Highly versatile cell-penetrating peptide loaded scaffold for efficient and localised gene delivery to multiple cell types: From development to application in tissue engineering.

Gene therapy has recently come of age with seven viral vector-based therapies gaining regulatory approval in recent years. In tissue engineering, non-viral vectors are preferred over viral vectors, however, lower transfection efficiencies and difficulties with delivery remain major limitations hampering clinical translation. This study describes the development of a novel multi-domain cell-penetrating peptide, GET, designed to enhance cell interaction and intracellular translocation of nucleic acids; combined with a series of porous collagen-based scaffolds with proven regenerative potential for different indications. GET was capable of transfecting cell types from all three germ layers, including stem cells, with an efficiency comparable to Lipofectamine® 3000, without inducing cytotoxicity. When implanted in vivo, GET gene-activated scaffolds allowed for host cell infiltration, transfection localized to the implantation site and sustained, but transient, changes in gene expression - demonstrating both the efficacy and safety of the approach. Finally, GET carrying osteogenic (pBMP-2) and angiogenic (pVEGF) genes were incorporated into collagen-hydroxyapatite scaffolds and with a single 2 µg dose of therapeutic pDNA, induced complete repair of critical-sized bone defects within 4 weeks. GET represents an exciting development in gene therapy and by combining it with a scaffold-based delivery system offers tissue engineering solutions for a myriad of regenerative indications.

Harnessing an Inhibitory Role of miR-16 in Osteogenesis by Human Mesenchymal Stem Cells for Advanced Scaffold-Based Bone Tissue Engineering.

MicroRNA (miRNA) therapeutics is increasingly being developed to either target bone-related diseases such as osteoporosis and osteoarthritis or as the basis for novel bone tissue engineering strategies. A number of miRNAs have been reported as potential osteo-therapeutics but no consensus has yet been established on the optimal target. miR-16 has been studied extensively in nonosteogenic functions and used as functionality reporter target in the development of nonviral miRNA delivery platforms. This study hypothesized that miR-16 may also play an inhibitory role in osteogenesis due to its ability to directly target Smad5 and AcvR2a. This study thus aimed to assess the potential of miR-16 inhibition to increase osteogenesis in human mesenchymal stem cells (hMSCs) using a previously established miRNA delivery platform composed of nanohydroxyapatite (nHA) particles as nonviral vectors in combination with collagen-nHA scaffolds designed specifically for bone repair. Initial results showed that antagomiR-16 delivery efficiently increased the relative levels of both putative targets and Runx2, the key transcription factor for osteogenesis, while also increasing osteocalcin levels. Furthermore, significant increases in mineral calcium deposition by hMSCs were found in both monolayer and most importantly in scaffold-based osteodifferentiation studies, ultimately demonstrating that miR-16 inhibition further enhances the therapeutic potential of a scaffold with known potential for bone repair applications and thus holds significant therapeutic potential as a novel bone tissue engineering strategy. Furthermore, we suggest that harnessing the additional functions known to miR-16 by incorporating either its enhancers or inhibitors to tissue-specific tailored scaffolds provides exciting opportunities for a diverse range of therapeutic indications.

Delivery of the improved BMP-2-Advanced plasmid DNA within a gene-activated scaffold accelerates mesenchymal stem cell osteogenesis and critical size defect repair.

Gene-activated scaffolds have been shown to induce controlled, sustained release of functional transgene both in vitro and in vivo. Bone morphogenetic proteins (BMPs) are potent mediators of osteogenesis however we found that the delivery of plasmid BMP-2 (pBMP-2) alone was not sufficient to enhance bone formation. Therefore, the aim of this study was to assess if the use of a series of modified BMP-2 plasmids could enhance the functionality of a pBMP-2 gene-activated scaffold and ultimately improve bone regeneration when implanted into a critical sized bone defect in vivo. A multi-cistronic plasmid encoding both BMP-2 and BMP-7 (BMP-2/7) was employed as was a BMP-2-Advanced plasmid containing a highly truncated intron sequence. With both plasmids, the highly efficient cytomegalovirus (CMV) promoter sequence was used. However, as there have been reports that the elongated factor 1-α promoter is more efficient, particularly in stem cells, a BMP-2-Advanced plasmid containing the EF1α promoter was also tested. Chitosan nanoparticles (CS) were used to deliver each plasmid to MSCs and induced transient up-regulation of BMP-2 protein expression, in turn significantly enhancing MSC-mediated osteogenesis when compared to untreated controls (p < 0.001). When incorporated into a bone mimicking collagen-hydroxyapatite scaffold, the BMP-2-Advanced plasmid, under the control of the CMV promotor, induced MSCs to produce approximately 2500 µg of calcium per scaffold, significantly higher (p < 0.001) than all other groups. Just 4 weeks post-implantation in vivo, this cell-free gene-activated scaffold induced significantly more bone tissue formation compared to a pBMP-2 gene-activated scaffold (p < 0.001) as indicated by microCT and histomorphometry. Immunohistochemistry revealed that the BMP-2-Advanced plasmid accelerated differentiation of osteoprogenitor cells to mature osteoblasts, thus causing rapid healing of the bone defects. This study confirms that optimising the plasmid construct can enhance the functionality of gene-activated scaffolds and translate to accelerated bone formation in a critical sized defect.

Translating the role of osteogenic-angiogenic coupling in bone formation: Highly efficient chitosan-pDNA activated scaffolds can accelerate bone regeneration in critical-sized bone defects.

The clinical translation of bioactive scaffolds for the treatment of large segmental bone defects has remained a challenge due to safety and efficacy concerns as well as prohibitive costs. The design of an implantable, biocompatible and resorbable device, which can fill the defect space, allow for cell infiltration, differentiation and neovascularisation, while also recapitulating the natural repair process and inducing cells to lay down new bone tissue, would alleviate the problems with existing treatments. We have developed a gene-activated scaffold platform using a bone-mimicking collagen hydroxyapatite scaffold loaded with chitosan nanoparticles carrying genes encoding osteogenic (BMP-2) and angiogenic (VEGF) proteins. With a single treatment, protein expression by mesenchymal stem cells (MSCs) seeded onto the scaffold is sustained for up to 28 days and is functional in inducing MSC osteogenesis. The in vivo safety and efficacy of this gene-activated scaffold platform was demonstrated resulting in the successful transfection of host cells, abrogating the requirement for multiple procedures to isolate cells or ex vivo cell culture. Furthermore, the level of bone formation at the exceptionally early time-point of 28 days was comparable to that achieved following recombinant BMP-2 protein delivery after 8 weeks in vivo, without the adverse side effects and at a fraction of the cost. This naturally derived cell-free gene-activated scaffold thus represents a new 'off-the-shelf' product capable of accelerating bone repair in critical-sized bone defects.

Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis.

Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

A novel collagen-nanohydroxyapatite microRNA-activated scaffold for tissue engineering applications capable of efficient delivery of both miR-mimics and antagomiRs to human mesenchymal stem cells.

Manipulation of gene expression through the use of microRNAs (miRNAs) offers tremendous potential for the field of tissue engineering. However, the lack of sufficient site-specific and bioactive delivery systems has severely hampered the clinical translation of miRNA-based therapies. In this study, we developed a novel non-viral bioactive delivery platform for miRNA mimics and antagomiRs to allow for a vast range of therapeutic applications. By combining nanohydroxyapatite (nHA) particles with reporter miRNAs (nanomiRs) and collagen-nanohydroxyapatite scaffolds, this work introduces the first non-viral, non-lipid platform to date, capable of efficient delivery of mature miRNA molecules to human mesenchymal stem cells (hMSCs), a particularly difficult cell type to transfect effectively, with minimal treatment-associated cytotoxicity. Firstly, miRNAs were successfully delivered to hMSCs in monolayer, with internalisation efficiencies of 17.4 and 39.6% for nanomiR-mimics and nanoantagomiRs respectively, and both nanomiR-mimics and nanoantagomiRs yielded sustained interfering activity of greater than 90% in monolayer over 7 days. When applied to 3D scaffolds, significant RNA interference of 20% for nanomiR-mimics and 88.4% for nanoantagomiRs was achieved with no cytotoxicity issues over a 7 day period. In summary, in-house synthesised non-viral nHA particles efficiently delivered reporter miRNAs both in monolayer and on scaffolds demonstrating the immense potential of this innovative miRNA-activated scaffold system for tissue engineering applications.