Development of a microRNA Delivery Scaffold System for Bone Tissue Engineering

Abstract

Although bone has an intrinsic capacity for self repair, the healing of large bone defects that typically present in humans often involves complications which result in failure to heal, leading to delayed or non-union of the defect. Due to limitations of current therapeutic approaches of autografting and allografting, the use of tissue engineered scaffolds has emerged. Despite some success with this approach, these scaffolds often require a further stimulus to promote complete healing of large bone defects. microRNAs (miRNAs) have recently emerged as promising therapeutics to stimulate bone repair, owing to their ability to intercept entire gene cohorts. However, the development of a safe and efficient localised delivery system is required for successful clinical translation of miRNA therapeutics to bone tissue engineering (TE). The overall goal of the research presented in this thesis was to determine the potential of using in-house synthesised nano-sized hydroxyapatite particles (nHA) to act as non-viral vectors for the delivery of a series of miRNAs to human (h)MSCs and to determine the combination leading to optimal osteogenesis and angiogenesis before ultimately producing miRNA-activated scaffolds capable of mediating enhanced osteogenesis by human MSCs. In the study presented in Chapter 2 of this thesis, nHA particles combined with reporter miRNAs demonstrated potential as highly efficient and minimally cytotoxic non-viral vectors for the delivery of miRNA enhancers and inhibitors (miR-mimics and antagomiRs) to human MSCs. Single administration of low miRNA doses rendered very pronounced silencing activities to a level comparable to viral and lipid-based vectors and ultimately, a 20 nM dose was brought forward for further study. In Chapter 3, efficient nHA-based delivery of antagomiR-133a, antagomiR-16 and a miR-210 mimic, three targets identified to have particular therapeutic potential, enhanced osteogenesis by human MSCs. AntagomiR-133a emerged as the optimal osteo-therapeutic, while both antagomiR-16 and the miR-210 mimic were deemed worthy of further investigation.In Chapter 4, the coll-nHA scaffolds demonstrated significant potential for the efficient localised delivery of both miR-mimics and antagomiRs to human MSCs, representing the first non-viral, non-lipid, ‘off-the shelf’ 3D system developed in the field. Additionally, antagomiR-133a activated scaffolds upregulated Runx2 and orchestrated accelerated calcium deposition, thus showcasing the osteo-therapeutic potential of this innovative strategy for bone TE applications. In Chapter 5, while the miR-210 mimic showed a limited pro-angiogenic therapeutic efficacy, the combinatorial delivery of the miR-210 mimic with antagomiR-16 demonstrated significant potential to simultaneously enhance the angiogenesis and osteogenesis capabilities of human MSCs. This dual formulation presents the first combinatorial miRNA approach harnessing these two processes and sits within seminal reports on the recently emergent field of combinatorial miRNA delivery. Collectively, this thesis has demonstrated that nHA particles are able to deliver miRNAs with superior efficiency than that reported for other non-viral systems. When applied in 3D, a miRNA-activated coll-nHA scaffold with significantly enhanced therapeutic potential was achieved. Together with the demonstration of successful combinatorial miRNA delivery to harness both angiogenesis and osteogenesis, this underlines the immense potential of extending this platform to different fields of TE beyond osteogenesis and bone repair.