Research ArticleCELL BIOLOGY

Tensile forces drive a reversible fibroblast-to-myofibroblast transition during tissue growth in engineered clefts

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Science Advances  17 Jan 2018:
Vol. 4, no. 1, eaao4881
DOI: 10.1126/sciadv.aao4881
  • Fig. 1 De novo microtissue growth rate increased with cytoskeletal tension.

    (A) Schematic of the microfabrication process used to produce thick 3D PDMS substrates with controlled macroscopic geometry. Detailed illustrations of the fabrication process are shown in fig. S5. (B) Illustration of 3D tissue growing in the corners of rectangular substrate pores. (C and D) Illustration of surface and interior regions and tissue dimensions. (E) Maximum intensity projection of the resulting 3D tissue containing cells (blue) and ECM (red, FN; green, Col-I; blue, nuclei). The tissue is rich in both FN and Col-I fibers. (F to H) Single slices through tissues (green, actin; blue, nuclei) at the indicated depth in xy and xz directions, under normal conditions (left) and under conditions of inhibited (middle) and elevated (right) cytoskeletal tension in the presence of 10 μM blebbistatin or TGF-β1 (1 ng/ml), respectively. The individual channels and an animated z fly-through are shown in fig. S1 and movie S1, respectively. (I) Quantification of tissue radius (distance between the scaffold corner and the growth front at 40 μm of z depth) after 19 days of growth under normal conditions and in the presence of 10 μM blebbistatin or TGF-β1 (1 ng/ml), respectively. Increased cytoskeletal tension results in increased tissue volume. Scale bars, 50 μm. Data points indicate the tissue radius for individual tissues.

  • Fig. 2 Spatial mapping of EdU incorporation reveals that cells preferentially proliferate in the surface growth front.

    (A to C) Single slices through tissues at the indicated z depth showing EdU signals from proliferating nuclei (green) and Hoechst counterstain of all nuclei (red) for all three conditions in xy and xz directions. Scale bars, 50 μm. The individual channels and an animated z fly-through are shown in fig. S2 and movie S2, respectively. (D) Time course of proliferation experiments. Tissues were cultured for 19 days under normal conditions and in the presence of 10 μM blebbistatin or TGF-β1 (1 ng/ml), respectively. (E) Percentage of EdU-positive volume compared to total nuclear volume as a function of tissue depth and age. (F) Ratio of EdU-positive cells in growth front compared to interior. (G) Nuclear volume fraction as a measure for cell density as a function of tissue depth and age. (H) Mean nuclear volume fraction for all three conditions. Error bars indicate SEM. Data points in (F) and (H) indicate ratios and fractions for individual tissues.

  • Fig. 3 FN fiber tension is highest in the growth front and decays as the tissue matures.

    (A) Time course showing the late supplementation of the medium by the FN-FRET probes. (B) Histograms of FN-FRET ratios of FN fibers within the ECM for three different medium supplementations. The FN-FRET probe was added to the medium 24 hours before fixing the cells. The FN-FRET ratios in solution for the same batch are also given in different concentrations of the denaturant GdnHCl (gray lines). (C to E) Single slices through tissues at the indicated z depth showing the FN-FRET ratio for all three conditions. Scale bars, 50 μm. Notice how overall tissue volume depends on up-regulation (TGF-β1, 1 ng/ml) or down-regulation (10 μM blebbistatin) of cell contractility. An animated z fly-through is shown in movie S3. (F) Median FRET ratio is significantly different between all three conditions. (G) Median FN-FRET ratio was always higher in the interior compared to the growth front, with no significant differences between the three conditions. (H) FN-FRET ratio as a function of distance to the growth front or, equivalently, tissue maturity, for all three conditions. Tissues were cultured for 19 days. Error bars indicate SEM. Data points in (F) and (G) indicate median values and ratios for individual tissues.

  • Fig. 4 α-SMA intensity and YAP nuclear localization were highest in the growth front.

    (A to C) Single slices through tissues at the indicated locations for all three conditions. F-actin is shown in green, and α-SMA fibers are shown in red. Individual channels are shown in fig. S3, and an animated z fly-through in movie S4. (D) Ratio of α-SMA signal density in fibers and in background as a function of tissue depth and age shows preferential α-SMA expression on the outermost layer within 10 μm from the tissue growth front. Addition of TGF-β1 (1 ng/ml) results in an increase of α-SMA signal, whereas blebbistatin (10 μM) reduces it. (E) Average α-SMA expression on surface and (F) ratio of α-SMA expression between growth front and interior. (G to I) Single slices through tissues at the indicated locations showing YAP signal (cyan) overlaid with nuclei (red) for all three conditions. Individual channels are shown in fig. S4, and an animated z fly-through is shown in movie S5. (J) Ratio of YAP signal in nuclei over total YAP signal shows a decrease from up to 60% nuclear localization on the tissue surface to nearly 25% deeper in the tissue. Addition of TGF-β results in a larger difference (gradient) in nuclear localization between tissue growth front and interior. (K) YAP nuclear localization in the growth front and (L) ratio of YAP nuclear localization between growth front and interior. Tissues were cultured for 19 days. Error bars indicate SEM. Data points in (E) and (F), and (K) and (L) indicate ratios and percentages for individual tissues. Scale bars, 50 μm.

  • Fig. 5 The myofibroblast-to-fibroblast transition illustrating how the highly tensed growth front rich in myofibroblasts drives tissue growth and how myofibroblasts revert to quiescent fibroblasts as the tissue matures.

    As the tissue grows tensed by cell contractility, and new cells and ECM are added at the growth front in a layer-by-layer process, a gradient emerges from the growth surface toward the tissue interior. The arrow of tissue maturation denotes the emerging gradients in tissue properties over space and time from the surface to the core. In the growth front, cells proliferate, are well spread and elongated, and deposit and stretch an early, 2D FN-rich ECM, whereas within the tissue interior, cells are more isotropic and embedded into a 3D Col-I–rich ECM. This gradient in ECM properties correlates with a gradient in cell phenotype, from activated myofibroblasts in the growth front toward a quiescent fibroblast phenotype that maintains tissue homeostasis in the interior.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/1/eaao4881/DC1

    fig. S1. Individual channels of images from Fig. 1, F to H.

    fig. S2. Individual channels of images from Fig. 2, A to C.

    fig. S3. Individual channels of images from Fig. 4, A to C.

    fig. S4. Individual channels of images from Fig. 4, G to I.

    fig. S5. Illustration of the scaffold fabrication method.

    fig. S6. Gradients of cell phenotype and FN stretch after 11 days.

    movie S1. Z stack showing actin (green) and nuclei (blue).

    movie S2. Z stack showing nuclei (red) and EdU (green).

    movie S3. Z stack showing color-coded FN-FRET signals.

    movie S4. Z stack showing actin (green) and α-SMA (red).

    movie S5. Z stack showing nuclei (red) and YAP (cyan).

    movie S6. Time lapse recording of a tissue with fluorescently labeled FN.

  • Supplementary Materials

    This PDF file includes: 

    • fig. S1. Individual channels of images from Fig. 1, F to H.    
    • fig. S2. Individual channels of images from Fig. 2, A to C.    
    • fig. S3. Individual channels of images from Fig. 4, A to C.    
    • fig. S4. Individual channels of images from Fig. 4, G to I.    
    • fig. S5. Illustration of the scaffold fabrication method.    
    • fig. S6. Gradients of cell phenotype and FN stretch after 11 days.
    • Legends for movies S1 to S6
    • Download PDF
    • Movie S1 -

       (.avi format). Z stack showing actin (green) and nuclei (blue).

    • Movie S2 -

      (.avi format). Z stack showing nuclei (red) and EdU (green).

    • Movie S3 -

       (.avi format). Z stack showing color-coded FN-FRET signals.

    • Movie S4 -

      (.avi format). Z stack showing actin (green) and α-SMA (red).

    • Movie S5 -

      (.avi format). Z stack showing nuclei (red) and YAP (cyan).

    • Movie S6 -

       (.avi format). Time lapse recording of a tissue with fluorescently labeled FN.



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