Early Intervention Strategies For Acute Cartilage Injury: Ex-vivo Porcine Knee Model

OBJECTIVES: Traumatic injury to cartilage has been shown to lead to Post-Traumatic Osteoarthritis (PTOA). The acute phase of PTOA is characterized with increased expression of aggrecanases and inflammatory cytokines in the injured cartilage. Early intervention therapies aim to be administered during the acute phase for the prevention of PTOA development. Our objective was to determine the effect of Interleukin Receptor Antagonist Protein (IRAP), Hyaluronan (HA), and Mesenchymal Stem Cell (MSC) treatment as early intervention strategies by examining the changes in microRNA (miRNA) and mRNA expression in cartilage at 8 hours after impact injury. METHODS: Custom impact device was used to create replicable injury ex-vivo to intact porcine knee joint. Injury was caused by dropping a 10kg weight one time from 1m directly above the knee in extension. One hour after impact 20µg/mL IRAP, 15mg/mL HA (MW 1.9 MDa), or 5x106 P4 MSCs in 1mL saline was intra-articularly injected. Control legs (no injury) and injury legs (injury, no treatment) received saline injection. At 8 hours post-injury, cartilage samples were harvested for genetic expression analysis. Genetic expression of miR-140 (regulates ADAMTS-5) miR-125b (regulates ADAMTS-4), ADAMTS-4, ADAMTS-5, MMP-3, IL-1β, and TNF-α were analyzed by RT-PCR. Groups were compared by one-way analysis of variance followed by Tukey's post-hoc test. A P-value <0.05 was considered significant (N=3 pigs/group). RESULTS: After IRAP treatment, expressions of ADAMTS-4, ADAMTS-5, IL-1β, and TNF-α in cartilage were significantly down-regulated from injury group (all P<0.001) (Figure 1A). Expressions of miR-140 and miR-125b were significantly up-regulated after IRAP treatment as compared to control and injury (both P<0.05 to control, P<0.001 to injury) (Figure 1B). MiR-27b expression was significantly up-regulated after treatment as compared to control (P<0.001). After HA treatment, expressions of ADAMTS-4, ADAMTS-5, MMP-3, and TNF-α were significantly up-regulated from control (all P<0.05) (Figure 1C). Expression of IL-1β after HA treatment tended for up-regulation (P=0.15). Expressions of miR-125b, miR-140, and miR-27b were significantly up-regulated after HA treatment as compared to control and injury groups (all P<0.05) (Figure 1D). After MSC treatment, relative expressions of ADAMTS-4, ADAMTS-5, and IL-1β were significantly up-regulated (all P<0.01) as compared to controls (Figure 1E). Expression of TNF-α after treatment tended for up-regulation (P=0.14). No significant differences were found in miRNA expressions after MSC treatment (Figure 1F). CONCLUSION: The results show that IRAP, HA, and MSC treatment administered during acute phase of cartilage injury each have a distinct effect on catabolic and inflammatory regulation. HA and MSC treatment did not have significant effect on the inflammatory and catabolic response of injured cartilage within the first 8 hours of injury. However, the beneficial effects of HA may take place beyond the acute time frame as suggested by increased miR-125b and miR-140 expressions after treatment. IRAP treatment increased expressions of miR-140, -125b, and -27b in cartilage, indicating increased inhibition of their respective enzymes, as shown by reduced aggrecanase and inflammatory genetic expressions. Clinically, these findings support the potential of IRAP and HA treatment as early intervention strategies for the prevention of cartilage degeneration after impact injury.