Co-administration of hyaluronic acid with local anaesthetics shows lower cytotoxicity than local anaesthetic treatment alone in bovine articular chondrocytes

Chondrocyte harvest

Articular chondrocytes were harvested by removing cartilage from weight-bearing portions of bovine femoral condyles (Rancho Veal, Petaluma, California). Cartilage was minced into 3-mm³ cubes and washed in sterile phosphate buffered saline (PBS; Hyclone, Logan, Utah) treated with 250 μg/ml Amphotericin B (MP Biomedicals, Solon, Ohio) and Penicillin-Streptomycin-Glutamine antibiotic (Pen-Strep; Hyclone). Chondrocytes were isolated by digesting cartilage in digestion media consisting of 500 ml 1:1 Dulbecco’s Modified Eagle Media (DMEM):F-12 (Hyclone), 50 ml Fetal Bovine Serum (FBS) (Axenia BioLogix, Sacramento, California), and 100 mg collagenase-P (Roche, Mannheim, Germany). The digested cartilage was filtered and the remaining chondrocytes in suspension were centrifuged at 500 g for 10 minutes. After discarding the supernatant and re-suspending the chondrocytes in a mixture of 10% FBS Media (500 ml 1:1 DMEM:F12, 50 ml FBS, 5 ml 100×Amphotericin B, 5 ml Pen-Strep), cells were counted using a haemacytometer.

Chondrocytes were then plated on flat-bottomed clear 75-cm² flasks at a concentration of 7.5×10⁵ cells/ml and allowed to grow to confluence. At 24 hours following plating, cells were treated with insulin transferrin selenium (ITS)-supplemented media until use. ITS media was prepared by combining 500 ml 1:1 DMEM:F12, 25 mg L-ascorbic acid 2-phospate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, Missouri), 5 ml 100×Pen-Strep antibiotic, 5 ml Amphotericin B, 500 mg bovine serum albumin (BSA) (Sigma Aldirich), 5 ml 10 mg/ml sodium pyruvate (Mediatech, Manassas, Virginia), 5 ml 1M HEPES Buffer (University of California, San Francisco Cell Culture Facility, San Francisco, California), and 5 ml of ITS premix (BD Biosciences, San Jose, California).

Seeding

Chondrocytes were removed from flasks, counted, and transferred to 96-well plates for treatment. A plate was seeded with 15 000 chondrocytes in each well. Each plate consisted of five conditions, described below, in replicates of six and a standard curve. The standard curve was prepared by serial dilution, starting with a density of 30 000 chondrocytes per well. The appropriate volume for each was calculated using the concentration found by methods described in the previous section. Additional ITS Media was added to each of the occupied wells to bring their total volume to 100 μl. Each plate was incubated at 37°C in standard cell culture conditions for 48 hours to allow the cells to rest and adhere to the wells.

Treatment

After 48 hours, the media was removed from the wells and the chondrocytes were treated with 70 μl of one of the seven following conditions: 1) ITS media; 2) PBS; 3) 1% lidocaine diluted from 2% stock solution (20 mg/ml lidocaine HCl, 6 mg/ml sodium chloride; APP Pharmaceuticals, Schaumburg, Illinois) and PBS; 4) 0.25% bupivacaine diluted from 0.5% stock (5 mg/ml bupivacaine HCl, 8.1 mg/ml sodium chloride; Hospira Inc., Lake Forest, Illinois) and PBS; 5) 1:1 2% Lidocaine and Supartz (Smith & Nephew); 6) 1:1 0.5% bupivacaine and Supartz; or 7) 1:1 Supartz and PBS. Following treatment, the conditions were removed from the wells and replaced with 70 μl of ITS media. Chondrocytes were allowed to recover by incubating for 24 hours at 37°C.

Quantification

After 24 hours, media was removed with from all wells and replaced with 50 μl of a 1:10 solution of PrestoBlue (Invitrogen, Frederick, Maryland) in 10% FBS media (500 ml 1:1 DMEM:F12, 50 ml FBS, 5 ml 100× Amphotecerin B, 5 ml Pen-Strep). Briefly, PrestoBlue is a non-cytotoxic cell viability fluorescence assay. The reagent measures viability by testing the cell’s ability to reduce nicotinamide adenine dinucleotide (NAD⁺). Following ten minutes of incubation at 37°C, the plate was read on the Synergy2 plate reader machine (BioTek Instruments Inc., Winooski, Virginia) with an excitation frequency of 535 nm and emission frequency of 595 nm, producing a fluorescence intensity read out in arbitrary units. The mean of the blank well readouts on each plate was calculated and subtracted from each experimental well. Fluorescence values were converted number of chondrocytes using the standard curve.

LIVE/DEAD stain

In order to confirm the results from the PrestoBlue staining, chondrocytes were visualised to qualitatively assess chondrocyte viability. Following staining with LIVE/DEAD Viability/Cytotoxicity Kit for Mammalian Cells (Invitrogen), representative images of chondrocytes under each treatment were taken using fluorescence microscopy. The stain was prepared by adding 5 μl calcein AM and 20 μl ethidium bromide homodimer-1 to 10 ml of 1× PBS. 100 μl of the LIVE/DEAD stain was added to each well and allowed to incubate at room temperature and protected from light for 35 minutes. Employing fluorescence microscopy, live cells were visualised using a 5× and 10× objective under a fluorescein isothiocyanate (FITC) filter (approximately 494 nm) and dead cells were visualised under a rhodamine filter (approximately 517 nm).

Statistical analysis

Statistical analysis was conducted using the computer software R (The R Foundation for Statistical Computing; University of Vienna, Vienna, Austria). Data were analysed for statistically significant differences between all conditions using analysis of variance (ANOVA). A post-hoc Tukey’s Honestly Significant Difference (HSD) test was conducted to make pair-wise comparisons between conditions in order to find statistical significance, indicated by p-values < 0.05.

Fluorescence assay for cell viability

The fluorescence using the PrestoBlue reagent was converted to number of chondrocytes using a standard curve. Combining results from the two experiments, the mean number of cells for each condition is given in Table I and Figure 1.

Fig. 1

Bar chart showing the mean chondrocyte viability by treatment group. The error bars denote the standard error of the mean (sem). All treatments showed significantly reduced viability compared with media-only controls (all < 0.003). †, statistically significant difference between the bupivacaine-only and bupivacaine and hyaluronic acid (HA) groups (p = 0.027) (PBS, phosphate buffered saline).

ANOVA comparison of all conditions against one another indicated a statistically significant difference within the data (p < 0.001). A post-hoc Tukey’s HSD test was conducted to test pair-wise differences in comparison with the media-only controls. All conditions resulted in significantly lower viability compared with the media-only controls.

Treating chondrocytes with bupivacaine and HA significantly increased cell viability when compared with bupivacaine only (p = 0.027). The lidocaine and HA treatment did not significantly increase chondrocyte viability compared with the lidocaine-only treatment (p = 0.999).

LIVE/DEAD staining and visualisation

Chondrocyte viability was also assessed microscopically by LIVE/DEAD staining. Representative pictures of LIVE-stained chondrocytes at both 5× and 10× magnification are shown in Figure 2.

Fig. 2

Representative LIVE staining at 5× and 10× magnification of chondrocytes treated with each condition, stained using 4 mM Calcien AM and visualised using fluorescence microscopy under a fluorescein isothiocyanate (FITC) filter. Chondrocytes co-treated with bupivacaine and hyaluronic acid (HA) showed improved density when compared with bupivacaine-only treated chondrocytes. There was no noticeable increase in cell density between cells treated with lidocaine and cells co-treated with lidocaine and HA (PBS, phosphate buffered saline).

At 5× magnification, the media only, PBS and HA groups show high, even live cell distribution and low dead signal. 1% lidocaine and 0.25% bupivacaine show lower live cell density, while also showing increased dead cell staining at 5× magnification. Under the 10× objective, the remaining live chondrocytes under these two conditions have a more ball-like shape.

As observed on microscopy, bupivacaine and HA co-treated groups show an increase in live stained chondrocytes at 5×. However, there is still a high density of dead staining chondrocytes as well, especially compared with the control groups. Comparing the LIVE/DEAD pictures from the 1% lidocaine treated and the lidocaine-HA co-treated chondrocytes, there does not appear to be a change in the number of LIVE/DEAD cells or in the shape of these cells.

Conclusions

Despite the scientific evidence that HA does have a positive effect on chondrocytes, there are no studies that have shown a clinical effect when used for osteoarthritis. More broadly, it is our hope that clinicians will consider more carefully non-surgical treatment and diagnosis. It may be that HA is better suited as an acute treatment following injury or inflammation. At the same time, clinical evidence of local anaesthetic toxicity is limited, suggesting that the in vivo effects of anaesthetics are missed or tempered by the normal articular environment. In clinical practice, the presence of normal synovial fluid, which is rich in HA, may mitigate some of this anaesthetic toxicity. In applying our growing foundation of knowledge to clinical practice, the risks and benefits of all our treatments must be weighted among individual patient factors. Understanding situations in which the normal joint environment is compromised, such as after haemarthrosis or injury, will help us to guide treatments to maximise benefit while minimising risks.