Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast‐derived extracellular vesicles to promote human bone marrow stem cell osteogenic differentiation

Abstract

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.

Development of a Bone-Mimetic 3D Printed Ti6Al4V Scaffold to Enhance Osteoblast-Derived Extracellular Vesicles’ Therapeutic Efficacy for Bone Regeneration

“…Additionally, in this study, we reported that osteoblasts cultured on triangle scaffolds elicited substantially altered epigenetic functionality, resulting in increased osteogenic gene activation. Previously we demonstrated that altering the epigenome of mineralising osteoblast via HDACi induced hyperacetylation, significantly enriched secreted EVs with several pro-osteogenic microRNAs and transcriptional regulators which augmented the epigenetic landscape of recipient hBMSCs and accelerated its osteogenic capacity ( Man et al, 2021a ). Therefore, it is likely the EVs derived from osteoblasts cultured on triangle pore scaffolds were enriched with bioactive factors which modified the epigenetic landscape at recipient hBMSCs increasing its differentiation capacity.…”

“…Relative gene expression was evaluated as previously described ( Man et al, 2021a ). Briefly, total RNA was extracted using the RNase mini kit (Qiagen, United Kingdom) from scaffold-cultured osteoblasts according to the manufacturer’s protocol.…”

“…Medium was isolated from osteoblasts cultured on titanium scaffolds every 2 days for 14 days. EVs were isolated from the collected conditioned medium by differential centrifugation as previously described ( Man et al, 2021a ): 2000 g for 20 min, 10,000 g for 30 min and 120,000 g for 70 min. The EV pellet was washed with sterile PBS and centrifuged at 120,000 g for 70 min and the resultant pellet was re-suspended in 500 μL PBS.…”

“…Immunoblotting was used to confirm the presence of EVs as previously described ( Man et al, 2021a ). Briefly, EV proteins from osteoblasts cultured on 2D or T1000 constructs were electrophoretically separated using precast gels (4–15% Mini-PROTEAN TBX, Biorad, United Kingdom).…”

“…The multitude of bioactive factors delivered by EVs has been shown to promote stem cell mineralisation when compared to the use of single growth factor treatment ( Davies et al, 2017 ). Although the therapeutic efficacy of these cell-derived nanoparticles has been reported ( Martins et al, 2016 ; Qin et al, 2016 ; Man et al, 2021a ), to date EVs are commonly harvested from cells cultured on 2D tissue culture plastic. Conventional 2D culture limits the surface area for cell growth as well as providing an artificial cell-cell and cell-extracellular matrix interactions, which do not replicate in situ conditions mechanically or biologically ( Abbott, 2003 ; Baker and Chen, 2012 ).…”