Necroptosis of stem cell aggregates suppresses bone regeneration via necroptosis-derived extracellular vesicles

Abstract

Despite the therapeutic promise of stem cell aggregates in bone regeneration, clinical outcomes remain unstable due to the complex and incompletely understood fate of transplanted cells. Here, we investigated the role of programmed cell death in this process, moving beyond the established paradigm of apoptosis. Our study demonstrates that stem cell aggregates (CAs) from human exfoliated deciduous teeth (SHEDs) implanted into a murine femoral defect model undergo necroptosis in a time-dependent manner. Pre-induction of necroptosis in CAs prior to transplantation profoundly impaired bone repair, significantly reducing new bone formation, osteogenic differentiation, and vascularization at the defect site. Conversely, pharmacological inhibition of necroptosis using Necrostatin-1 rescued the regenerative capacity. Crucially, we identified that this inhibitory effect is primarily mediated by extracellular vesicles released during necroptosis (Nec-EVs). Inhibition of Nec-EV biogenesis with GW4869 restored bone healing. Characterization confirmed the successful isolation of phosphorylated mixed lineage kinase domain-like protein (pMLKL)-enriched Nec-EVs. Proteomic analysis revealed a distinct cargo profile in Nec-EVs, notably enriched in proteins involved in translation regulation and RNA metabolism. Functionally, Nec-EVs directly suppressed the proliferation and osteogenic potential of bone marrow stem cells (BMSCs) in vitro, and their local application in vivo was sufficient to recapitulate the impaired bone regeneration phenotype. Collectively, this work establishes necroptosis and its associated vesicular signaling as novel detrimental axes in cell-based bone regeneration, providing mechanistic insights and potential therapeutic targets for enhancing the reliability of regenerative therapies.

Keywords

Necroptosis / Extracellular vesicles / Stem cell aggregates / Bone regeneration / SHED

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