Synergistic aligned neuronal and vascular growth inside 3D-PEG-Anisogels utilizing a triple-co-culture

Over the past few decades, researchers in tissue engineering and regenerative medicine have developed numerous strategies for cell growth and regeneration, both in vitro and in vivo. However, these strategies often focus on only one specific biomaterial, cellular composition, or molecular guidance cue, while their combination could lead to undesired cross-interactions. This study aims to understand the combined effects of several design parameters on joint neuro-vascular regeneration, including hydrogel stiffness, cell adhesive molecules, media conditions, and anisotropic guiding elements. We used a fully synthetic PEG-hydrogel system to establish a tri-culture of dorsal root ganglia from chicken embryos, human umbilical vein endothelial cells, and human mesenchymal stem cells. All cell types grew well inside the PEG-hydrogels, independent of the tested hydrogel stiffness and the coupled cell-adhesive peptides IKVAV and RGD. Only fibronectin led to increased formation of vascular-like structures. We observed that adding specific growth factors to enhance vascular-like structure formation, such as angiopoietins or platelet-derived growth factor, decreased overall neuronal growth in stiffer hydrogels. This adverse effect on neurite extension was mitigated by using an Anisogel containing thin, high-aspect-ratio, magnetically aligned microgels. On the other hand, the addition of aligned microgels to a hydrogel complicated the formation of vascular-like structures, likely due to steric hindrance, which was countered by increasing the cell concentration, thereby promoting endothelial cell-cell interactions. This demonstrates that cross-interaction of regeneration strategies has to be studied more thoroughly to enable their success.