Design and characterization of microporous hyaluronic acid hydrogels for in vitro gene transfer to mMSCs.

The effective and sustained delivery of DNA locally could increase the applicability of gene therapy in tissue regeneration and therapeutic angiogenesis. One promising approach is to use porous hydrogel scaffolds to encapsulate and deliver nucleotides in the form of nanoparticles to the affected sites. We have designed and characterized microporous (µ-pore) hyaluronic acid hydrogels which allow for effective cell seeding in vitro post-scaffold fabrication and allow for cell spreading and proliferation without requiring high levels of degradation. These factors, coupled with high loading efficiency of DNA polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique, then allowed for long-term sustained transfection and transgene expression of incorporated mMSCs. In this study, we examined the effect of pore size on gene transfer efficiency and the kinetics of transgene expression. For all investigated pore sizes (30, 60, and 100 µm), encapsulated DNA polyplexes were released steadily, starting by day 4 for up to 10 days. Likewise, transgene expression was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. The knowledge acquired through this in vitro model can be utilized to design and better predict scaffold-mediated gene delivery for local gene therapy in an in vivo model where host cells infiltrate the scaffold over time.

The effect of vascular endothelial growth factor (VEGF) presentation within fibrin matrices on endothelial cell branching.

Vascular endothelial growth factor (VEGF) has been extensively investigated to promote vascularization at damaged or diseased sites and in tissue implants. Here we are interested in determining if the manner in which VEGF is presented from a scaffold to endothelial cells influences the architecture of the blood vessels formed. We bound VEGF to nanoparticles and placed these nanoparticles inside fibrin hydrogels, which contained human umbilical vein endothelial cells (HUVECs) bound to cytodex beads. Fibroblast cells are plated on top of the fibrin gel to further mimic a physiologic environment. In addition, we used a chorioallantoic membrane (CAM) assay to determine the role of VEGF presentation on angiogenesis in vivo. We tested VEGF bound in high density and low density to study differences between growth factor presentation in heterogeneous nanodomains and homogenous distribution. VEGF covalently bound to nanoparticles at high density led to an increase in HUVEC tube branching, thickness, and total vessel network length compared to soluble VEGF. While VEGF bound electrostatically exhibited no significant difference with covalently bound VEGF in the tube formation assay, this method failed to promote host vessel infiltration into the fibrin implant on the CAM. Together our data suggest that the mode of VEGF presentation to endothelial cells influences the vessel architecture and vascularization of implants in vivo.