Crosslinking of fibrous hydrogels

Abstract

In contrast to most synthetic hydrogels, biological gels are made of fibrous networks. This architecture gives rise to unique properties, like low concentration, high porosity gels with a high mechanical responsiveness as a result of strain-stiffening. Here, we used a synthetic polymer model system, based on polyisocyanides, that we crosslinked selectively inside the bundles. This approach allows us to lock in the fibrous network present at the crosslinking conditions. At minimum crosslink densities, we are able to freeze in the architecture, as well as the associated mechanical properties. Rheology and X-ray scattering experiments show that we able to accurately tailor network mechanics, not by changing the gel composition or architecture, but rather by tuning its (thermal) history. Selective crosslinking is a crucial step in making biomimetic networks with a controlled architecture.

Fig. 1

Crosslinking a bundled network. a Schematic representation of the crosslinking method. Azide (orange) decorated polymers (blue) are gelled and crosslinked selectively within the bundles by a crosslinker (pink), stabilizing the architecture. b Gel components: PIC 1 (yield: 94%, 3.3-mol-% N₃, M_v = 599 kg mol⁻¹) and crosslinkers 2a and 2b. c–f Freeze-fractured cryoSEM micrographs of PIC gels (scale bars=1 µm): crosslinked (c) and not-crosslinked (d) gel at T = 37 °C and crosslinked (e) and not-crosslinked (f) gel at T = 5 °C. Note that in the final cryoSEM image, the network structure has disappeared

Fig. 2

Mechanical properties of intra-bundle crosslinked PIC hydrogels. a Storage modulus G′ of a crosslinked (1 + 2a, blue data) and a not-crosslinked (only 1, orange data) PIC gel. The heating (solid lines) curves shows gel formation at 15–20 °C. After crosslinking at 37 °C, the cooling (dashed lines) curve of 1 shows ‘melting’, while 1 + 2a remains a gel. The thermal stiffening exponent β originates from single chain stiffening. b Strain-stiffening: differential modulus K′ = ∂σ/∂γ as a function of applied stress σ for the 1 + 2a gel (crosslinked at T_cl = 37 °C) and measured at 5 °C (open diamonds) and 37 °C (solid circles). Below a critical stress σ_c, K′ = G′, above it K′ ~ σ^m, with m the stiffening index, quantifying the responsiveness to stress. c Crosslinking in the absence of bundles (at T_cl = 5 °C, light blue data) does not lead to network formation, but as soon as the temperature is raised and bundles form, the gel forms permanent crosslinks that are stable on cooling, similar to that of the immediately crosslinked sample (blue data). d Storage moduli G′ of 1 + 2a gels crosslinked at T_cl = 25 °C (blue), T_cl = 30 °C (violet), T_cl = 40 °C (purple), T_cl = 50 °C (red) and T_cl = 65 °C (pink) when cooling from the crosslinking temperature T_cl to 5 °C (cooling rate: 1 °C min^–1). e G′ of crosslinked PIC gels (T_cl = 25–50 °C), reheating from 5 °C to 50 °C with a heating rate of 1 °C min^–1. f The moduli of crosslinked PIC gels (T_cl = 25–50 °C), cooling from 50 °C to 5 °C with a heating rate of 1 °C min⁻¹ fully overlap. g The moduli of crosslinked gels at T = 20 °C, measured after cooling from their respective crosslinking temperature (red data) and after cooling from T = 50 °C (black data). h Effect of the length of the crosslinker: G′ of gels crosslinked with a ‘long’ crosslinker 2b (green) and ‘short’ crosslinker 2a (blue); both cooling from T_cl = 37 °C to 5 °C (−1 °C min^–1). i Differential modulus of the hydrogels crosslinked with the ‘long’ (2b, green) and the ‘short’ (2a, blue) spacer, crosslinked at T_cl = 37 °C and measured at T = 5 °C. For all experiments in Fig. 2, the concentrations were identical: [1] = 1 mg mL⁻¹, equivalent to [N₃] = 104 µM; crosslinkers: [2a] or [2b] = 52 µM (or 0 µM in panel a)

Fig. 3

Architectural analysis with SAXS. a The scattering curve of the 1 + 2a crosslinked gel at 5 °C (orange) is a combination of the bundled hydrogel pattern (red, 1 + 2a, 50 °C) and the dissolved polymer pattern (black, 1, 5 °C). b, c Contributions of the models for crosslinked 1 + 2a gel at 5 °C and model interpretation of architectural length scales. d, e SAXS curves of 1 + 2a gels, crosslinked at 25, 30, 40, and 50 °C at the crosslinking temperatures (d) and after cooling to 5 °C (e) with the best fit to the model (solid lines). The curves were shifted vertically to enhance clarity; un-shifted curves are given in Supplementary Fig. 5. f Average bundle radii of gels crosslinked gels 1 + 2a (blue to red) and 1 + 2b (green) at the crosslinking temperature T_cl, at 5 °C and at 50 °C, as well as the bundle radius of 1 at 50 °C for comparison. All fits include a 40% distribution on the bundle diameter; the error bars are an estimate for the range where the fit yields acceptable results. The concentrations for all samples in the figure are [1] = 4 mg mL⁻¹ and [2a] = [2b] = [N₃]/2 = 208 µM

Fig. 4

Crosslinker ratios and conversions. a Relative stiffness of crosslinked 1 + 2a hydrogels (T_cl= 37 °C, [1] = 1 mg mL⁻¹), after 11 h stabilization at 5 °C as compared to the stiffness of the same gel at 37 °C as a function of the [DBCO]/[N₃] ratio (blue data). The error bars represent the standard deviation of n = 2 or 3 measurements. The ratio of [DBCO]/[N₃] was varied between 0 and 2. Note that the graph shows the relative decrease of the modulus at 5 °C that is normalized to the modulus at 37 °C, because the DBCO units that are present at different concentrations for different crosslinking ratios shift the gel transition temperature slightly, and with that the stiffness of the gel. The absolute values of the moduli at T = 5 °C and 37 °C are given in Supplementary Table 1. Estimated crosslink concentration based on statistical reactions between the crosslinker and the azide and the experimentally determined conversion values (red dotted line). b DBCO conversion for gels crosslinked with 2a or 2b, at 37 °C and at 5 °C in time measured using UV–vis spectrometry. Colors are explained in the panel