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 (1.3‐fold) 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.
Highlights
There is a tremendous need for bone tissue due to numerous clinical situations (Baroli, 2009; Dimitriou et al, 2011), with currently 10 million people in the UK affected by musculoskeletal disorders costing the National Health Service £4.76 billion annually (Chance-Larsen et al, 2019)
We demonstrated that Extracellular vesicles (EVs) isolated from Trichostatin A (TSA)-treated osteoblasts substantially promoted the mineralisation capacity of human bone marrow stem cells (hBMSCs) through the early, mid and late-stages of osteogenic differentiation exhibited by enhanced osteoblast-related gene/protein expression, alkaline phosphatase (ALP) activity and extracellular matrix mineralisation compared to MO-EVs treated and the untreated cells
In addition to conducting in vivo testing, to move this novel approach towards the clinic, demonstration of TSA-EVs efficacy against a positive treatment control would be a valuable pursuit, for example bone morphogenic protein 2 (BMP2). These findings demonstrate that altering osteoblasts epigenetic functionality via TSA induced hyperacetylation, enhanced the differentiation capacity of the parental cell and the osteoinductive potency of their EVs
Summary
There is a tremendous need for bone tissue due to numerous clinical situations (Baroli, 2009; Dimitriou et al, 2011), with currently 10 million people in the UK affected by musculoskeletal disorders costing the National Health Service £4.76 billion annually (Chance-Larsen et al, 2019). This is anticipated to increase further in the future as a result of the growing ageing population and demand for continued quality of life in the older years. There is a growing demand to develop acellular approaches for bone repair (Burdick et al, 2013)
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