Cartilage explant model reflects resolvin D1 increase during recovery from LPS stimulation

Abstract

Purpose: Osteoarthritis (OA) is a low-grade chronic inflammatory condition wherein the homeostatic balance of catabolic and anabolic processes favour the former, leading to a net decline in structural integrity of cartilage. The dissimilating pathways contributing to this decline have attracted much scientific scrutiny, with a host of pro-inflammatory cytokines and catabolic enzymes now known to orchestrate the protracted but eventual loss in joint function. Yet, amid the homeostatic badinage between repair and decline, there lies a wealth of reparative physiology which delays the tissue failure which characterizes clinical OA. An improved understanding of the interplay between pro-inflammatory and pro-resolving mediators can inform better management of early-stage OA, which can thus prolong healthy tissue function. Resolvin D1 (RvD1) is a pro-resolving mediator, which has been found to be dysregulated in rheumatoid arthritis and assists with resolving acute inflammation. The role of RvD1 in cartilage recovery from pro-inflammatory stimulation with lipopolysaccharide (LPS) is not known. We hypothesized that after an acute bout of inflammation in the articulating joint, cartilage-derived RvD1 is increased during the recovery phase, and thus participates in initiating inflammation resolution. The aim of this pilot study was to quantify production of RvD1 by cartilage explants following in vitro exposure to LPS. Methods: A single front limb from seven market weight pigs were collected and aseptically dissected extracting 0.4mm articular cartilage explants from the intercarpal joint. Explants were cultured in media (DMEM, antibiotics, amino acids) and incubated at 37°C and 7%CO2 for 168h. Media was removed and refreshed every 24h, following the first 48h of acclimatization explants were stimulated with LPS (0 or 10μg/mL) for 48h (collection time: 0h-S, 24h-S, 48h-S), followed by 48h of recovery (72h-R, 96h-R) in which media was refreshed without LPS stimulation. Media samples from the final 96h (0h-S to 96h-R) were collected every 24h during media changes and analyzed for nitric oxide (NO) (Greiss Reaction) and RvD1 (ELISA). Data were analyzed using a PROC GLIMMIX model (SAS Studio) looking at time-treatment interactions and visualized with two scatter plot graphs. Data are shown as mean±SEM and significance was accepted at p<0.05. Results: LPS stimulated explants elicited an increase in NO compared to controls from 0h-S to 48h-S and continued to increase until 72h-R (p=0.0006), as shown in Figure 1. RvD1 did not elicit a significant response to LPS stimulation but when graphed over time (0h-S to 96h-R) as seen in Figure 2, did visually show a change in response to stimulation and in comparison to its unstimulated controls. RvD1 response in unstimulated explants was elevated at 24h-R, (2109.40±188.56pg/mL) before continuously decreasing until 96h-R (1514.63±203.50pg/mL). RvD1 release in LPS stimulated explants decreased from 0h-S to 48h-S (0h: 1790.83±188.56pg/mL;48h:1489.11±203.50pg/mL) then increased until 96h-R (72h: 1743.28±177.16pg/mL; 96h:1814.47±177,17pg/mL).View Large Image Figure ViewerDownload Hi-res image Download (PPT)