We developed an injectable gelatin/hyaluronic acid hydrogel with slow degradability, which consisted of carbohydrazide-modified gelatin (Gel-CDH) and hyaluronic acid monoaldehyde (HA-mCHO). Gel-CDH/HA-mCHO hydrogels were degraded much more slowly in phosphate-buffered saline than the other Schiff's base cross-linked gelatin/hyaluronic acid hydrogels that were comprised of native gelatin, adipic acid dihydrazide-modified gelatin, or hyaluronic acid dialdehyde because of stable Schiff's base formation between aldehyde and carbohydrazide groups, and suppression of ring-opening oxidation by monoaldehyde modification. This prolonged degradation would be suitable for inducing angiogenesis. Therefore, the Gel-CDH/HA-mCHO hydrogels were sufficiently stable during the angiogenesis process. In addition, the hydrogel had a pore size of 15-55 μm and a shear storage modulus of 0.1-1 kPa, which were appropriate for scaffold application. Ex vivo rat aortic-ring assay demonstrated the concentration dependency of microvascular extension in the Gel-CDH/HA-mCHO hydrogel. These results demonstrated the potential usefulness of Gel-CDH/HA-mCHO hydrogel for tissue-engineering scaffolds.

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Fabrication of perfusable vascular networks in vitro is one of the most critical challenges in the advancement of tissue engineering. Because cells consume oxygen and nutrients during the fabrication process, a rapid fabrication approach is necessary to construct cell-dense vital tissues and organs, such as the liver. In this study, we propose a rapid molding process using an in situ crosslinkable hydrogel and electrochemical cell transfer for the fabrication of perfusable vascular structures. The in situ crosslinkable hydrogel was composed of hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified hyaluronic acid (HA-CHO). By simply mixing these two solutions, the gelation occurred in less than 20 s through the formation of a stable hydrazone bond. To rapidly transfer cells from a culture surface to the hydrogel, we utilized a zwitterionic oligopeptide, which forms a self-assembled molecular layer on a gold surface. Human umbilical vein endothelial cells adhering on a gold surface via the oligopeptide layer were transferred to the hydrogel within 5 min, along with electrochemical desorption of the oligopeptides. This approach was applicable to cylindrical needles 200-700 µm in diameter, resulting in the formation of perfusable microchannels where the internal surface was fully enveloped with the transferred endothelial cells. The entire fabrication process was completed within 10 min, including 20 s for the hydrogel crosslinking and 5 min for the electrochemical cell transfer. This rapid fabrication approach may provide a promising strategy to construct perfusable vasculatures in cell-dense tissue constructs and subsequently allow cells to organize complicated and fully vascularized tissues while preventing hypoxic cell injury.