IRW-Functionalized Milk-Derived Extracellular Vesicles Promote Osteogenic Recovery in Inflammatory Osteolysis via Modulation of the TXNIP–NRF2 Axis

Background: Inflammatory osteolysis features persistent inflammation and impaired bone formation, requiring therapeutics that concurrently suppress inflammation and promote osteogenesis. Ile-Arg-Trp (IRW), a bioactive tripeptide with anti-inflammatory and pro-osteogenic properties, suffers from poor cellular delivery. Milk-derived extracellular vesicles (MEVs) provide a biocompatible delivery platform to enhance intracellular bioavailability of IRW. We engineered IRW-functionalized MEVs (M-IRW), tested whether MEV delivery amplifies IRW’s dual bioactivity, and elucidated the molecular mechanism. Methods: The therapeutic effects of M-IRW were evaluated in LPS-stimulated MC3T3-E1 cells and a murine calvarial osteolysis model. Transcriptomic analysis and Geneformer-based virtual knockout identified candidate regulatory pathways, validated by gene silencing, rescue experiments, and reciprocal co-immunoprecipitation. A chronic apical periodontitis model assessed the disease relevance of this pathway. Results: Nano-flow cytometry and quantitative fluorescence analysis confirmed successful preparation and IRW loading of M-IRW, and M-IRW achieved enhanced cellular association with intracellular accumulation of the labeled IRW formulation relative to free peptide. In LPS-treated MC3T3-E1 cells, M-IRW more potently elevated ALP, RUNX2, and OPN expression than free IRW (p < 0.05). In vivo, M-IRW significantly enhanced osteogenic recovery in calvarial osteolysis, surpassing free IRW (p < 0.05). Transcriptomic analysis identified thioredoxin-interacting protein (Txnip) as a candidate mediator; virtual Txnip knockout predicted NRF2-associated transcriptional enrichment (p < 0.05). Depletion of NRF2 attenuated the osteogenic benefit conferred by both TXNIP knockdown and M-IRW treatment. Conclusions: M-IRW enables efficient intracellular delivery of IRW and promotes osteogenic repair in inflammatory osteolysis, at least partly through modulation of the TXNIP–NRF2 axis.