Elevated Lipid Metabolites in Stored Clinical OCA Media Correlate With Chondrocyte Death

Abstract

Background: A major limitation of osteochondral allografts (OCA) is the deterioration of cartilage health associated with cell death during prolonged storage. However, little is known about the mechanisms that contribute to chondrocyte death during storage. Purpose/Hypothesis: This study aimed to determine whether bioactive lipid metabolites accumulate in the storage media of OCA and whether they are associated with a loss of chondrocyte viability during prolonged storage. It was hypothesized that free fatty acids (FFAs) would accumulate over time in the storage media of OCA and adversely affect cartilage health during storage. Study Design: Controlled laboratory study. Methods: A group of 21 (n = 6-8 OCA/treatment group) fresh human hemicondylar OCA tissues and media were analyzed after 7, 28, and 68 days of prolonged cold (4°C) storage. Targeted mass spectrometry analysis was used to quantify bioactive FFAs, as well as primary (lipid hydroperoxide [ROOH]) and secondary (malondialdehyde) lipid oxidation products. Chondrocyte viability was measured using a fluorescence-based live/dead assay and confocal microscopy. Results: The concentration of all targeted fatty acid metabolites in storage media was significantly increased with increased cold storage time ( P < .05). ROOH was significantly higher on day 28 of cold storage. No difference in secondary ROOH products in storage media was observed. Chondrocyte viability significantly declined in both the en face and the vertical cross-sectional analysis with increased cold storage time and inversely correlated with fatty acid metabolites ( P < .05). Conclusion: It is well established that elevated levels of certain FFAs and lipid oxidation products can alter cell function and cause cell death via lipotoxicity and other mechanisms. This work is the first to identify elevated levels of FFA metabolites and primary oxidation lipid products in the storage media from clinical OCA. The concentrations of FFA metabolites were measured at levels (>100 µM) known to induce cell death and were directly correlated with chondrocyte viability. Clinical Relevance: These findings provide important targets for understanding why cartilage health declines during cold storage, which can be used to optimize media formulations and improve graft health.