Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Interpenetrating Networks

Abstract

Biomechanical contributions of the extracellular matrix underpin cell growth and proliferation, differentiation, signal transduction, and other fate decisions. As such, biomaterials whose mechanics can be spatiotemporally altered- particularly in a reversible manner- are extremely valuable for studying these mechanobiological phenomena. Herein, a poly(ethylene glycol) (PEG)-based hydrogel model consisting of two interpenetrating step-growth networks is introduced that are independently formed via largely orthogonal bioorthogonal chemistries and sequentially degraded with distinct recombinant sortases, affording reversibly tunable stiffness ranges that span healthy and diseased soft tissues (e.g., 500 Pa-6 kPa) alongside terminal cell recovery for pooled and/or single-cell analysis in a near "biologically invisible" manner. Spatiotemporal control of gelation within the primary supporting network is achieved via mask-based and two-photon lithography; these stiffened patterned regions can be subsequently returned to the original soft state following sortase-based secondary network degradation. Using this approach, the effects of 4D-triggered network mechanical changes on human mesenchymal stem cell morphology and Hippo signaling, as well as Caco-2 colorectal cancer cell mechanomemory using transcriptomics and metabolic assays are investigated. This platform is expected to be of broad utility for studying and directing mechanobiological phenomena, patterned cell fate, and disease resolution in softer matrices.

Keywords: bioorthogonal; hydrogels; interpenetrating polymer networks; mechanomemory; sortase; stimuli‐responsive.

Figure 1.

Interpenetrating networks are tunably formed. (a-b) IPNs are reversibly formulated with spatiotemporal control using a collection of orthogonal network formation and degradation chemistries. (c-d) IPNs are composed of two distinct PEG-based networks formed via two popular bioorthogonal reactions: (b) strain-promoted azide-alkyne cycloaddition (SPAAC) and (c) the radical-mediated and light-driven thiol-ene reaction. (e) In situ photorheology demonstrates stepwise IPN formation; SPAAC network formation proceeds spontaneously in the presence of thiol-ene network precursors, the latter of which is rapidly photopolymerized. Inset shows dramatic increase in storage modulus upon light exposure. (f) Young’s moduli as determined by AFM of swollen in PBS gels (initial concentrations 3 mM PEG-BCN: 6 mM diazide: 3 mM PEG-NB: 12 mM dicysteine: 1 mM LAP), post various light exposure times (0 – 2 min, 10 mW cm⁻²). SPAAC gels—1196 ± 370 Pa; 10 s—3381 ± 670Pa; 30 s—3829 ± 970 Pa; 1 min—5079 ± 690 Pa; 2 min—4648 ± 750Pa. One-Way ANOVA, Tukey’s post-hoc test. *p = 0.025, **p = 0.0078, ***p = 0. 0004. (g) Young’s moduli as determined by AFM of swollen gels, keeping SPAAC network constant, but varying molarity of thiol-ene network. 3mM PEG-NB—4648 ± 750 Pa; 4 mM PEG-NB—5352 ± 320 Pa; 5 mM PEG-NB—5687 ± 450 Pa.

Figure 2.

Interpenetrating networks can be independently degraded in a stepwise manner. (a) IPNs 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 CaCl₂ were added to the solution the IPN 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. N = 5 gels per treatment. (e) AFM measurements of IPN gels pre- and post-4S9 treatment. IPN pre 4S9 treatment: 4648 ± 750 Pa; IPN post 4S9 treatment: 908 ± 550 Pa. Unpaired t-test, **p = 0.0024.

Figure 3.

IPNs can be formed and dynamically softened in a cytocompatible manner. (a) Experimental set-up for viability measurements. Static controls of thiol-ene, IPN, and SPAAC gels were compared against dynamic IPN 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.

Figure 4.

Interpenetrating networks can be reversibly and spatiotemporally patterned. (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 bars = 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) IPN 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 IPN formations can be patterned using multiphoton laser-scanning lithography and visualized both in fluorescent channels, as well as in the brightfield view. Bottom left is a depth-coded image, with yellow representing closer to the top of the z-stack, and blue representing bottom of the z-stack. Scale bars = 200 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. Scale bar = 100 μm. (h) Quantification of cell area in soft and stiff regions in line patterns of different thickness. Unpaired t-test, ****p < 0.0001. N = 3 gels per patterning condition.

Figure 5.

Time-dependent softening controls hMSC spreading behavior. (a) Experimental set up. hMSCs were encapsulated in static or dynamic IPNs, or SPAAC networks. Dynamic IPNs underwent softening on day 3 (“softened d3”) or day 5 (“softened d5”). (b) Actin and nuclear staining reveals distinct morphologies amidst experimental groups. Scale bar = 100 μm. (c) Quantification of cell area. Small dots indicate individual cell values, whereas larger circles indicate per gel average. Statistics were conducted on the per-gel averages. One-Way ANOVA, Tukey’s post-hoc test, *p = 0.0105, **p = 0.0021. (d) Quantification of eccentricity. One-Way ANOVA, Tukey’s post-hoc test, *p = 0.0123. (e) Live imaging of tdTomato intensity as a function of time in line (left) and bar (right) plot form. Dynamic IPN gels were imaged prior to degradation on their respective degradation days. Two-Way ANOVA, Tukey’s post-hoc test. *p < 0.05, **p < 0.01 (f) Representative images of hMSCs expressing tdTomato upon TEAD binding events occurring on day 7 of encapsulation. Scale bar = 100 μm. (g) Representative photos of hMSCs on day 14 stained for Oil Red O (adipogenesis; top) and Alizarin Red (osteogenesis; bottom). Black arrows denote fat droplets, white arrows denote calcium deposits. Scale bar = 50 μm. (h) Quantification of Oil Red O positive cells on days 7 and 14 of culture. One-Way ANOVA, Tukey’s post-hoc test, *p < 0.05.

Figure 6.

Transcriptomic and metabolic analysis of Caco-2 cells in static and dynamically softened hydrogels. (a) Experimental set up. (b) PCA plot from bulk RNAseq data. (c) Volcano plot of gene expression profile in SPAAC vs. IPN hydrogels. Red = upregulated genes in SPAAC condition, blue = downregulated genes in SPAAC condition. (d) Top upregulated and downregulated genes for dynamic vs. static comparison. (e) Enriched MSigDB gene sets among differentially expressed genes for dynamic vs. static comparison. (f) Day 7 RealTime-Glo^™ in gel luminescence measurements as a proxy for cellular redox capacity. Raw luminescence values were normalized to total lysed protein content. One-Way ANOVA, Tukey’s post-hoc test, *p < 0.05. (g) Bioenergetic profiles of released Caco-2 cells (ECAR: extracellular acidification rate, PER: proton efflux rate, OCR: oxygen consumption rate). N ≥ 3 for all conditions.