman RD. Buprenorphine formulations clinical best practice strategies recommendations for perioperative management of patients undergoing surgical or interventional pain procedures. Pain Physician. 2018;21(1):E1E12. doi:10.36076/ppj.2018.1.e1

    • Search Google Scholar
    • Export Citation
  • 17.

    Präger TM, Mischkowski RA, Zöller JE. Effect of intra-articular administration of buprenorphine after arthrocentesis of the temporomandibular joint: a pilot study. Quintessence Int. 2007;38(8):e484e489.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18.

    Varrassi G, Marinangeli F, Ciccozzi A, et al. Intra-articular buprenorphine after knee arthroscopy. A randomised, prospective, double-blind study. Acta Anaesthesiol Scand. 1999;43(1):5155. doi:10.1034/j.1399-6576.1999.430112.x

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19.

    Gowdra Sugandarajappa S, Sumitha RN, Sumitha CS Chengode S. Comparison of analgesic effect of intra-articular buprenorphine and morphine following arthroscopic surgery of knee. J Evol Med Dental Sci. 2016;5:41754180. doi:10.14260/jemds/2016/953

    • Search Google Scholar
    • Export Citation
  • 20.

    Nixon A, Lust G, Vernier-Singer M. Isolation, propagation, and cryopreservation of equine articular chondrocytes. Am J Vet Res. 1992;53(12):23642370.

  • 21.

    Rubio-Martinez LM, Rioja E, Castro Martins M, Wipawee S, Clegg P, Peffers MJ. Local anaesthetics or their combination with morphine and/or magnesium sulphate are toxic for equine chondrocytes and synoviocytes in vitro. BMC Vet Res. 2017;13(1):318. doi:10.1186/s12917-017-1244-8

    • Search Google Scholar
    • Export Citation
  • 22.

    Rengert R, Snider D, Gilbert PJ. Effect of bupivacaine concentration and formulation on canine chondrocyte viability in vitro. Vet Surg. 2021;50(3):633640. doi:10.1111/vsu.13590

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23.

    Breu A, Rosenmeier K, Kujat R, Angele P, Zink W. The cytotoxicity of bupivacaine, ropivacaine, and mepivacaine on human chondrocytes and cartilage. Anesth Analg. 2013;117(2):514522. doi:10.1213/ANE.0b013e31829481ed

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24.

    Ekman L, Nilsson G, Persson L, Lumsden JH. Volume of the synovia in certain joint cavities in the horse. Acta Vet Scand. 1981;22(1):2331. doi:10.1186/BF03547202

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25.

    Taylor PM, Hoare HR, de Vries A, et al. A multicentre, prospective, randomised, blinded clinical trial to compare some perioperative effects of buprenorphine or butorphanol premedication before equine elective general anaesthesia and surgery. Equine Vet J. 2016;48(4):442450. doi:10.1111/evj.12442

    • Search Google Scholar
    • Export Citation
  • 26.

    Dahan A, Yassen A, Romberg R, et al. Buprenorphine induces ceiling in respiratory depression but not in analgesia. Br J Anaesth. 2006;96(5):627632. doi:10.1093/bja/ael051

    • Search Google Scholar
    • Export Citation
  • 27.

    Abrams GD, Chang W, Dragoo JL. In vitro chondrotoxicity of nonsteroidal anti-inflammatory drugs and opioid medications. Am J Sports Med. 2017;45(14):33453350. doi:10.1177/0363546517724423

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28.

    Adler D, Frellesen JF, Karlsen CV, Jensen LD, Dahm ASQ, Berg LC. Evaluation of the in vitro effects of local anesthetics on equine chondrocytes and fibroblast-like synoviocytes. Am J Vet Res. 2021;82(6):478486. doi:10.2460/ajvr.82.6.478

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 29.

    Helgesen KG, Ellingsen O, Ilebekk A. Inotropic effect of meperidine influence of receptor and ion channel blockers in the rat atrium. Anesth Analg. 1990;70(5):499506. doi:10.1213/00000539-199005000-00006

    • PubMed
    • Search Google Scholar
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