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Hydraulic fracture during epithelial stretching.

Casares L, Vincent R, Zalvidea D, Campillo N, Navajas D, Arroyo M, Trepat X - Nat Mater (2015)

Bottom Line: Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension.We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres.Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioengineering of Catalonia, 08028 Barcelona, Spain.

ABSTRACT
The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells' cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

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The origin of cracks is hydraulica, (left) Clusters of MDCK cells expressing LifeAct-GFP on PAA hydrogels of different thickness. (right) Time-lapse evolution of the regions highlighted in orange on left panels before and after a 10 min pulse of 10% biaxial stretch. The acquisition time of each snap shot is marked by a black dot on the time axis (top). Arrowheads point to a subset of cracks. b, Time evolution of the thickness of three PAA hydrogels of same stiffness (12 kPa) but different initial thickness (CT=168 μm, thin=54 μm, thick=358 μm) during and after application of a 10 min stretch pulse. Solid lines are fits of the poroelastic model described in Supplementary Note 1. c, Percentage of the initial crack area that remains open 2 and 5 min after unstretching hydrogels of different thickness (n=4 per condition, CT=156.3± 9.8 μm, thin=59.3±3.8 μm, thick=345.0±15.3 μm, mean±SEM). d, (left) Clusters of MDCK expressing LifeAct-GFP on PAA hydrogels of same initial thickness but different stiffness (0.2, 12, 200kPa). (right) Time-lapse evolution of the regions highlighted on left panels before and after a 10 min 10% biaxial stretch. e, Distribution of crack size 45 s after unstretch in clusters attached to hydrogels of different stiffness (0.2kPa, 12kPa and 200kPa). f, Percentage of crack area 45s after stretch cessation in clusters attached to substrates of different stiffness. Values on the x-axis are hydraulic pressures at the cell-gel interface estimated from the poroelastic theory (Supplementary Note 1). Error bars show SEM. Epithelial clusters are 80 μm in diameter.
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Figure 4: The origin of cracks is hydraulica, (left) Clusters of MDCK cells expressing LifeAct-GFP on PAA hydrogels of different thickness. (right) Time-lapse evolution of the regions highlighted in orange on left panels before and after a 10 min pulse of 10% biaxial stretch. The acquisition time of each snap shot is marked by a black dot on the time axis (top). Arrowheads point to a subset of cracks. b, Time evolution of the thickness of three PAA hydrogels of same stiffness (12 kPa) but different initial thickness (CT=168 μm, thin=54 μm, thick=358 μm) during and after application of a 10 min stretch pulse. Solid lines are fits of the poroelastic model described in Supplementary Note 1. c, Percentage of the initial crack area that remains open 2 and 5 min after unstretching hydrogels of different thickness (n=4 per condition, CT=156.3± 9.8 μm, thin=59.3±3.8 μm, thick=345.0±15.3 μm, mean±SEM). d, (left) Clusters of MDCK expressing LifeAct-GFP on PAA hydrogels of same initial thickness but different stiffness (0.2, 12, 200kPa). (right) Time-lapse evolution of the regions highlighted on left panels before and after a 10 min 10% biaxial stretch. e, Distribution of crack size 45 s after unstretch in clusters attached to hydrogels of different stiffness (0.2kPa, 12kPa and 200kPa). f, Percentage of crack area 45s after stretch cessation in clusters attached to substrates of different stiffness. Values on the x-axis are hydraulic pressures at the cell-gel interface estimated from the poroelastic theory (Supplementary Note 1). Error bars show SEM. Epithelial clusters are 80 μm in diameter.

Mentions: We next used the theory to produce additional testable predictions. A first set of predictions concerns hydrogel geometry. The theory predicts that the pressure that builds up immediately after stretch cessation is independent of the initial hydrogel thickness, but that the characteristic time for pressure relaxation is proportional to the square of the thickness. We confirmed these predictions in hydrogels of different initial thickness (68 μm to 358 μm) (Fig. 4a, Supplementary Notes 1,2). When we applied stretch pulses of 10 min duration and 10% strain, cracks appeared in all cell clusters regardless of the hydrogel thickness. However, the healing dynamics were much faster in thinner hydrogels, consistent with the fact that the relaxation of transepithelial pressure was faster (Fig. 4b,c).


Hydraulic fracture during epithelial stretching.

Casares L, Vincent R, Zalvidea D, Campillo N, Navajas D, Arroyo M, Trepat X - Nat Mater (2015)

The origin of cracks is hydraulica, (left) Clusters of MDCK cells expressing LifeAct-GFP on PAA hydrogels of different thickness. (right) Time-lapse evolution of the regions highlighted in orange on left panels before and after a 10 min pulse of 10% biaxial stretch. The acquisition time of each snap shot is marked by a black dot on the time axis (top). Arrowheads point to a subset of cracks. b, Time evolution of the thickness of three PAA hydrogels of same stiffness (12 kPa) but different initial thickness (CT=168 μm, thin=54 μm, thick=358 μm) during and after application of a 10 min stretch pulse. Solid lines are fits of the poroelastic model described in Supplementary Note 1. c, Percentage of the initial crack area that remains open 2 and 5 min after unstretching hydrogels of different thickness (n=4 per condition, CT=156.3± 9.8 μm, thin=59.3±3.8 μm, thick=345.0±15.3 μm, mean±SEM). d, (left) Clusters of MDCK expressing LifeAct-GFP on PAA hydrogels of same initial thickness but different stiffness (0.2, 12, 200kPa). (right) Time-lapse evolution of the regions highlighted on left panels before and after a 10 min 10% biaxial stretch. e, Distribution of crack size 45 s after unstretch in clusters attached to hydrogels of different stiffness (0.2kPa, 12kPa and 200kPa). f, Percentage of crack area 45s after stretch cessation in clusters attached to substrates of different stiffness. Values on the x-axis are hydraulic pressures at the cell-gel interface estimated from the poroelastic theory (Supplementary Note 1). Error bars show SEM. Epithelial clusters are 80 μm in diameter.
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Figure 4: The origin of cracks is hydraulica, (left) Clusters of MDCK cells expressing LifeAct-GFP on PAA hydrogels of different thickness. (right) Time-lapse evolution of the regions highlighted in orange on left panels before and after a 10 min pulse of 10% biaxial stretch. The acquisition time of each snap shot is marked by a black dot on the time axis (top). Arrowheads point to a subset of cracks. b, Time evolution of the thickness of three PAA hydrogels of same stiffness (12 kPa) but different initial thickness (CT=168 μm, thin=54 μm, thick=358 μm) during and after application of a 10 min stretch pulse. Solid lines are fits of the poroelastic model described in Supplementary Note 1. c, Percentage of the initial crack area that remains open 2 and 5 min after unstretching hydrogels of different thickness (n=4 per condition, CT=156.3± 9.8 μm, thin=59.3±3.8 μm, thick=345.0±15.3 μm, mean±SEM). d, (left) Clusters of MDCK expressing LifeAct-GFP on PAA hydrogels of same initial thickness but different stiffness (0.2, 12, 200kPa). (right) Time-lapse evolution of the regions highlighted on left panels before and after a 10 min 10% biaxial stretch. e, Distribution of crack size 45 s after unstretch in clusters attached to hydrogels of different stiffness (0.2kPa, 12kPa and 200kPa). f, Percentage of crack area 45s after stretch cessation in clusters attached to substrates of different stiffness. Values on the x-axis are hydraulic pressures at the cell-gel interface estimated from the poroelastic theory (Supplementary Note 1). Error bars show SEM. Epithelial clusters are 80 μm in diameter.
Mentions: We next used the theory to produce additional testable predictions. A first set of predictions concerns hydrogel geometry. The theory predicts that the pressure that builds up immediately after stretch cessation is independent of the initial hydrogel thickness, but that the characteristic time for pressure relaxation is proportional to the square of the thickness. We confirmed these predictions in hydrogels of different initial thickness (68 μm to 358 μm) (Fig. 4a, Supplementary Notes 1,2). When we applied stretch pulses of 10 min duration and 10% strain, cracks appeared in all cell clusters regardless of the hydrogel thickness. However, the healing dynamics were much faster in thinner hydrogels, consistent with the fact that the relaxation of transepithelial pressure was faster (Fig. 4b,c).

Bottom Line: Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension.We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres.Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioengineering of Catalonia, 08028 Barcelona, Spain.

ABSTRACT
The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells' cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

Show MeSH
Related in: MedlinePlus