# Iron toxicity undermines microfracture-induced cartilage regeneration by predisposing a pre-ferroptotic niche

## TL;DR

Iron toxicity from microfracture surgery harms cartilage regeneration by creating a pre-ferroptotic environment, which can be mitigated with iron chelators or antioxidants.

## Contribution

The study reveals that iron toxicity creates a pre-ferroptotic niche in cartilage regeneration after microfracture.

## Key findings

- Regenerated cartilage showed disrupted cellular organization and a poor extracellular matrix.
- Iron chelators and lipid ROS scavengers improved joint mobility and tissue quality.
- Iron toxicity was linked to increased sphingolipid signaling and mitochondrial/lysosomal changes.

## Abstract

Microfracture (MF) often yields regenerated cartilage that resembles scar tissue and is prone to rapid deterioration. This outcome may be linked to elevated iron levels, which upregulate sphingolipid (SP) signaling and increase lipid exposure to reactive oxygen species (ROS), thereby heightening cellular sensitivity to iron. In this study, we analyze whether heme-derived iron released during clinical MF undermines cartilage regeneration. We compared regenerated and intact cartilage using histomorphological, proteomic, metabolomic, and transcriptional analyses. Regenerated tissue exhibited disrupted cellular organization and a deficient extracellular matrix. Omics profiling highlighted transferrin-mediated iron transfer, striking SP signaling, and increased oxidized glutathione tripeptide in cartilage regeneration. Integrated analysis further revealed a pre-ferroptotic microenvironment in newborn chondrocytes after MF, which is characterized by extracellular Fe3+ accumulation, moderately increased Fe2+ levels, heterogeneous expression of ferroptotic markers, and altered mitochondrial and lysosomal structures. To assess the role of iron toxicity and iron-dependent oxidative stress, we administered intra-articular injections of the iron chelator deferoxamine (DFO) or the lipid ROS scavenger ferrostatin-1 (FER-1). Both treatments improved joint mobility, increased regenerated tissue thickness, elevated proteoglycan content, reduced sphingomyelin levels, preserved mitochondrial structure, and decreased lysosome abundance. These findings demonstrate that iron toxicity establishes a pre-ferroptotic niche that compromises cartilage regeneration following MF. In this study, we provide new mechanistic insights for developing targeted therapeutic strategies to enhance cartilage restoration.