Development of Antibiotic-Free Antimicrobial Scaffolds for Effective Delivery of Osteogenic & Angiogenic MicroRNAs to Simultaneously Treat and Repair Infected Bone Tissue

<b>INTRODUCTION:</b> The treatment of bacterial infection of bone while regenerating bone at the same time is extraordinarily challenging in clinical settings (1). The escalating problem of antimicrobial resistance necessitates the development of novel antibiotic-free strategies to eradicate bacteria. One such approach is the use of metal-doped nanoparticles (2). To address bone regeneration, the approaches include the local delivery of gene therapies, such as microRNA in scaffold systems. A scaffold provides structural support while microRNA induces host cells to produce multiple angiogenic and osteogenic proteins essential for bone healing (3). The present study combines these two promising approaches to develop a multifunctional biomaterial-based platform with angiogenic, osteogenic and antimicrobial properties. The scaffold delivers osteogenic genes (by delivering antagomiR-138, a microRNA targeting ERK pathway) (4) and antibiotic-free antimicrobial nanoparticles (copper-doped bioactive glass, CuBG) (2), leading to a novel platform with superior therapeutic potential and angiogenic, osteogenic and antimicrobial features.<br/><br/><b>METHODS: </b>Freeze-dried collagen-based scaffolds, previously optimized in our lab for bone regeneration (3), were enriched with antagomiR-138 and Cu-BG nanoparticles and assessed <i>in vitro</i> using human mesenchymal stem cells (transfection efficacy, osteogenic and angiogenic gene expression, metabolic activity, DNA content, alkaline phosphatase activity, calcium deposition). The antimicrobial properties of the scaffolds were assessed using gram-positive (<i>S.aureus</i>) and gram-negative (<i>E.coli</i>). The angiogenic and osteogenic properties of the scaffolds were assessed in two <i>in vivo</i> models: a load-bearing 5 mm femoral defect in female Sprague Dawley rats and a chick chorioallantoic membrane model (CAM).<br/><br/><b>RESULTS SECTION:</b> The CuBG and antagomiR-138 particles were effectively incorporated into collagen scaffolds without affecting the mechanical and structural properties of the scaffolds. The CuBG scaffolds reduced the adhesion of <i>S.aureus</i> and <i>E.coli</i> 5-fold and 2-fold, respectively. The antagomiR138 and-CuBG functionalised scaffolds effectively transfected hMSCs <i>in vitro</i>, enhancing the osteogenic (COLL1A2, NOCH4, SMAD5) and angiogenic genes (VEGFA) and calcium production. The <i>in vivo</i> assessment showed that antagomiR-138 scaffolds promoted enhanced healing in the femoral defect model by producing endochondral ossification. The antagomiR138-CuBG scaffold promoted vasculogenesis, improving the number of branches and the length of blood vessels in <i>in vivo</i> CAM model.<br/><br/><b>DISCUSSION:</b> The study demonstrated the therapeutic potential of a multifunctional, antagomiR138 antimicrobial scaffold system. The system delivered antagomiR138 to hMSCs <i>in vitro, </i>enhancing the osteogenic genes and mineralisation. The incorporation of CuBG nanoparticles enhanced antimicrobial mechanisms (production of ROS) in <i>S.aureus</i> and <i>E.coli, </i>inhibiting the attachment to the scaffold. The antagomiR138 scaffolds improved osteogenesis and vasculogenesis <i>in vivo</i> in a femoral defect model and a chick chorioallantoic membrane model, respectively. Our work demonstrates, for the first time, the feasibility of combining non-antibiotic biomaterial based-approaches with gene therapeutics for developing a platform which not only regenerates bone but also fights rising problems of antimicrobial resistance. This approach opens the door to new possibilities in a myriad of indications beyond bone repair.<br/><br/><b>REFERENCES:</b> <b>1)</b> Sadowska <i>et al. Mat. Today</i>, <b>2021</b>, 46:136-154; <b>2)</b> Ryan <i>et al. Biomaterials</i>, <b>2019</b>, 197:405-416; <b>3)</b> Mencía Castaño <i>et al., Acta Biomater</i>., <b>2020</b> 109: 267-279; <b>4) </b>Eskildsen <i>et al.</i> <i>PNAS</i>, 2011, 108: 6139-6144.<br/><br/><b>ACKNOWLEDGEMENTS:</b> Marie Sklodowska Curie Individual Fellowship, the European Commission, the H2020 project GAMBBa (892389), the ON Fundation (21-053), Science Foundation Ireland under the US-Ireland Research and Development Partnership (17/US/3437)