Red blood cell exposure increases chondrocyte susceptibility to oxidative stress following hemarthrosis

Objective The detrimental effects of blood exposure on articular tissues are well characterized, but the individual contributions of specific whole blood components are yet to be fully elucidated. Better understanding of mechanisms that drive cell and tissue damage in hemophilic arthropathy will inform novel therapeutic strategies. The studies here aimed to identify the specific contributions of intact and lysed red blood cells (RBCs) on cartilage and the therapeutic potential of Ferrostatin-1 in the context of lipid changes, oxidative stress, and ferroptosis. Methods Changes to biochemical and mechanical properties following intact RBC treatment were assessed in human chondrocyte-based tissue-engineered cartilage constructs and validated against human cartilage explants. Chondrocyte monolayers were assayed for changes to intracellular lipid profiles and the presence of oxidative and ferroptotic mechanisms. Results Markers of tissue breakdown were observed in cartilage constructs without parallel losses in DNA (control: 786.3 (102.2) ng/mg; RBCINT: 751 (126.4) ng/mg; P = 0.6279), implicating nonlethal chondrocyte responses to intact RBCs. Dose-dependent loss of viability in response to intact and lysed RBCs was observed in chondrocyte monolayers, with greater toxicity observed with lysates. Intact RBCs induced changes to chondrocyte lipid profiles, upregulating highly oxidizable fatty acids (e.g., FA 18:2) and matrix disrupting ceramides. RBC lysates induced cell death via oxidative mechanisms that resemble ferroptosis. Conclusions Intact RBCs induce intracellular phenotypic changes to chondrocytes that increase vulnerability to tissue damage while lysed RBCs have a more direct influence on chondrocyte death by mechanisms that are representative of ferroptosis.