Three-dimensional Chemical Patterning of Transparent Hydrogels

Abstract

Covalent modification of agarose with a 6-bromo-7-hydroxycoumarin (Bhc) sulfide derivative yields a hydrogel that generates bound thiol groups upon excitation with either UV light or a pulsed infrared laser. Using a multiphoton confocal microscope as the patterning platform, intricate internal chemically modified volumes of stable nucleophilic thiol groups are created within these hydrogel samples, which are in turn modified with biomolecules without causing hydrogel cross-linking or changes in its physical properties. The use of automated scripts and microscope stage control allows, for the first time, biologically relevant molecules of interest to be photochemically immobilized within the hydrogel in complex patterns with feature sizes comparable to those of mammalian cells. The resulting chemically patterned hydrogels have applications in tissue engineering, where they can be used to control cell behavior.

Diels−Alder Click Cross-Linked Hyaluronic Acid Hydrogels for Tissue Engineering

“…A particularly relevant example is thiol-maleimide click coupling (Scheme 1E), which is frequently exploited for protein conjugation 92,93,99,100 . Hydrogels prepared with polyfunctional PVA formed higher modulus gels with reduced swelling than those synthesized with the bifunctional PEG crosslinker.…”

“…Shoichet and co-workers have exploited this click reaction for 3D patterning of agarose hydrogels 92,93,100,121 . Covalent modification of agarose with S-2-nitrobenzyl-cysteine (S-NBC) renders a photolabile matrix, which upon UV irradiation, releases free thiols capable of reacting with any thiol-reactive biomolecule through Michael addition.…”

“…In order to advance this click technology towards more sophisticated architectures that better mimic the native extracellular matrix, agarose was later modified with a coumarin derivative, which upon exposure to a pulsed infrared laser yields free thiols 100 . The use of this photolabile group, allows for intricate 3D control by way of multi-photon excitation.…”

“…The stringent spatial resolution and controlled biochemical heterogeneity required for 3D patterning make simple bioorthogonal chemistries paramount. The use of click reactions for 3D patterning of scaffolds allows significant spatial control when combined with multi-photon processing 93,100 . Additionally, many of these hydrogel scaffolds are click crosslinked, and sequentially click patterned 38,39,124 .…”

In situ cell manipulation through enzymatic hydrogel photopatterning

“…Whereas native microenvironments are frequently degraded and remodelled by cellular activity (for example, during proteolysis in wound healing), locally modular microenvironments may emerge most frequently from the secretion of biological signals from a cellular source (for example, gradients of morphogens in embryonic development). Thus, as a second approach, this additive mode of local microenvironmental patterning was achieved by other groups using chemical crosslinking strategies [11][12][13][14][15] . Indeed, approaches employing radical reactions of acrylates or thiol-enes, or photoprotected thiol residues in Michael-type addition, have demonstrated successful local patterning of hydrogel matrices with simple cell-adhesion peptides 11,12,14 .…”