Dual scaffold delivery of miR-210 mimic and miR-16 inhibitor enhances angiogenesis and osteogenesis to accelerate bone healing.

Angiogenesis is critical for successful bone repair, and interestingly, miR-210 and miR-16 possess counter-active targets involved in both angiogenesis and osteogenesis: miR-210 acts as an activator by silencing EFNA3 & AcvR1b, while miR-16 inhibits both pathways by silencing VEGF & Smad5. It was thus hypothesized that dual delivery of both a miR-210 mimic and a miR-16 inhibitor from a collagen-nanohydroxyapatite scaffold system may hold significant potential for bone repair. Therefore, this systems potential to rapidly accelerate bone repair by directing enhanced angiogenic-osteogenic coupling in host cells in a rat calvarial defect model at a very early 4 week timepoint was assessed. In vitro, the treatment significantly enhanced angiogenic-osteogenic coupling of human mesenchymal stem cells, with enhanced calcium deposition after just 10 days in 2D and 14 days on scaffolds. In vivo, these dual-miRNA loaded scaffolds showed more than double bone volume and vessel recruitment increased 2.3 fold over the miRNA-free scaffolds. Overall, this study demonstrates the successful development of a dual-miRNA mimic/inhibitor scaffold for enhanced in vivo bone repair for the first time, and the possibility of extending this 'off-the-shelf' platform system to applications beyond bone offers immense potential to impact a myriad of other tissue engineering areas. STATEMENT OF SIGNIFICANCE: miRNAs have potential as a new class of bone healing therapeutics as they can enhance the regenerative capacity of bone-forming cells. However, angiogenic-osteogenic coupling is critical for successful bone repair. Therefore, this study harnesses the delivery of miR-210, known to be an activator of both angiogenesis and osteogenesis, and miR-16 inhibitor, as miR-16 is known to inhibit both pathways, from a collagen-nanohydroxyapatite scaffold system to rapidly enhance osteogenesis in vitro and bone repair in vivo in a rat calvarial defect model. Overall, it describes the successful development of the first dual-miRNA mimic/inhibitor scaffold for enhanced in vivo bone repair. This 'off-the-shelf' platform system offers immense potential to extend beyond bone applications and impact a myriad of other tissue engineering areas.

Collagen/GAG scaffolds activated by RALA-siMMP-9 complexes with potential for improved diabetic foot ulcer healing.

Impaired wound healing of diabetic foot ulcers has been linked to high MMP-9 levels at the wound site. Strategies aimed at the simultaneous downregulation of the MMP-9 level in situ and the regeneration of impaired tissue are critical for improved diabetic foot ulcer (DFU) healing. To fulfil this aim, collagen/GAG (Col/GAG) scaffolds activated by MMP-9-targeting siRNA (siMMP-9) were developed in this study. The siMMP-9 complexes were successfully formed by mixing the RALA cell penetrating peptide with siMMP-9. The complexes formulated at N:P ratios of 6 to 15 had a diameter around 100 nm and a positive zeta potential about 40 mV, making them ideal for cellular uptake. In 2 dimensional (2D) culture of human fibroblasts, the cellular uptake of the complexes surpassed 60% and corresponded to a 60% reduction in MMP-9 gene expression in low glucose culture. In high glucose culture, which induces over-expression of MMP-9 and therefore serves as an in vitro model mimicking conditions in DFU, the MMP-9 gene could be downregulated by around 90%. In the 3D culture of fibroblasts, the siMMP-9 activated Col/GAG scaffolds displayed excellent cytocompatibility and ~60% and 40% MMP-9 gene downregulation in low and high glucose culture, respectively. When the siMMP-9 complexes were applied to THP-1 macrophages, the primary cell type producing MMP-9 in DFU, MMP-9 gene expression was significantly reduced by 70% and 50% for M0 and M1 subsets, in 2D culture. In the scaffolds, the MMP-9 gene and protein level of M1 macrophages decreased by around 50% and 30% respectively. Taken together, this study demonstrates that the RALA-siMMP-9 activated Col/GAG scaffolds possess high potential as a promising regenerative platform for improved DFU healing.

Rapid bone repair with the recruitment of CD206+M2-like macrophages using non-viral scaffold-mediated miR-133a inhibition of host cells.

microRNAs offer vast therapeutic potential for multiple disciplines. From a bone perspective, inhibition of miR-133a may offer potential to enhance Runx2 activity and increase bone repair. This study aims to assess the therapeutic capability of antagomiR-133a delivery from collagen-nanohydroxyapatite (coll-nHA) scaffolds following cell-free implantation in rat calvarial defects (7 mm diameter). This is, to the best of our knowledge, the first report of successful in vivo antagomiR uptake in host cells of fully immunocompetent animals without distribution to other off-target tissues. Our results demonstrate the localized release of antagomiR-133a to the implant site at 1 week post-implantation with increased calcium deposits already evident in the antagomiR-133a loaded scaffolds at this early timepoint. This was followed by an approximate 2-fold increase in bone volume versus antagomiR-free scaffolds and a significant 10-fold increase over the empty defect controls, after just 4 weeks. An increase in host CD206+ cells suggests an accelerated pro-remodeling response by M2-like macrophages accompanying bone repair with this treatment. Overall, this non-viral scaffold-mediated antagomiR-133a delivery platform demonstrates capability to accelerate bone repair in vivo - without the addition of exogenous cells - and underlines the role of M2 macrophage-like cells in directing accelerated bone repair. Expanding the repertoire of this platform to deliver alternative miRNAs offers exciting possibilities for a variety of therapeutic indications. STATEMENT OF SIGNIFICANCE: microRNAs, small non-coding RNA molecules involved in gene regulation, may have potential as a new class of bone healing therapeutics as they can enhance the regenerative capacity of bone-forming cells. We developed a collagen-nanohydroxyapatite-microRNA scaffold system to investigate whether miR133a inhibition can enhance osteogenesis in rat MSCs and ultimately accelerate endogenous bone repair by host cells in vivo without pre-seeding cells prior to implantation. Overall, this off-the-shelf, non-viral scaffold-mediated antagomiR-133a delivery platform demonstrates capability to accelerate bone repair in vivo - without the requirement of exogenous cells - and highlights the role of CD206+M2 macrophage-like cells in guiding accelerated bone repair. Translating the repertoire of this platform to deliver alternative miRNAs offers exciting possibilities for a vast myriad of therapeutic indications.

Transfection of autologous host cells in vivo using gene activated collagen scaffolds incorporating star-polypeptides.

It is increasingly being recognised within the field of tissue engineering that the regenerative capacity of biomaterial scaffolds can be augmented via the incorporation of gene therapeutics. However, the field still lacks a biocompatible gene delivery vector which is capable of functionalizing scaffolds for tailored nucleic acid delivery. Herein, we describe a versatile, collagen based, gene-activated scaffold platform which can transfect autologous host cells in vivo via incorporation of star-shaped poly(˪-lysine) polypeptides (star-PLLs) and a plasmid DNA (pDNA) cargo. Two star-PLL vectors with varying number and length of poly(˪-lysine) arms were assessed. In vitro, the functionalization of a range of collagen based scaffolds containing either glycosaminoglycans (chondroitin sulfate or hyaluronic acid) or ceramics (hydroxyapatite or nano-hydroxyapatite) with star-PLL-pDNA nanomedicines facilitated prolonged, non-toxic transgene expression by mesenchymal stem cells (MSCs). We demonstrate that the star-PLL structure confers enhanced spatiotemporal control of nanomedicine release from functionalized scaffolds over a 28-day period compared to naked pDNA. Furthermore, we identify a star-PLL composition with 64 poly(˪-lysine) arms and 5 (˪-lysine) subunits per arm as a particularly effective vector, capable of facilitating a 2-fold increase in reporter transgene expression compared to the widely used vector polyethylenimine (PEI), a 44-fold increase compared to a 32 poly(˪-lysine) armed star-PLL and a 130-fold increase compared to its linear analogue, linear poly(˪-lysine) (L-PLL) from a collagen-chondroitin sulfate gene activated scaffold. In an in vivo subcutaneous implant model, star-PLL-pDNA gene activated scaffolds which were implanted cell-free exhibited extensive infiltration of autologous host cells, nanomedicine retention within the implanted construct and successful host cell transfection at the very early time point of just seven days. Overall, this article illustrates for the first time the significant ability of the star-PLL polymeric structure to transfect autologous host cells in vivo from an implanted biomaterial scaffold thereby forming a versatile platform with potential in numerous tissue engineering applications.

Scaffold-Based microRNA Therapies in Regenerative Medicine and Cancer.

microRNA-based therapies are an advantageous strategy with applications in both regenerative medicine (RM) and cancer treatments. microRNAs (miRNAs) are an evolutionary conserved class of small RNA molecules that modulate up to one third of the human nonprotein coding genome. Thus, synthetic miRNA activators and inhibitors hold immense potential to finely balance gene expression and reestablish tissue health. Ongoing industry-sponsored clinical trials inspire a new miRNA therapeutics era, but progress largely relies on the development of safe and efficient delivery systems. The emerging application of biomaterial scaffolds for this purpose offers spatiotemporal control and circumvents biological and mechanical barriers that impede successful miRNA delivery. The nascent research in scaffold-mediated miRNA therapies translates know-how learnt from studies in antitumoral and genetic disorders as well as work on plasmid (p)DNA/siRNA delivery to expand the miRNA therapies arena. In this progress report, the state of the art methods of regulating miRNAs are reviewed. Relevant miRNA delivery vectors and scaffold systems applied to-date for RM and cancer treatment applications are discussed, as well as the challenges involved in their design. Overall, this progress report demonstrates the opportunity that exists for the application of miRNA-activated scaffolds in the future of RM and cancer treatments.

Nanoparticle-mediated siRNA delivery assessed in a 3D co-culture model simulating prostate cancer bone metastasis.

siRNA has emerged as a potential therapeutic for the treatment of prostate cancer but effective delivery remains a major barrier to its clinical application. This study aimed to develop and characterise a 3D in vitro co-culture model to simulate prostate cancer bone metastasis and to assess the ability of the model to investigate nanoparticle-mediated siRNA delivery and gene knockdown. PC3 or LNCaP prostate cancer cells were co-cultured with hFOB 1.19 osteoblast cells in 2D on plastic tissue culture plates and in 3D on collagen scaffolds mimicking the bone microenvironment. To characterise the co-culture model, cell proliferation, enzyme secretion and the utility of two different gene delivery vectors to mediate siRNA uptake and gene knockdown were assessed. Cell proliferation was reduced by∼50% by day 7 in the co-culture system relative to monoculture (PC3 and LNCaP co-cultures, in 2D and 3D) and an enhanced level of MMP9 (a marker of bone metastasis) was secreted into the media (1.2-4-fold increase depending on the co-culture system). A cationic cyclodextrin gene delivery vector proved significantly less toxic in the co-culture system relative to the commercially available vector Lipofectamine 2000(®). In addition, knockdown of both the GAPDH gene (minimum 15%) and RelA subunit of the NF-κB transcription factor (minimum 20%) was achieved in 2D and 3D cell co-cultures. Results indicate that the prostate cancer-osteoblast in vitro co-culture model was more physiologically relevant vs the monoculture. This model has the potential to help improve the design and efficacy of gene delivery formulations, to more accurately predict in vivo performance and, therefore, to reduce the risk of product failure in late-stage clinical development.

Delivering Nucleic-Acid Based Nanomedicines on Biomaterial Scaffolds for Orthopedic Tissue Repair: Challenges, Progress and Future Perspectives.

As well as acting to fill defects and allow for cell infiltration and proliferation in regenerative medicine, biomaterial scaffolds can also act as carriers for therapeutics, further enhancing their efficacy. Drug and protein delivery on scaffolds have shown potential, however, supraphysiological quantities of therapeutic are often released at the defect site, causing off-target side effects and cytotoxicity. Gene therapy involves the introduction of foreign genes into a cell in order to exert an effect; either replacing a missing gene or modulating expression of a protein. State of the art gene therapy also encompasses manipulation of the transcriptome by harnessing RNA interference (RNAi) therapy. The delivery of nucleic acid nanomedicines on biomaterial scaffolds - gene-activated scaffolds -has shown potential for use in a variety of tissue engineering applications, but as of yet, have not reached clinical use. The current state of the art in terms of biomaterial scaffolds and delivery vector materials for gene therapy is reviewed, and the limitations of current procedures discussed. Future directions in the clinical translation of gene-activated scaffolds are also considered, with a particular focus on bone and cartilage tissue regeneration.