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. 2017 Jul 7;28(14):2010–2022. doi: 10.1091/mbc.E17-01-0010

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© 2017 Ivanovska et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Analysis of cell morphology and protein organization in MSCs cultured on pristine and cross-linked collagen-I films shows the equivalence of cross-linking to increased matrix stiffness. (A) Thin collagen films allowed an in vitro study of the effects on MSC morphology of culture on (i) pristine and (iii) cross-linked collagen-I films; (ii) nuclear height on the two films. (B) AFM amplitude-mode topographical images of molecular films of highly ordered collagen-1 fibrils, showing D-periodic structure, self-assembled on hard substrates in the absence (i, ii) and presence (iii, iv) of cross-linking. Collagen fibrils were deformed by using the AFM stylus in lithography mode to apply a low dragging force across the film (ii, iv), thus showing the flexibility of the fibrils and the deconstruction of the ribbons to monomers in the absence of cross-linking (ii). (C) Immunofluorescence images of MSCs cultured for 24 h on (i) pristine and (iii) cross-linked collagen-I films with staining against F-actin (red) and DNA (Hoechst, blue). (ii, iv) Staining against collagen showed that cross-linked films are not as severely deformed as the native ones. The random orientation of the dense regions of collagen protein suggests that the films are mechanically anisotropic, despite the topographically isotropic orientation of the fibrils evident from AFM images. (D) Comparison of matrix E–dependent cell morphologies of MSCs cultured on thick, isotropic polyacrylamide (PA) gels or thin collagen-I films. Plots of (i) cellular and (ii) nuclear spread area were fitted to hyperbolic functions, with free parameters obtained from cells on gels of controlled stiffness (blue) and cells on thin films (red squares), with the unknown effective stiffness felt by the cells on thin films superimposed to obtain the effective stiffness (10 kPa for pristine and 50 kPa for cross-linked collagen-I films). (E) Cellular aspect ratio (i, left axis), order parameter (i, right axis), and nuclear aspect ratio (ii) as functions of the gel matrix elasticity also showed consistency between morphology of pristine and cross-linked films with 10- and 40-kPa PA gels, respectively. All experiments, n ≥ 3 (mean + SEM).