Extracellular vesicles (EVs) are emerging in tissue engineering as promising acellular tools, circumventing many of the limitations associated with cell-based therapies. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Therefore, this study aimed to investigate the potential of augmenting osteoblast epigenetic functionality using the HDAC inhibitor Trichostatin A (TSA) to enhance the therapeutic efficacy of osteoblast-derived EVs for bone regeneration. TSA was found to substantially alter osteoblast epigenetic function through reduced HDAC activity and increased histone acetylation. Treatment with TSA also significantly enhanced osteoblast alkaline phosphatase activity (1.35-fold), collagen production (2.8-fold) and calcium deposition (1.55-fold) during osteogenic culture (P ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) exhibited reduced particle size (1-05-fold) (P > 0.05), concentration (1.4-fold) (P > 0.05) and protein content (1.16-fold) (P ≤ 0.001) when compared to untreated EVs. TSA-EVs significantly enhanced the proliferation (1.13-fold) and migration (1.3fold) of human bone marrow stem cells (hBMSCs) when compared to untreated EVs (P ≤ 0.05). Moreover, TSA-EVs upregulated hBMSCs osteoblast-related gene and protein expression (ALP, Col1a, BSP1 and OCN) when compared to cells cultured with untreated EVs. Importantly, TSA-EVs elicited a time-dose dependent increase in hBMSCs extracellular matrix mineralisation. MicroRNA profiling revealed a set of differentially expressed microRNAs from TSA-EVs, which were osteogenic-related. Target prediction demonstrated these microRNAs were involved in regulating pathways such as 'endocytosis' and 'Wnt signalling pathway' . Moreover, proteomics analysis identified the enrichment of proteins involved in transcriptional regulation within TSA-EVs. Taken together, our findings suggest that altering osteoblasts' epigenome accelerates their mineralisation and promotes the osteoinductive potency of secreted EVs partly due to the delivery of pro-osteogenic microRNAs and transcriptional regulating proteins. As such, for the first time we demonstrate the potential to harness epigenetic regulation as a novel engineering approach to enhance EVs therapeutic efficacy for bone repair. K E Y WO R D S bone, epigenetics, extracellular vesicles, histone deacetylase, microRNAs, tissue engineering, trichostatin AThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Extracellular Vesicles (EVs) are considered promising nanoscale therapeutics for bone regeneration. To date, EVs are typically procured from cells on 2D tissue culture plastic, an artificial environment that limits cell growth and does not replicate in situ biochemical or biophysical conditions. This study investigated the potential of 3D printed titanium scaffolds coated with hydroxyapatite to promote the therapeutic efficacy of osteoblast-derived EVs. Ti6Al4V titanium scaffolds with different pore sizes (500 and 1000 µm) and shapes (square and triangle) were fabricated by selective laser melting. A bone-mimetic nano-needle hydroxyapatite (nnHA) coating was then applied. EVs were procured from scaffold-cultured osteoblasts over 2 weeks and vesicle concentration was determined using the CD63 ELISA. Osteogenic differentiation of human bone marrow stromal cells (hBMSCs) following treatment with primed EVs was evaluated by assessing alkaline phosphatase activity, collagen production and calcium deposition. Triangle pore scaffolds significantly increased osteoblast mineralisation (1.5-fold) when compared to square architectures (P ≤ 0.001). Interestingly, EV yield was also significantly enhanced on these higher permeability structures (P ≤ 0.001), in particular (2.2-fold) for the larger pore structures (1000 µm). Furthermore osteoblast-derived EVs isolated from triangular pore scaffolds significantly increased hBMSCs mineralisation when compared to EVs acquired from square pore scaffolds (1.7-fold) and 2D culture (2.2-fold) (P ≤ 0.001). Coating with nnHA significantly improved osteoblast mineralisation (>2.6-fold) and EV production (4.5-fold) when compared to uncoated scaffolds (P ≤ 0.001). Together, these findings demonstrate the potential of harnessing bone-mimetic culture platforms to enhance the production of pro-regenerative EVs as an acellular tool for bone repair.

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Extracellular vesicles (EVs) have the potential to provide new insights into skeletal muscle (SM) physiology and pathophysiology. However, current isolation protocols often do not eliminate co-isolated components such as lipoproteins and RNA binding proteins that could confound outcomes and hinder downstream clinical translation. In this study, we validated an EV isolation protocol that combined size-exclusion chromatography (SEC) with ultrafiltration (UF) to increase sample throughput, scalability and purity, while providing the very first analysis of the effects of UF column choice and fraction window on EV recovery. C2C12 myotube conditioned medium was pre-concentrated using either Amicon ® Ultra 15 or Vivaspin ® 20 100 KDa UF columns and processed by SEC (IZON, qEV 70 nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. The EV marker TSG101 could be detected from fractions 5 to 14, while CD9 and Annexin A2 only up to fraction 6. ApoA1 + lipoprotein co-isolates were detected from fraction 6 onwards for both protocols. Strikingly, Amicon and Vivaspin UF concentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co-isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. In conclusion, our study developed an effective UF+SEC protocol for the isolation of EVs based on sample purity (fractions 1-5) and total EV abundance (fractions 2-10). We provide evidence to demonstrate that the choice of UF column can affect the composition of the resulting EV preparation and needs to be considered when being applied in EV isolation studies in SM. The resulting protocols will be valuable in isolating highly pure EV preparations for applications in a range of therapeutic and diagnostic studies.

While extracellular vesicles (EVs) continue to gain interest for therapeutic applications, their clinical translation is limited by a lack of optimal isolation methods. We sought to determine how universally applied isolation methods impact EV purity and yield. EVs were isolated by ultracentrifugation (UC), polyethylene glycol precipitation, Total Exosome Isolation Reagent, an aqueous two-phase system with and without repeat washes or size exclusion chromatography (SEC). EV-like particles could be detected for all isolation methods but varied in their purity and relative expression of surface markers (Alix, Annexin A2, CD9, CD63 and CD81). Assessments of sample purity were dependent on the specificity of characterisation method applied, with total particle counts and particle to protein (PtP) ratios often not aligning with quantitative measures of tetraspanin surface markers obtained using high-resolution nano-flow cytometry. While SEC resulted in the isolation of fewer particles with a relatively low PtP ratio (1.12 × 107 ± 1.43 × 106 vs highest recorded; ATPS/R 2.01 × 108 ± 1.15 × 109, p ⩽ 0.05), EVs isolated using this method displayed a comparatively high level of tetraspanin positivity (e.g. ExoELISA CD63⁺ particles; 1.36 × 1011  ± 1.18 × 1010 vs ATPS/R 2.58 × 1010  ± 1.92 × 109, p ⩽ 0.001). Results originating from an accompanying survey designed to evaluate pragmatic considerations surrounding method implementation (e.g. scalability and cost) identified that SEC and UC were favoured for overall efficiency. However, reservations were highlighted in the scalability of these methods, which could potentially hinder downstream therapeutic applications. In conclusion, variations in sample purity and yield were evident between isolation methods, while standard non-specific assessments of sample purity did not align with advanced quantitative high-resolution analysis of EV surface markers. Reproducible and specific assessments of EV purity will be critical for informing therapeutic studies.