2.1 Cell culture

Ethical approval was granted by the UK National Research Ethics Service (NRES 14-ES-1044) in accordance with the Declaration of Helsinki (World Medical Association), and was institutionally approved and sponsored by the University of Birmingham as required under the UK Research Governance Framework. Study participants were provided in advance with a participant information sheet, and a participant consent form.

Following patient's written consent, femoral heads were collected from patients undergoing total joint replacement surgery for hip osteoarthritis. Osteoblasts were cultured out from bone chips as previously described.¹¹ In brief, the articular cartilage was removed from the femoral head subchondral bone cut into small chips. The bone chips were then washed thoroughly in serum-free primary osteoblast media to remove any excess blood, connective or adipose tissue and then incubated in differentiation media (10% FCS, penicillin (100units/ml), streptomycin (100 μg/ml), l-glutamine (2 mM), β-glycerophosphate (2 mM), Ascorbic acid (50 μg/ml) and Dexamethasone (10 nM)) in a culture flask at 37 °C (5% CO2). Differentiation media was replaced with fresh media 2x per week, and the bone chips removed upon the appearance of osteoblast cells. Primary human osteoblast cells were cultured in differentiation media.

2.2 Osteoblast proliferation and morphology

Primary human osteoblasts were seeded at a density of 10,000 cells per well in a 96 well-plate. At confluency the cells were exposed for 30 min to either PBS control or to a clinically relevant LIA treatment (Table 1). After 30 min of treatment, wells were aspirated, cells washed with PBS and then placed back into 200 μl of cell culture media and cultured at 37 °C. After 24 h and 7 days of culture, cellular proliferation was determined by MTS assay (Cell Titer Aqueous One Solution Cell Proliferation Assay, Promega, Southampton, UK) performed as per manufacturer's instructions as a measure of osteoblast proliferation. To assess osteoblast morphology, primary human osteoblasts were seeded at a density of 50,000 cells per well in a 24 well-plate. Following PBS or LIA treatment cells were visualised using a confocal microscope and images of the cells captured.

2.3 Osteoprotegerin and RANKL ELISAs

Primary human osteoblasts cultured in a 96-well plate at 10,000 cells/well were treated with either PBS or LIA treatment for 30 min as previously described., After 24 h and 7 days, OPG and RANKL in the cell supernatants were determined using commercially available ELISAs (DuoSet ELISA Development Series) performed as per the manufacturer's instructions.

2.4 Alkaline phosphatase assay

Primary human osteoblasts were seeded at a density of 10,000 cells per well in a 96 well-plate. Cells were allowed to reach confluence, before being exposed to 100 μl of either PBS or to a LIA treatment (Table 1) for 30 min. Wells were then aspirated, washed with PBS and placed in 200 μl of culture media and cultured as before at 37 °C. After 24 h and 7 days at 37 °C, cells were lysed in 100 μl of RIPA buffer containing protease inhibitors. Alkaline phosphatase activity in the cell lysates was determined as previously described,¹² with absorbance measured at 405 nm against alkaline phosphatase standards. Total protein content was determined by Pierce BCA assay (ThermoFisher, UK) and Alkaline phosphatase activity expressed as units of activity per mg of total protein.

2.5 Statistical analysis

Data was analysed by 1-way ANOVA with Dunnett's post-hoc tests. Data within figures represents mean ± SEM, with significance accepted as p < 0.05.

3.1 The effect of LIA treatments on human hip OA osteoblast morphology and proliferation

We first examined the effect of the LIA reagents and their combinations (Table 1) on osteoblast morphology. Osteoblasts exposed for 30 min to the PBS control maintained a spindle like morphology and aligned to the surface of the culture plates over the course of 7 days of culture. Conversely, cells which were exposed for 30 min to 0.5% Bupivacaine showed a clear reduction in confluency and cell homogeneity after 7 days of culture. Furthermore, the combination of Bupivacaine with either 0.0005% Adrenaline or 0.5% Ketorolac, and the combination of 0.5% Ropivacaine with Ketorolac also reduced cell confluence after 7 days post-exposure (Fig. 1). The effects of cell morphology and confluence were particularly pronounced in the combination of LIA reagents. Osteoblasts exposed to 1% Lidocaine either with or without Adrenaline appeared to maintain a similar morphology to the control PBS treated cells (Fig. 1).

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Fig. 1 Human osteoblast morphology following acute exposure to LIA reagents. Primary human osteoblasts were seeded in into 24-well plates at 50,000 cells per well and exposed to either PBS or LIA reagents (n = 3) for 30 min. Cells were viewed by confocal microscopy 7 days after exposure to reagents.

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We next examined the effect of the LIA regimens on osteoblast proliferation as determined by MTA assay. At 24 h post-exposure to LIA reagents, there was no significant reduction in cell proliferation in osteoblasts exposed to any of the LIA monotherapies at clinically relevant concentrations, namely 0.5% Bupivacaine, 1% Lidocaine, 0.5% Ropivacaine or 0.5% Ketorolac (Fig. 2A). However, osteoblasts that were exposed to the combination treatments of either Bupivacaine/Adrenaline or Bupivacaine/Ketorolac showed a significant reduction in proliferation after 24 h compared to PBS treated cells (p < 0.05; Fig. 2A).

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Fig. 2 The effect of acute LIA exposure on human hip OA osteoblast proliferation. Primary osteoblasts cultured in 96-well plates were exposed to either PBS or LIA reagents for 30 min. Cellular proliferation was determined by MTS assay at (A) 24 h and (B) 7 days post exposure to reagents. Bars represent mean ± SEM (n = 4). * = p < 0.05, ** = p < 0.0, *** = p < 0.001, significantly different from PBS control cells, as determined by 1-way ANOVA using Dunnett's post-hoc tests.

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At 7 days post-exposure, osteoblasts exposed to either Bupivacaine alone (p < 0.001), or the combination treatments of either Bupivacaine/Adrenaline (p < 0.001), Bupivacaine/Ketorolac (p < 0.001) or Ropivacaine/Ketorolac (p < 0.01) showed a significant reduction in proliferation compared to control cells (Fig. 2B).

3.2 The effect of LIA treatments on human hip OA osteoblast function

We next investigated whether the LIA treatments affected osteoblast function by first examining their effect on the secretion of OPG and RANKL, which are known mediators of bone remodelling. Osteoblasts treated with either 0.5% Bupivacaine or 0.5% Ketorolac exhibited a significant reduction in OPG production after only 24 h post exposure (p < 0.0001). Furthermore, the combination treatments of either Bupivacaine or Ropivacaine with either Adrenaline or Ketorolac were also found to significantly reduce OPG production at 24 h post exposure (Fig. 3A). The reduction in OPG production was sustained at 7 days post-exposure in osteoblasts treated with either Ropivacaine alone or the combination treatments of Bupivacaine/Adrenaline, Ropivacaine/Adrenaline, Bupivacaine/Ketorolac and Ropivacaine/Ketorolac (Fig. 3B). Production of RANKL was not significantly altered by any of the LIA treatments (Fig. 3B), and therefore LIA treatments that significantly reduced OPG production also increased the RANKL:OPG ratio (Fig. 3C).

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Fig. 3 The effect of acute LIA exposure on human hip OA osteoblast OPG and RANKL secretion. Primary osteoblasts cultured in 96-well plates were exposed to either PBS or LIA reagents for 30 min. (A) OPG and (B) RANKL production was determined by ELISA at 24 h post-exposure and 7 days post-exposure. (C) Represents the RANKL/OPG ratio. Bars represent mean ± SEM (n = 3). * = p < 0.05, ** = p < 0.0, *** = p < 0.001, significantly different from PBS control cells, as determined by 1-way ANOVA using Dunnett's post-hoc tests.

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Given the differential effects of Bupivacaine and Lidocaine on both osteoblast proliferation and OPG production we then further examined the effect of these reagents on osteoblast function by determining alkaline phosphatase activity. At 24 h post-exposure, 0.5% Bupivacaine, but not 1% Lidocaine, significantly reduced alkaline phosphatase activity, compared to control cells (p < 0.01). However, both the combination of either Bupivacaine/Adrenaline (p < 0.01) or Lidocaine/Adrenaline (p < 0.001) also significantly reduced alkaline phosphatase activity, compared to control cells (Fig. 4A). At 7 days post-exposure, osteoblast alkaline phosphatase activity in cells treated with either Lidocaine alone, or the combination of Lidocaine/Adrenaline was no different to control cells (Fig. 4B). By comparison, osteoblasts exposed to either Bupivacaine alone, or the combination of Bupivacaine/Adrenaline exhibited a sustained reduction in alkaline phosphatase activity at 7 days, comparted to control cells (Fig. 4B).

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Fig. 4 The effect of acute anaesthetic exposure on human osteoblast alkaline phosphatase activity. Primary osteoblasts cultured in 96-well plates were exposed to either PBS or LIA reagents for 30 min. Alkaline phosphatase activity in cell lysates was determined at (A) 24 h post-exposure and (B) 7 days post-exposure. Bars represent mean ± SEM (n = 4). ** = p < 0.0, *** = p < 0.001, significantly different from PBS control cells, as determined by 1-way ANOVA using Dunnett's post-hoc tests.

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4.1 Conclusion

In summary, this study shows that LIA reagents at concentrations advocated for clinical use, particularly when used in combination, have detrimental effects on osteoblast viability and function in vitro. Clearly, further studies, both in vitro and in vivo are warranted in order to provide definitive recommendations. While the ability to progress a patient through a recovery pathway at an accelerated rate is undoubtedly attractive, it must not come at the cost of long-term wellbeing. For this reason, extensive study is required into the potential long-term side effects of LIA.