Poster 124: Elevated Lipid Metabolites in Stored Clinical OCA Media are Associated with Chondrocyte Death

OBJECTIVES: A major limitation of osteochondral allografts (OCA) is the deterioration of cartilage health associated with cell death during prolonged storage. It is well established that chondrocyte viability in OCA is a critical factor for repair and clinical outcomes, but little is known about the mechanisms that contribute to chondrocyte death during storage. Elevated bioactive lipid metabolites and free fatty acids (FFAs) have been associated with cell death and tissue damage for various pathologies through lipotoxicity. However, the levels and effect of FFA accumulation in OCA media during prolonged storage are unknown. The purpose of this study was to determine if bioactive lipid metabolites accumulate in the storage media of OCA and are associated with a loss of chondrocyte viability during prolonged storage. We hypothesized that FFAs will accumulate over time in the storage media of OCA and adversely impact cartilage health during storage. METHODS: To elucidate potential mechanisms of cartilage health decline during storage, 21 fresh human hemi-condyle osteochondral allograft tissues and media provided by a US tissue bank were analyzed after 7, 28, and 68 days of prolonged cold storage. Targeted metabolomics analysis measured bioactive FFAs, and primary (lipid hydroperoxide, ROOH) and secondary (malondialdehyde, MDA) lipid oxidation products. Chondrocyte viability was measured using standard LIVE/DEAD assay and confocal microscopy. RESULTS: All fatty acid metabolites in storage media significantly increased with increased storage time (Figure 1; p<0.05). ROOH was significantly higher at Day 28 of storage. No difference in secondary lipid hydroperoxide products in storage media were observed. Chondrocyte viability significantly declined in both the en face and vertical cross-section analysis with increased storage time (Figure 2) and inversely correlated with fatty acid metabolites (Figure 3; p<0.05). CONCLUSIONS: It is well established that elevated levels of certain free fatty acids and lipid oxidation products can alter cell function and cause cell death through lipotoxicity and other mechanisms. This work is the first to identify elevated levels of FFA metabolites and primary oxidation lipid product in the storage media from clinical OCA. The concentrations of FFA metabolites were measured at levels (>100µM) known to induce cell death and were directly correlated with chondrocyte viability. These findings provide important targets for understanding why cartilage health declines during storage to better optimize media formulations and improve graft health.