Tetrazine ligation is a rapid and bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) which can occur under physiological conditions. The fast kinetics (k2 &gt; 105 M-1s-1) and cyto-compatibility of the tetrazine ligation makes it a particularly attractive chemistry for the fabrication of spatially patterned hydrogel matrices. By introducing aqueous solutions of TCO flanked PEG or matrix metalloproteinase degradable peptide (bis-TCO) to a reservoir of tetrazine modified hyaluronic acid (HA-Tz), covalently crosslinked, liquid-filled hydrogel channels of desired lengths can be fabricated. At the interface between the two aqueous solutions, an instantaneous crosslinked channel wall forms. As the reaction is allowed to proceed, the smaller bis-TCO molecules (2kDa) will diffuse across the crosslinked wall, reacting with the HA-Tz (500kDa) at the gel-liquid interface, growing the channel wall outward until fully exhausted. This interfacial, diffusion controlled crosslinking enables the creation of 3D spatial patterns of ligands and other biomolecules to modulate the biochemical and biomechanical environment within the hydrogel channel. To create patterns, the desired TCO conjugated molecules are perfused into the lumen of the channel at predetermined times where they will diffuse through the channel wall and react on its outer edge as the wall continues to grow. This chemistry also allows for the spatial patterning of different cell populations. By alternating cell laden HA-Tz reservoirs during the channel wall formation, cells can be spatially patterned into the channel wall in desired orders. Human aortic vascular endothelial, smooth muscle and fibroblasts were spatially patterned into the hydrogel channels in anatomical order with high viability. The interfacial diffusion driven process, which can be utilized to create not only patterns of ligands but also of different cell populations, will enable the construction of in vitro vasculature models.

Patterned liquid-filled hydrogel channels formed via tetrazine ligation for in vitro vasculature models

254th National Meeting (2017)

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