Design of Extrudable Click Hydrogel Biomaterials for Three-Dimensional Bioprinting and Injection

Abstract

Hydrogels are widely used as scaffolds in tissue engineering. Designing biocompatible hydrogel platforms requires consideration of fabrication techniques and routes of administration. Click chemistries facilitate crosslinking of hydrogel networks in the presence of biological systems. Moreover, hydrogel extrusion is an advantageous delivery mechanism that enables fabrication of hydrogel constructs with complex geometries and direct injection of hydrogels at their site of application. Nevertheless, the material properties of extrudable hydrogel systems must be tailored to accommodate extrusion without sacrificing biocompatibility. Herein is reported the design of two extrudable click hydrogel systems for fabrication of tissue engineered scaffolds.

The first extrudable hydrogel platform was applied to extrusion bioprinting of gelatin-based bioinks. Gelatin was functionalized with norbornene groups (GelNB) and incorporated into bioink formulations, enabling crosslinking by thiol-norbornene click chemistry. The rheological properties and applicability of GelNB-bioinks to extrusion bioprinting were directly compared to bioinks formulated from gelatin-methacryloyl (GelMA). Moreover, the efficacy of Irgacure-2959 (I2959) and lithium acyl phosphinate (LAP) as bioink photoinitiators were compared. GelNB-bioinks were more viscous than GelMA-bioinks and exhibited faster crosslink kinetics at 365 nm light, which translated to printed constructs with rougher surfaces but better stability. LAP greatly increased crosslink kinetics relative to I2959. GelNB and LAP are suitable bioink components.

The second extrudable hydrogel platform was designed as an injectable microparticle hydrogel (microgel) platform that supports neural progenitor cell (NPC) grafts in spinal cord injury (SCI). Norbornene-functionalized poly(ethylene glycol) (PEG) microgels were synthesized using an aqueous two-phase system (ATPS) of PEG and dextran. The influence of ATPS composition on microgel properties was investigated. The results suggest that ATPSs are homogenized by mixing and that phase separation kinetics may influence microgel properties. The norbornene-functionalized microgels were then delivered with a linear bifunctional PEG-tetrazine linker molecule to SCIs in mice for in situ assembly by tetrazine-norbornene click reaction. Microgel scaffolds did not exacerbate secondary injury or inflammatory responses when delivered alone. When applied with NPC grafts, microgel scaffolds did not impede neuronal or astrocytic differentiation, inhibit outgrowth of graft-derived axons, or exacerbate immune responses to NPC grafts. Thus, the biocompatibility of the microgel scaffolds with NPC grafts was demonstrated.