(637c) Development and Characterization of Injectable, Guest-Host Hydrogels for Neural Tissue Engineering Applications

INTRODUCTION: Traumatic brain injury (TBI) is a global health concern that affects millions of people each year. Primary and secondary injury sequences associated with a sustained TBI lead to acute and chronic cellular processes that exacerbate the damage caused by the initial injury. While current research focuses on the use of tissue engineering strategies to modulate the injury pathology, there are no approved therapies to treat the underlying cellular mechanisms involved in the progression of TBI-related tissue damage. Tissue engineering approaches use a combination of cells, signaling molecules, and biomaterials to facilitate repair and regeneration. A hyaluronic acid (HA)-based hydrogel that is crosslinked via hydrophobic interactions between adamantane groups and cyclodextrin groups that combine to form a guest-host complex in solution was chosen for this study. The reversible nature of guest-host crosslinks provides the hydrogel with shear-thinning and self-healing capabilities that allow it to be injected into the damaged tissue with minimal diffusion and ejection. In this study, we synthesize and characterize the guest-host hydrogel for a novel application in the treatment of traumatic brain injury.

METHODS: Adamantane (guest group) and cyclodextrin (host group) modifications of HA were performed through esterification and amidation reactions, respectively. The cyclodextrin-HA was further modified with a methacrylate group to facilitate the covalent attachment of the cell adhesion motifs, RGD and IKVAV, through Michael-type addition reactions. Tethering of the cell adhesion peptides was confirmed using a bicinchoninic acid (BCA) assay to determine the presence of peptide in a purified sample. The guest and host-modified HA polymers were then combined in aqueous solution to form a hydrogel. Rheology was performed on hydrogels of varying HA weight percentage to determine the appropriate formulation for central nervous system (CNS) tissue. Afterwards, a lactate dehydrogenase (LDH) assay was used to assess the cytotoxicity of the guest-host hydrogels towards primary mouse astrocytes after 24 hours of exposure. The hydrogel formulation determined to be ideal for CNS tissue applications will be used for subsequent in vivo biocompatibility studies in a mouse model of TBI.

RESULTS AND DISCUSSION: Nuclear magnetic resonance analysis of the hydrogel components confirmed the modification of the HA with the adamantane, or cyclodextrin and methacrylate groups. An approximate 30% functionalization was achieved for each of the respective groups on the backbone of the HA. RGD and IKVAV conjugation was confirmed with the BCA assay as peptide remained on the HA backbone after extensive dialysis of the samples. Mixing of the two adamantane-HA and cyclodextrin-HA components in aqueous solution resulted in the formation of a hydrogel whose rheological properties could be characterized. Modulating the weight percentage of the hydrogel from 5% to 7.5% caused a shift in the mechanical properties of the hydrogel (Figure 1), with higher weight percentage hydrogels exhibiting increased relaxation times (0.999 seconds vs 2.512 seconds) and overall stiffness values (~1,100 pascals vs ~4,000 pascals). For future in vivo studies, a hydrogel formulation near 5 weight percent HA will be selected as this formulation results in mechanical properties similar to endogenous neural tissue. Importantly, altering the hydrogel weight percentage did not impact the shear-thinning or self-healing capabilities of the material. Hydrogel weight percentage did not impact the toxicity of the material towards primary mouse astrocytes after 24 hours of indirect exposure via Transwell. Altogether, the provided data indicates potential utility as a tissue engineering scaffold and bioactive agent delivery vehicle in the treatment of TBI.

Figure 1. Material properties are altered by modulating the hyaluronic acid weight percentage in the hydrogel formulations. (A) frequency sweep, (B) strain sweep.