In regenerative medicine, extracellular vesicles (EVs) have been increasingly studied as alternative acellular therapies overcoming the limitations of cell-based strategies. Derived from mineralising osteoblasts, EVs demonstrated their osteogenic potency suggesting their potential as a novel bone regenerative therapy. However, the clinical translation of EVs remains limited by issues associated with the scalability, reproducibility and purity of these naturally-derived nanoparticles. In this study, the characterisation of mineralising-osteoblast-derived EVs (MO-EVs) was performed to inspire the development of osteogenic synthetic EVs.
EVs were collected from cultures of mineralising osteoblasts over a 2-week period and the EV-isolation was performed by ultracentrifugation. Subsequently, the size, ζ-potential, morphology and particle concentration of these nanovesicles was characterised and the presence of tetraspanin markers (CD9, CD63 and CD81) was confirmed using nano-flow cytometry. Furthermore, the pro-osteogenic capacity of MO-EVs was assessed in vitro via quantifying alkaline phosphatase (ALP) activity and calcium deposition. From the composition analysis of MO-EVs, bio-inspired proteoliposomes harboring ALP and/or annexin VI were formulated via the thin-film hydration method followed by extrusion. Both the activity of the proteins post-insertion and their resulting incorporation efficiency in proteoliposomes were then determined. Additionally, cell-derived nanovesicles (CDNs) were produced by the serial extrusion of mineralising osteoblasts and the resulting synthetic EVs were similarly characterised.
The isolation of MO-EVs was validated as positivity for all tetraspanin markers was reported for these sub-100 nm vesicles. Notably, their osteogenic potency was confirmed in vitro on osteoblasts as MO-EVs increased significantly ALP activity, calcium deposition and collagen production after a 2-week treatment. MO-EVs were found enriched in several annexin proteins which guided the formulation of bio-inspired proteoliposomes. The insertion of both ALP and annexin VI was successful with >30% incorporation efficiency obtained for all formulations. Importantly, EV-inspired liposomes harboring annexin VI or ALP were found to be functional with the validation of the mediation of Ca2+-influx by annexin VI inside proteoliposomes and the confirmation of ALP enzymatic activity. Moreover, CDNs were also successfully produced as a nanoparticle population with an EV-size was obtained after serial extrusion. Both EV-inspired proteoliposomes and CDNs’ osteogenic potencies were then compared to MO-EVs after 14 days in osteogenic conditions.
Taken together, these results shows the potential of the development of synthetic EVs as biomimetic nanocarriers to accelerate the clinical translation of EV-based therapies for bone regeneration.