(267f) An Injectable and Anisotropic Hydrogel with Biomimetic Structures for Directed Cell and Nerve Growth

Many biological tissues consist of complex, anisotropic, and hierarchical structures, such as nerve, bone, enamel, and heart. For these tissues, regenerative matrices have to mimic the biological architecture to guide cell organization during the healing process. However, up to now, most injectable materials, which allow a minimal-invasive application, are isotropic and therefore, lack the ability to template complex tissues with directionally organized functions and mechanical properties. Here, we demonstrate a new injectable composite material, which has the ability to direct cell growth. The composite hydrogel contains rod-shaped microgel objects with variable mechanical properties, geometry, and porosity, which are incorporated into a surrounding biocompatible hydrogel. The microgels are doped with a low amount of superparamagnetic iron oxide nanoparticles (SPIONs), which induce longitudinal alignment in situ within a magnetic field in the milli tesla range. Moreover, defined amounts of cell adhesive peptides can be covalently coupled to the microgel precursor to foster a cell-specific interaction and thus mimics the features of endogenous structural proteins. Both nonadhesive and biofunctionalized microgels are applied to create a low-invasive, injectable, anisotropic hydrogel (‘Anisogel’). This study encloses the development and characterization of the Anisogel in regard to the cellular response of fibroblasts, primary nerve cells and dorsal root ganglions (DRGs).¹

Rod-shaped microgels were fabricated with a mold-based soft lithography approach. The microgels consist of a UV-crosslinking star-shaped poly(EO-stat-PO)-acrylate (sPEG-A), which was supplemented with SPIONs. The harvested microgels can be tuned in regard to their size, aspect ratio, stiffness, and porosity, as well as modified in their interaction with cells by covalently attached cell adhesion peptides. For coupling of cell-adhesive peptides the Michael-type addition between thiol-containing biomolecules and the acrylate end group of the sPEG-A in basic conditions was utilized. Furthermore, doping microgels with SPIONs allows their orientation within less than 60 s in a magnetic field of ~100 mT. Interestingly, microgels that have an aspect ratio of 10 exhibit an ultrahigh magnetic response, which allows significant microgel alignment in only 1.9 mT, which corresponds to only 19 times the earth’s magnetic field. In order to create a regenerative anisotropic hydrogel, microgels were aligned in a fibrin gel, which enzymatically polymerizes within 120 s, allowing prior microgel alignment. After fibrin gelation, the microgels maintained their position and orientation in the absence of a magnetic field. To investigate the material’s functionality in regard to neural tissue, fibroblasts, primary neurons or DRGs were inserted into the Anisogel. When the microgels were randomly oriented, cells infiltrated the matrix less and showed isotropic morphologies. In the case of oriented microgels, extending cells were affected by the aligned physical barrier, resulting in linear cell outgrowth. Interestingly, very low amounts between 1 to 3 vol % of structural guidance elements were sufficient to align the cells. In some cases a single contact with an oriented microgel was enough to align the nerve cells over tens of micrometer distances, propelling the hypothesis that the cells decide to orient inside the Anisogel. The attachment of adhesion peptides to microgels led to a stronger interaction with the surrounding cells, while maintaining the induced cellular directionality. Therefore, the Anisogel with biointeractive microgels can superiorly mimic the ECM of complex tisssues.

By applying magnetoceptive, tailorable microgel rods in a fibrin hydrogel, a global material anisotropy was created after injection. The biomaterial is the first that can achieve highly controlled and ordered structures in situ and demonstrated to guide neurite growth in a linear manner. This feature could be groundbreaking as supporting therapeutic material for spinal cord repair.

1. Rose, J. C.; Cámara-Torres, M.; Rahimi, K.; Köhler, J.; Möller, M.; De Laporte, L., Nerve Cells Decide to Orient inside an Injectable Hydrogel with Minimal Structural Guidance. Nano Lett. 2017.