"INTRODUCTION: There remains a substantial unmet clinical need for tissue engineered strategies to heal large volume bone defects. The delivery of microRNAs from biomaterial-based scaffolds presents a promising approach: whereby the scaffold provides a structural support to bone tissue while the microRNAs (miRs) induce the endogenous cells to produce relevant therapeutic proteins and genes at physiological levels while shutting off aberrant effects 1,2. However, the effective delivery of miRs is frequently jeopardized by their poor stability, requiring a suitable vector which would protect them from degradation guaranteeing their effective intracellular delivery and transient secretion of osteogenic proteins by host cells. In this study, collagen-nanohydroxyapatite (coll-nHA) scaffolds1,2,3, previously optimized for bone repair within our lab, were coupled with self-assembling, amphiphilic, cell-penetrating RALA peptide4 as a delivery non-viral vector yielding a scaffold-based system for simultaneous delivery of miR-26a mimic1,2 and miR-133a inhibitor5 for bone repair.
METHODS: miRs were complexed with cationic RALA peptide5, incorporated (1μg or 3 μg) into coll-nHA scaffolds1 which were assessed in terms of calcium release, loading efficacy, distribution and release of nanoparticles (NPs). 3×105 human mesenchymal stem cells (hMSC) were seeded onto the miR-activated scaffolds and the expression of miRs, metabolic activity, DNA content, ALP activity and calcium deposition were quantified. The scaffolds were implanted into calvarial defect in male rats, the total bone volume and tissue mineral density were assessed at week 4, 8 and 12 of the study.
RESULTS: The NPs were successfully incorporated into scaffolds and worked effectively delivering miRs to the hMSCs in controlled manner. The miR-activated scaffolds cultured in cell-free media showed sustained release of miRs, uptake of calcium, and an increase in compressive modulus. The scaffolds delivered the miR-26a mimic or miR-133a inhibitor, either alone or combined, to the hMSCs resulting in a silencing effect and an enhanced ALP activity. The miR-activated scaffolds enhanced the healing in rat calvaria generating greater amount of bone compared to the scaffold alone.
CONCLUSION: This study describes the development of scaffold system using self-assembling, amphiphilic, cell-penetrating peptide for sustained delivery of therapeutic microRNAs for treatment of bone defects. The miR-activated scaffolds transfected the hMSCs with miRs enhancing the osteogenesis of the cells3,5. The miR-scaffold system has potential to be used as a next generation therapeutic for repair of large bone defects offering precise and transient gene editing with minimal immunogenicity. The novel miR co-delivery scaffold-based system is versatile and has the potential for a myriad of applications beyond bone repair by tailoring the individual miRs delivered – as well as the scaffold composition.
ACKNOWLEDGEMENTS: National Science Foundation- Science Foundation Ireland (NSF-SFI) US-Ireland R&D Partnership Programme (NSF_ 17_US_3437). JMS benefits from a Marie Skłodowska-Curie Individual Fellowship from the European Commission through the H2020 project GAMBBa (Project ID: 892389).
REFERENCES: 1)Mencía Castaño I. et al., J. Control. Release., 2015 200: 42-51, 2)Raftery R. et al., Adv. Mater., 2016 28: 5447-5469, 3)Mencía Castaño I. et al., Sci. Rep., 2016 6: 2794, 4)McCarthy H et al., J Control Release, 2014 189: 141-9, 5)Li L. et al., Biomaterials, 2013 34: 5048-58."