Interfacial bioorthogonal crosslinking for the fabrication of functional hydrogels
Date
2018
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Tetrazine ligation, the cycloaddition of s-tetrazine (Tz) with trans-cyclooctene (TCO) derivatives, is particularly attractive for the synthesis of cell-instructive hydrogel matrices. This reaction is high yielding, does not require a catalyst, does not produce any toxic side products, and is the fastest bioorthogonal reaction (>106 M-1s-1 faster). Using tetrazine-modified hyaluronic acid (HA-Tz) and polyethylene glycol (PEG) flanked with TCO (bisTCO), the formation of hydrogel spheres via a diffusion-controlled process at the gel-liquid interface was demonstrated. Syringe delivery of HA-Tz into a reservoir of bisTCO resulted in the instantaneous formation of a crosslinked shell, through which bisTCO diffused inwards to introduce further crosslinking. Prostate cancer LNCaP cells were encapsulated in the hydrogel spheres with high viability, proliferated readily in the HA matrix and formed 3D, tissue-like cell aggregates. ☐ The interfacial diffusion-controlled process has also permitted the creation of hydrogel spheres with pre-determined spatial distribution of TCO-tagged biomolecules. Through temporally controlled introduction of TCO conjugates during the crosslinking process, the enzymatic degradability, cell adhesivity, and mechanical properties of the synthetic microenvironment was tuned with spatial precision. Using human mesenchymal stem cells (hMSCs) and hydrogels with a core-shell structure, the ability of the synthetic ECM with spatially defined guidance cues to modulate cell morphology in a biomimetic fashion was demonstrated. ☐ Introduction of aqueous solutions of bisTCO to a reservoir of HA-Tz led to the fabrication of liquid-filled hydrogel channels of desired lengths. An instantaneous covalently-crosslinked channel wall formed at the interface between the two aqueous solutions. bisTCO molecules diffuse through the crosslinked wall, reacting with the HA-Tz at the gel-liquid interface and subsequently growing the channel wall outward until fully exhausted. This interfacial, diffusion controlled crosslinking enabled the creation of 3D spatial patterns of ligands and other biomolecules to modulate the biochemical environment within the hydrogel channel walls via perfusion of TCO conjugates into the lumen of the channel at predetermined times. This chemistry also permitted the 3D spatial patterning of different cell populations by systematically alternating cell laden HA-Tz reservoirs during crosslinking. Vascular endothelial cells, smooth muscle cells and adventitial fibroblasts were spatially patterned into the hydrogel channels in anatomical order with high viability. ☐ Tetrazine ligation is suitable for the construction of crosslinked tissue-mimetic hydrogel networks with 3D spatial patterns without the necessity of external triggers or templates. The novel bioorthogonal, interfacial, diffusion-driven crosslinking was utilized to fabricate hydrogels with 3D spatial patterns of biochemical and biomechanical signals which can be employed to study tissue with layered structures or interfaces between tissue. Liquid-filled hydrogel channels with spatial patterns of biomolecules and different cell populations will enable the construction of complex, physiologically relevant in vitro vasculature models.
Description
Keywords
Bioorthogonal, Hyaluronic acid, Hydrogel, Interfacial crosslinking, Spatial patterning, Tetrazine ligation