Cytoskeletal stiffening in synthetic hydrogels

Abstract

Although common in biology, controlled stiffening of hydrogels in vitro is difficult to achieve; the required stimuli are commonly large and/or the stiffening amplitudes small. Here, we describe the hierarchical mechanics of ultra-responsive hybrid hydrogels composed of two synthetic networks, one semi-flexible and stress-responsive, the other flexible and thermoresponsive. Heating collapses the flexible network, which generates internal stress that causes the hybrid gel to stiffen up to 50 times its original modulus; an effect that is instantaneous and fully reversible. The average generated forces amount to ~1 pN per network fibre, which are similar to values found for stiffening resulting from myosin molecular motors in actin. The excellent control, reversible nature and large response gives access to many biological and bio-like applications, including tissue engineering with truly dynamic mechanics and life-like matter.

Fig. 1

Controlling the stiffening response. a Schematic overview of the structure of PIC-PNIPAM hybrid hydrogels. The flexible PNIPAM network (green) is generated in the presence of the pre-formed semi-flexible PIC network (red), resulting in two interpenetrating networks. Heating the hybrid gel (transparent gel in the vial) beyond the LCST of PNIPAM (T ≈ 33 °C) leads to a transition in the PNIPAM network. The hybrid becomes opaque, but does not shrink (no volumetric change). b Thermoresponsive mechanical properties of PIC-PNIPAM hybrid hydrogel with 1.0 mg mL⁻¹ PIC and 17 mg mL⁻¹ PNIPAM, and the single networks at the same concentrations. Arrows indicate PIC gelation and the PNIPAM LCST. The stiffness of the hybrid gel increases by more than an order of magnitude at the LCST of PNIPAM compared with the single component PIC hydrogel. The moduli of the PIC and hybrid gels are dominated by the elastic contributions. Loss data are provided in Supplementary Fig. 7

Fig. 2

Controlling the stiffening temperature. a Shifting transition temperatures with salts; NaCl (orange data) shifts the PIC T_gel and the PNIPAM LCST to lower temperatures. At 30 °C, the difference between deionised water (blue data) and 0.5 M NaCl is nearly a factor 100 in G'. NaClO₄ (red data) increases T_gel and decreases the LCST, such that they reverse, which impedes the stiffening effect. The transition temperatures are indicated by the arrows. b Copolymerisation with NEAM shifts the LCST to higher T, whilst T_gel remains constant (not shown). In addition, the transition broadens and the low-temperature modulus increases. At high T, the G' reaches the same plateau for all copolymer compositions

Fig. 3

Stiffening of the PIC/PNIPAM hybrids. a Storage modulus G′ from different architecture hybrids. The stiffening response is identical for a covalently cross-linked network (green), IPN (blue) and a semi-IPN (orange). b Reversibility and small hysteresis observed after 10 cycles (only even cycles displayed). c Stiffening transition at different heating rates (0.1–10 °C min⁻¹). d Stiffening rates G′_T+1/G′_T (orange) and transition temperature T (red) as a function of heating rate. The concentrations in a, b and c, d are PIC/PNIPAM 1/17, 2/17 and 1/40 mg mL⁻¹. e, f Increasing the PNIPAM (e) or PIC (f) concentration increases the storage modulus G′ of the PIC/PNIPAM hybrid after thermally induced stiffening. g Differential modulus K' against the external pre-stress σ for a 1 mg mL⁻¹ PIC hydrogel at T = 33 °C. The dotted lines correspond to the modulus of the PIC/PNIPAM hybrid gels at T = 37 °C and the external pre-stress σ corresponding to this modulus. Direct comparison between G′ and K′ is allowed, because of the strong similarity between the experiments: in both cases, we determine δσ/δγ as a function of static pre-stress that is induced either by PNIPAM or by externally applied stress. h The average internal stress σ_int generated by the PNIPAM network scales linearly with the product c_PNIPAM·c_PIC for all studied samples. The dashed line is a power law fit to the experimental data (slope 0.97). i, j The average force per fibre as a function of c_PNIPAM (i, c_PIC = 1 mg mL⁻¹) and c_PIC (j, c_PNIPAM = 17 mg mL⁻¹) only depends (linearly) on the PNIPAM concentration (note the logarithmic x-axes). Lines are power law fits

Fig. 4

Microstructural characterisation. a Small-angle X-ray scattering profiles of PIC/PNIPAM hybrids (4/17 mg mL⁻¹ and 4/6 mg mL⁻¹) and single PIC networks at T = 30 (blue fit) and 40 °C (red fit) with PNIPAM contributions (Supplementary Fig. 13) subtracted. The high-angle concave curvature (in all samples) originates from the scattering contribution of the fraction of dissolved PIC chains not incorporated in the gel that is well described by a Kholodenko worm-like chain term with contour length L = 72 nm, persistence length l_p = 8 nm and radius R = 1.0 nm. The fits (solid lines) follow the correlation length model, with the exception of 4/6 at 40 °C, which also contains a Kholodenko bundle term with L_B > 200 nm, l_p,B > 200 nm (both outside the experimental window) and bundle radius R_B = 6.7 nm. The inset tabulates the key fitting parameters, correlation length ξ (in nm) and Porod exponent p. Data are vertically offset for better visualisation (Supplementary Fig. 11, without offset). Note that in the SAXS experiments, the PIC network (4 mg mL⁻¹) scatters much stronger than the PNIPAM network (6 or 17 mg mL⁻¹), despite its lower concentration (Supplementary Fig. 13). To be sure that we only see the architectural changes in the PIC network, we subtract the (small) PNIPAM contribution. b, c AFM images of a PIC/PNIPAM (4 and 6 mg mL⁻¹) in water: at 25 °C with root mean square roughness R_q = 20 nm (b); and at 40 °C with R_q = 325 nm (c). Note the difference in z-scale for both images