Lidocaine potentiates the chondrotoxicity of methylprednisolone.

PURPOSE: This study examined the viability of bovine articular chondrocytes after exposure to methylprednisolone, methylprednisolone with lidocaine, and methylprednisolone in a simulated inflammatory environment. METHODS: Bovine articular chondrocytes were suspended in alginate beads and cultured in Dulbecco's modified Eagle's medium/F-12 for 1 week before experimentation. Suspended chondrocytes were exposed to 0.9% saline solution (negative control), methylprednisolone (4, 8, and 16 mg/mL), methylprednisolone (8 mg/mL) with 1% lidocaine, or methylprednisolone (8 mg/mL) and saline solution in a simulated inflammatory environment (interleukin [IL] 1beta exposure, 10 ng/mL) for 15, 30, and 60 minutes. Flow cytometry was performed 1 day, 4 days, and 7 days after exposure by use of annexin V and propidium iodide to assess chondrocyte viability. RESULTS: Chondrocyte viability decreased from 84% in saline solution to 62%, 38%, and 2.4% 1 day after 60 minutes of exposure to 4, 8, and 16 mg/mL of methylprednisolone, respectively (n = 7, P < .05). Chondrotoxicity increased with increasing time of exposure to methylprednisolone and with increasing time after exposure. In IL-1beta-activated chondrocytes, viability decreased from 76% in saline solution to 2.9% after 60 minutes of methylprednisolone exposure (8 mg/mL) (n = 4, P < .05). The combination of 8 mg/mL of methylprednisolone and 1% lidocaine further reduced viability to 1.0% after 60 minutes (n = 4, P < .05). CONCLUSIONS: These results show a dose- and time-dependent decrease in chondrocyte viability after exposure to clinically relevant doses of methylprednisolone. The combination of methylprednisolone and lidocaine was toxic, with virtually no cells surviving after treatment. In addition, methylprednisolone did not mitigate the inflammatory effects of IL-1beta; rather, it further potentiated the chondrotoxicity. CLINICAL RELEVANCE: Intra-articular injections of corticosteroids and local anesthetics are widely used in clinical practice. This in vitro study provides information on the potential effects of these drugs on articular cartilage.

An osteopontin-NADPH oxidase signaling cascade promotes pro-matrix metalloproteinase 9 activation in aortic mesenchymal cells.

Osteopontin (OPN) is a cytokine upregulated in diabetic vascular disease. To better understand its role in vascular remodeling, we assessed how OPN controls metalloproteinase (MMP) activation in aortic adventitial myofibroblasts (AMFs) and A7r5 vascular smooth muscle cells (VSMCs). By zymography, OPN and tumor necrosis factor (TNF)-alpha preferentially upregulate pro-matrix metalloproteinase 9 (pro-MMP9) activity. TNF-alpha upregulated pro-MMP9 in AMFs isolated from wild-type (OPN(+/+)) mice, but pro-MMP9 induction was abrogated in AMFs from OPN(-/-) mice. OPN treatment of VSMCs enhanced pro-MMP9 activity, and TNF-alpha induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F(2) alpha-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-alpha, 8-IsoP preferentially activated pro-MMP9. Superoxide, 8-IsoP, and NADPH oxidase 2 (Nox2) subunits were reduced in OPN(-/-) AMFs. Treatment of A7r5 VSMCs with OPN upregulated NADPH oxidase subunit accumulation. OPN structure/function studies mapped these activities to the SVVYGLR heptapeptide motif in the thrombin-liberated human OPN N-terminal domain (SLAYGLR in mouse OPN). Treatment of aortic VSMCs with SVVYGLR upregulated pro-MMP9 activity and restored TNF-alpha activation of pro-MMP9 in OPN(-/-) AMFs. Injection of OPN-deficient OPN(+/-) mice with SVVYGLR peptide upregulated pro-MMP9 activity, 8-IsoP levels, and Nox2 protein levels in aorta and increased panmural superoxide production (dihydroethidium staining). At equivalent hyperglycemia and dyslipidemia, 8-IsoP levels and aortic pro-MMP9 were reduced with complete OPN deficiency in a model of diet-induced diabetes, achieved by comparing OPN(-/-)/LDLR(-/-) versus OPN(+/-)/LDLR(-/-) siblings. Thus, OPN provides a paracrine signal that augments vascular pro-MMP9 activity, mediated in part via superoxide generation and oxylipid formation.

In vivo effects of single intra-articular injection of 0.5% bupivacaine on articular cartilage.

BACKGROUND: Single intra-articular injections of local anesthetics are commonly used clinically. Recent in vitro studies have demonstrated chondrotoxic effects of local anesthetics, with the greatest emphasis on bupivacaine toxicity. This in vivo study was conducted to determine whether a single intra-articular injection of 0.5% bupivacaine results in chondrocyte morbidity and rapid chondrolysis. METHODS: Forty-eight Sprague-Dawley rats received a 100-microL injection of sterile 0.9% saline solution (negative control) into one stifle joint and 100 microL of either preservative-free 0.5% bupivacaine (experimental group) or 0.6 mg/mL monoiodoacetate (positive control) into the contralateral joint. The rats were killed at one week, four weeks, twelve weeks, or six months. Live and dead cells were quantified with use of three-dimensional confocal reconstructions of fluorescent-stained tissues at standardized locations on the distal part of the femur. Histological findings were graded with use of a modified Mankin score, and cell density was quantified with use of custom image-analysis software. RESULTS: In the specimens injected with bupivacaine, the chondral surfaces remained intact as seen with gross and histological examination. No differences in superficial chondrocyte viability or modified Mankin scores were observed between the saline-solution and bupivacaine groups at any location or time point (p > 0.05). Quantitative histological analysis of the bupivacaine-treated knees at six months revealed an up to 50% reduction in chondrocyte density compared with that of the saline-solution-treated knees (p < or = 0.01). Monoiodoacetate injection resulted in death of up to 87% of the superficial chondrocyte cells at one week and chondrolysis at six months. Despite severe histological abnormalities by four weeks after monoiodoacetate injection, cartilage injury was not evident on gross inspection until six months. CONCLUSIONS: This in vivo study showing reduced chondrocyte density without cartilage tissue loss six months after a single intra-articular injection of 0.5% bupivacaine suggests bupivacaine toxicity. The effects of bupivacaine were milder than those of an injection of 0.6% monoiodoacetate, which resulted in chondrolysis over the same time period.