The extracellular matrix (ECM) constitutes a viscoelastic environment for cells. A growing body of evidence suggests that the behavior of cells cultured in naturally-derived or synthetic ECM mimics is influenced by the viscoelastic properties of these substrates. Adaptable crosslinking strategies provide a means to capture the viscoelasticity found in native soft tissues. In this work, we present a covalent adaptable hydrogel based on thioester exchange as a biomaterial for the in vitro culture of human mesenchymal stem cells. Through control of pH, gel stoichiometry, and crosslinker structure, viscoelastic properties in these crosslinked networks can be modulated across several orders of magnitude. We also propose a strategy to alter these properties in existing networks by the photo-uncaging of the catalyst 4-mercaptophenylacetic acid. Mesenchymal stem cells encapsulated in thioester hydrogels are able to elongate in 3D and display increased proliferation relative to those in static networks.

This work examined and quantitatively predicted the degradation of thioester-containing networks facilitated by base-catalyzed thiol−thioester exchange. A statistical model was developed that incorporated polymer structure, thiol−thioester exchange reaction kinetics, and mass gain resulting from dynamic bond exchange, and this model was compared to mass loss studies. Experimental results matched model predictions, showing that degradation times could be controlled from 2.5 to 12 h with optimal conditions by varying the free thiol butyl 3-mercaptopropionate concentration from 0.0 to 4.9 M and the base-catalyst triethylamine molar ratio from 0 to 40 mol %. Furthermore, thioester-based composite materials were formed by stereolithography (SLA) three dimensional (3-D) printing and subsequently degraded, achieving 91% recovery of the composite filler. This work provides insight into thioester-facilitated degradation and its future use in selective material release or encapsulated filler recovery applications.

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Biofabrication allows for the templating of structural features in materials on cellularly-relevant size scales, enabling the generation of tissue-like structures with controlled form and function. This is particularly relevant for growing organoids, where the application of biochemical and biomechanical stimuli can be used to guide the assembly and differentiation of stem cells and form architectures similar to the parent tissue or organ. Recently, ablative laser-scanning techniques was used to create 3D overhang features in collagen hydrogels at size scales of 10–100 µm and supported the crypt-villus architecture in intestinal organoids. As a complementary method, providing advantages for high-throughput patterning, we printed thioester functionalized poly(ethylene glycol) (PEG) elastomers using digital light processing (DLP) and created sacrificial, 3D shapes that could be molded into soft (G′ < 1000 Pa) hydrogel substrates. Specifically, three-arm 1.3 kDa PEG thiol and three-arm 1.6 kDa PEG norbornene, containing internal thioester groups, were photopolymerized to yield degradable elastomers. When incubated in a solution of 300 mM 2-mercaptoethanol (pH 9.0), 1 mm thick 10 mm diameter elastomer discs degraded in <2 h. Using DLP, arrays of features with critical dimensions of 37 ± 4 µm, resolutions of 22 ± 5 µm, and overhang structures as small as 50 µm, were printed on the order of minutes. These sacrificial thioester molds with physiologically relevant features were cast-molded into Matrigel and subsequently degraded to create patterned void spaces with high fidelity. Intestinal stem cells (ISCs) cultured on the patterned Matrigel matrices formed confluent monolayers that conformed to the underlying pattern. DLP printed sacrificial thioester elastomer constructs provide a robust and rapid method to fabricate arrays of 3D organoid-sized features in soft tissue culture substrates and should enable investigations into the effect of epithelial geometry and spacing on the growth and differentiation of ISCs.

Stimuli-responsive degradation of hydrogels has established and emerging utilities ranging from controlled release of biological products to sacrificial molding. Although covalent adaptable networks are particularly amenable for these applications, their degradation kinetics have yet to be clearly elucidated in these swollen material systems. In this work, the thiol−thioester exchange reaction in cross-linked poly(ethylene glycol) (PEG) hydrogels is characterized to determine the relative effects of thiol concentration, pK a , and pH on network mass loss and mechanical properties. From these measurements, we identify that network thiols liberated from exchanged thioester cross-links play a significant role in hydrogel degradation, resulting in predictable changes in the storage modulus but nonideal mass loss profiles. These findings highlight how the mechanism of thioester exchange degradation can give rise to useful deviations from ideal degradation behavior, such as amplified degradation rates and linear mass loss profiles. Furthermore, this work empowers the design of on-demand degradable hydrogels with uniquely tunable degradation and mass loss.