Effects of Premixing Betamethasone and Lidocaine on Chondrocyte Inflammation in an In Vitro Model Camille Pinpin1, Michael J. Sayegh2, Christopher G. Larsen2, Pooja Swami3, Andrew Wong3, Jessica M. Intravia2, Kate W. Nellans2, Lewis B. Lane2, Daniel Grande3 1Donald and Barbara Zucker School of Medicine at Hofstra/Northwell 2Department of Orthopedic Surgery, Northwell Health, Manhasset, NY 3Feinstein Institute for Medical Research at Northwell Health, Manhasset, NY
Background
Results
In orthopedics and hand surgery, corticosteroid injections remain an essential intervention for non-operative treatment of inflammatory conditions such as trigger finger and carpal tunnel syndrome. The cellular mechanism involves nuclear steroid-glucocorticoid receptor complexes that prevent the synthesis of pro-inflammatory prostaglandins and leukotrienes [1]. The most common synthetic preparations used in orthopedics are derivates of prednisolone. Betamethasone acetate/betamethasone sodium phosphate (BSP) suspension (Celestone Soluspan) is preferred amongst hand surgeons. Betamethasone is routinely mixed with anesthetics, such as lidocaine hydrochloride, for diagnostic and therapeutic purposes. Lidocaine causes a reversible conduction block along sensory nerve fibers [2]. It is common practice for hand surgeons to premix these medications in pre-loaded syringes to increase efficiency during clinic.
Results
TNF 200
ADAM-TS4
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Chondrocytes treated with BSP alone and with BSP/lidocaine mixtures prepared at 0 hr., 4 hours, and 24 hours prior to injection showed significantly decreased levels of TNF (p <0.0001, 0.0053, <0.0001, <0.0001), ADAMTS4 (p <0.0001, 0.0001, 0.0002,<0.0001) and MMP3 (p<0.0001) compared to the positive control. There were no significant differences in ADAMTS4 and MMP3 between all experimental groups. There was a significant decrease in TNF with both the 24-hour premixture (p=0.0457) and BSP alone (p=0.0435) compared to the 0 hr. mixture. MMP1 reduced with BSP alone and with mixtures prepared at 0 hr. and 4 hours prior to injection (p=0.0021, 0.0071, 0.0130) compared to the positive control. MMP1 was not significantly different between experimental groups. IL-8 decreased with BSP alone and with mixtures prepared at 4 hours and 24 hours prior to injection (p=0.0005, 0.0341, 0.0010) compared to the positive control. IL-8 was also not significantly different between experimental groups. IL-6 and MMP13 in the experimental groups were not significantly different from the positive control or from each other.
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Figure 2: Glucocorticoid Intracellular Mechanism. Active steroid-glucocorticoid receptor complexes translocate to the cell nucleus and bind to promoter regions of target genes, particularly resulting in upregulation of annexin-1. Annexin-1 strongly inhibits phospholipase-A2 and therefore prevents the synthesis of prostaglandins and leukotrienes, the chief mediators of inflammation.
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Hypothesis We hypothesize that BSP and lidocaine mixtures prepared within an acute time interval (0-24 hours) from injection will not have significantly different anti-inflammatory effects on chondrocytes in an in vitro model.
Methods
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Conclusions This study is the first to report on the interaction between betamethasone and lidocaine as well as the efficacy of premixing them in an in vitro model. Our results demonstrate that treatment with BSP alone and with BSP/lidocaine mixtures prepared 0-24 hours before injection do not significantly differ in reduction of certain key inflammatory mediators, particularly ADAMTS4 and MMP3. Further studies will investigate the anti-inflammatory effects of premixing BSP and lidocaine within a longer time interval (i.e. 1 day to 1 week) from injection. The results from this foundational study and future studies can help to improve the quality and efficiency of delivering corticosteroid injections, an important diagnostic and therapeutic intervention in orthopedics and hand surgery.
IL-8
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Figure 7: Independent CRISPR knockout of CDK4 or CDK6 does not cause dropout in most breast cancer cell lines studied.
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Human chondrocytes were plated in monolayer culture at a concentration of 100,000 cells per well. The positive control (Ctrl) group and experimental groups were treated with inflammatory cytokines,10 ng/ml of IL-1β and 50 ng/ml of oncostatin-M (OSM). Four experimental groups were additionally treated with BSP alone or BSP mixed with lidocaine (LE) at the time of treatment (0 hr.), 4 hours, or 24 hours prior to treatment. The final concentrations of BSP and lidocaine were 6 mg/ml and 0.2 mg/ml, respectively. Mixtures were stored at room temperature in the dark to mimic storage of pre-loaded syringes in clinic. After one hour of exposure, the chondrocytes were collected for relative gene expression analysis via isolation of mRNA followed by synthesis of cDNA and quantitative polymerase chain reaction (qPCR).
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Figure 1. Gene expression following 60 minutes of inflammatory stimulation (IL-1β and OSM) with BSP alone or mixed with lidocaine at 0 hr. (time of treatment), 4 hr. before treatment, or 24 hr. before treatment. Positive control received inflammatory stimulation only. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001.
References [1] Dahl et al., J Hand Surg Am. 2012. [2] Bräu et al., Anesth Analg. 1998.