Femtosecond laser photoporation enables efficient delivery of apoptotic vesicles for enhanced osteogenesis

Abstract

Abstract A major barrier to the translation of vesicle-based therapeutics in regenerative medicine is the ability to achieve efficient intracellular delivery without compromising, and ideally amplifying, biological activity. As a bioactive class of cell-derived vesicles with pro-regenerative potential, apoptotic vesicles remain functionally limited by inefficient intracellular delivery. Here, we present a femtosecond laser photoporation-enabled strategy that overcomes this bottleneck and transforms apoVs delivery into a programmable osteogenic stimulus in bone marrow mesenchymal stem cells (BMSCs). Leveraging the non-invasive nature, high biocompatibility, precise controllability, and ultrahigh temporal and spatial resolution of femtosecond laser photoporation, this platform induces localized and transient membrane permeabilization, enabling rapid and efficient apoVs entry with minimal cellular damage. Under the optimized condition, laser-assisted photoporation markedly enhances apoVs internalization and robustly promotes BMSCs migration, osteogenic differentiation, matrix mineralization, and ectopic bone formation in vivo. Mechanistically, femtosecond laser photoporation functions not only as a delivery-enhancing approach but also as an upstream physical trigger that induces early intracellular biophysical responses and subsequently activates focal adhesion-associated ERK signaling, ultimately amplifying the pro-osteogenic bioactivity of apoptotic vesicles. These findings establish femtosecond laser photoporation not only as a high-precision intracellular delivery modality but also as a biophysical regulator of stem cell fate determination. More broadly, this work establishes a mechanistically grounded framework for integrating precision physical stimulation with vesicle therapeutics, with potential implications for bone regeneration and next-generation regenerative nanomedicine. Graphical abstract