Fig. 1: StemBond hydrogels provide control over ECM density and stability independent of stiffness.

a Sketch of StemBond hydrogels. PAAm are synthesised with co-factor 6-Acrylamidohexanoic acid (AHA). The AHA chains terminate with a carboxyl group which then binds primary amines of ECM proteins. b Schematic of variations in ECM tethering strength with AHA concentrations. The estimated values of the surface density σ of carboxyl groups (black dots) is given for three different concentrations (see the “Methods” section). Each ECM protein fibre (red lines) can create many covalent bonds so different AHA concentrations will lead to different tethering strengths while not affecting the stiffness. c Young’s modulus of StemBond hydrogels with different proportions of acrylamide, bis-acrylamide and AHA co-factor was measured using AFM. The modulus in Pa is reported (mean ± standard deviation) (30 indentations/gel over 5 independent samples) for soft (black), intermediate (blue) and stiff (red) hydrogels. d Quantification of protein coverage on StemBond hydrogels, estimated by coating gels with FITC-labelled BSA and measuring the mean fluorescence intensity at the gel surface (n = 2–3 fields of view for three independent samples). Bars show mean ± standard deviation. P-values computed using a one-way ANOVA with Tukey–Kramer’s multiple comparison test. e Rupture force (log₁₀) from significant binding events between fibronectin bound onto hydrogels and an AFM probe coated with anti-Fibronectin antibody. ‘○’: average of n = 3 or 4 independent experiments; ‘□’: overall mean; bars: standard error. P-values correspond to pairwise comparison to sulfo-SANPAH substrates from two-way ANOVA linear model. f Rhodamine-Fibronectin layer on different stiff substrates after 3 days of MEFs culture. (Top) Holes in the ECM layer are sites of ECM remodelling by MEFs. (Bottom) Mean ± standard deviation of fluorescence intensity for four replicates (n = 12–17 frames/data point taken over two independent experiments). Mean intensities were normalised for each batch and background was subtracted (measured on uncoated substrates) before averaging. P-values computed using a one-way ANOVA with Tukey–Kramer’s multiple comparison test. In panels e and f, all hydrogels were coated with 200 µg ml⁻¹ fibronectin. Source data are provided as a Source Data file.