Journal: bioRxiv

Article Title: Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks

doi: 10.1101/2024.04.04.588191

Figure Lengend Snippet: Double networks can be independently degraded in a stepwise manner. (a) Double networks are first treated with 4S9 to remove the thiol-ene network, and then fully degraded by treatment with 2A9 to yield fully soluble macromolecular building blocks. (b) Peptide recognition sequences for 2A9 and 4S9 included in hydrogel crosslinkers and degradation reaction post sortase treatment. (c) Schematic depicting individual labeling of each network with distinct fluorophores, and the monomeric component released upon each sortase treatment, tracked by increases in supernatant fluorescence. (d) Fluorophore release studies. At time = 0 min, 18 mM GGG, the respective sortase, and 1 mM CaCl 2 were added to the solution the DN hydrogels were in. Hydrogel degradation was tracked by monitoring supernatant fluorescence, with values normalized to those obtained from 100% degraded gels 12 hours post reaction. (e) AFM measurements of DN gels pre- and post-4S9 treatment. DN pre 4S9 treatment: 4648 ± 750 Pa; DN post 4S9 treatment: 908 ± 550 Pa. Unpaired t-test, **p = 0.0024.

Article Snippet: [ , , , ] pET29b expression plasmids for 2A9 and 4S9 were a generous gift from Dr. David Liu at Harvard University (Addgene plasmids #75145 and #75146).

Techniques: Labeling, Fluorescence

Journal: bioRxiv

Article Title: Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks

doi: 10.1101/2024.04.04.588191

Figure Lengend Snippet: DNs can be formed and dynamically softened in a cytocompatible manner. (a) Experimental set-up for viability measurements. Static controls of thiol-ene, DN, and SPAAC gels were compared against dynamic DN gels treated with 4S9 on day 3 of culture. (b) Maximum Image Projection (MIP) of representative images (z = 250 µm). Live/Dead staining of encapsulated 10T1/2 fibroblasts shows excellent cytocompatibility of all possible network types on day 7 of culture. Scale bar = 100 µm. (c) Quantification of viability.

Article Snippet: [ , , , ] pET29b expression plasmids for 2A9 and 4S9 were a generous gift from Dr. David Liu at Harvard University (Addgene plasmids #75145 and #75146).

Techniques: Staining

Journal: bioRxiv

Article Title: Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks

doi: 10.1101/2024.04.04.588191

Figure Lengend Snippet: Double networks can be reversibly and spatiotemporally patterned to drive changes in encapsulated cell morphology. (a) Schematic depicting stepwise patterning and pattern removal. Soluble monomeric precursors can be mixed together in a one-pot mixture. SPAAC stepwise network formation occurs spontaneously, while thiol-ene polymerization can be spatially controlled photolithographically. Subsequently, thiol-ene patterns are removed with sortase 4S9 treatment. (b) Stiff patterns in a bulk hydrogel are enabled by localized thiol-ene polymerization and can be removed by 4S9 treatment. Insets depict no fluorescence is visible in the FAM channel (thiol-ene network) post enzymatic treatment. Top scale bar = 200 µm, bottom scale bar = 1 mm. (c) AFM measurements of half-patterned gels. “In” denotes a stiff region exposed to light, whereas “out” denotes the covered, non-exposed region. Two-Way ANOVA, ***p = 0.0002. (d) DN design allows for reversible patterning of mechanics. Thiol-ene gel components can be diffused into single network at later time points for mechanical patterning and can be reversibly removed and reinstated by rounds of 4S9 degradation and photopolymerization. Scale bar = 250 µm. (e) Intricate DN formations can be patterned using multiphoton laser-scanning lithography. Scale bar = 100 µm. (f) hMSCs encapsulated in stiffness-patterned hydrogels. Image shows the interface of stiff and soft regions. Scale bar = 100 µm. (g) hMSCs in soft (left) vs stiff (right) regions of patterned hydrogel. (h) Quantification of cell area in soft and stiff regions. Unpaired t-test, ****p < 0.0001.

Article Snippet: [ , , , ] pET29b expression plasmids for 2A9 and 4S9 were a generous gift from Dr. David Liu at Harvard University (Addgene plasmids #75145 and #75146).

Techniques: Fluorescence