"Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Although EVs efficacy has been shown, their short half-life in vivo hinders their therapeutic potency. Gelatin methacryloyl (GelMA) hydrogels functionalised with synthetic nanoclays have been demonstrated to improve growth factor retention. This study investigated the potential of combining epigenetically activated osteoblast-derived EVs with the GelMA nanocomposite hydrogel to stimulate bone repair.
GelMA/nanoclay composites were fabricated by combining 5wt% GelMA with different concentrations of LAP (0.5, 1 and 2 wt%) prior to visible light crosslinking. The hydrogels compressive modulus, shear-thinning behaviour, 3D printing fidelity and osteogenic potency was evaluated. EVs were derived from 5 nM TSA-treated or untreated osteoblasts over a 2-week period. The EVs size, morphology and concentration were assessed via nanoflow cytometry and transmission electron microscopy. The isolated EVs were incorporated within the composites and their release kinetics were determined using the CD63 ELISA. The osteogenic differentiation of human bone marrow stromal cells (hBMSCs) within the EV-functionalised hydrogel was evaluated by qPCR, biochemistry and histological analysis.
LAP improved GelMA compressive modulus and shear-thinning properties in a dose-dependent manner. Nanoclay incorporation enhanced the shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP exhibited increased mineralisation capacity (1.41-fold) over 14 days. EV release kinetics from these nanocomposites were strongly influenced by LAP concentration with significantly more vesicles released from LAP-free constructs. EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), and mineralisation (1.87-fold) of hBMSCs when released from the GelMA-LAP hydrogel compared to the untreated EV gels. Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs.
Taken together, these findings demonstrate the potential of combining epigenetically-activated osteoblast-derived EVs with a nanocomposite photocurable hydrogel to enhance the therapeutic efficacy of acellular vesicle approaches for bone regeneration."