Introduction: Impaired fracture healing, specifically non-union, has been found to occur up to 14% in tibial shaft fractures. The current standard of care to treat non-union often requires additional surgeries which can result in long recovery times. Injectable-based therapies to accelerate fracture healing have the potential to mitigate the need for additional surgeries. Gene therapies have recently undergone significant advancements due to developments in nanotechnology, which improve mRNA stability while reducing immunogenicity. Methods: In this study, we tested the efficacy of mineral coated microparticles (MCM) and fluoride-doped MCM (FMCM) to effectively deliver firefly luciferase (FLuc) mRNA lipoplexes (LPX) to the fracture site. Here, adult mice underwent a tibia fracture and stabilization method and all treatments were locally injected into the fracture. Level of osteogenesis and amount of bone formation were assessed using gene expression and histomorphometry respectively. Localized and systemic inflammation were measured through gene expression, histopathology scoring and measuring C-reactive protein (CRP) in the serum. Lastly, daily IVIS images were taken to track and measure transfection over time. Results: MCM-LPX-FLuc and FMCM-LPX-FLuc were not found to cause any cytotoxic effects when tested in vitro. When measuring the osteogenic potential of each mineral composition, FMCM-LPX-FLuc trended higher in osteogenic markers through qRT-PCR than the other groups tested in a murine fracture and stabilization model. Despite FMCM-LPX-FLuc showing slightly elevated il-1β and il-4 levels in the fracture callus, inflammation scoring of the fracture callus did not result in any differences. Additionally, an acute systemic inflammatory response was not observed in any of the samples tested. The concentration of MCM-LPX-FLuc and FMCM-LPX-FLuc that was used in the murine fracture model did not stimulate bone when analyzed through stereological principles. Transfection efficacy and kinetics of delivery platforms revealed that FMCM-LPX-FLuc prolongs the luciferase signal both in vitro and in vivo. Discussion: These data together reveal that FMCM-LPX-FLuc could serve as a promising mRNA delivery platform for fracture healing applications.

Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based transfection is an advancement in gene delivery technology, as it represents a non-viral approach that enables highly efficient, localized transfection and allows for transfection of three-dimensional cell constructs.

Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based transfection is an advancement in gene delivery technology, as it represents a non-viral approach that enables highly efficient, localized transfection and allows for transfection of three-dimensional cell constructs.

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 10⁴ cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).