Mechanical and failure properties of extracellular matrix sheets as a function of structural protein composition

Abstract

The goal of this study was to determine how alterations in protein composition of the extracellular matrix (ECM) affect its functional properties. To achieve this, we investigated the changes in the mechanical and failure properties of ECM sheets generated by neonatal rat aortic smooth muscle cells engineered to contain varying amounts of collagen and elastin. Samples underwent static and dynamic mechanical measurements before, during, and after 30 min of elastase digestion followed by a failure test. Microscopic imaging was used to measure thickness at two strain levels to estimate the true stress and moduli in the ECM sheets. We found that adding collagen to the ECM increased the stiffness. However, further increasing collagen content altered matrix organization with a subsequent decrease in the failure strain. We also introduced collagen-related percolation in a nonlinear elastic network model to interpret these results. Additionally, linear elastic moduli correlated with failure stress which may allow the in vivo estimation of the stress tolerance of ECM. We conclude that, in engineered replacement tissues, there is a tradeoff between improved mechanical properties and decreased extensibility, which can impact their effectiveness and how well they match the mechanical properties of native tissue.

Figure 1

(A) Confocal image examples of autofluorescence taken from the bottom layer of each of the three ECM sheet types while still encased in gelatin. (B) The average thickness data for all three ECM sheet groups at 0% strain (left) and ∼20% strain (right). The asterisk denotes a significant difference compared to 0% strain (p < 0.05). The pound sign denotes a significant difference between groups at a given strain (p < 0.05).

Figure 2

Time course of the mean ± SD of the stiffness parameter, H, and the damping parameter, G, for the control (solid shapes) and elastase-digested (open shapes) samples. Panels A–C display H-values for the EO, EC10, and EC6 groups, respectively. Panels D–F display G-values for the EO, EC10, and EC6 groups, respectively. The asterisk denotes a significant difference within group compared to the value at 0 min (p < 0.05). The pound sign denotes a significant difference between digested and control samples at a given time point (p < 0.05).

Figure 3

Representative examples of the stress-strain curves for the three sample groups.

Figure 4

Time course of the mean ± SD of the amplitude parameter A and the nonlinearity parameter b for the control (solid shapes) and elastase-digested (open shapes) samples. Panels A–C display A-values for the EO, EC10, and EC6 groups, respectively. Panels D–F display b-values for the EO, EC10, and EC6 groups, respectively. The asterisk denotes a significant difference within group compared to the value at 0 min (p < 0.05). The pound sign denotes a significant difference between digested and control samples at a given time point (p < 0.05).

Figure 5

(A) The mean ± SD values of the maximum stress during a failure test for control samples (left) and elastase-digested samples (right) for all three ECM sheet types. (B) The mean ± SD of the failure strain for control samples (left) and elastase-digested samples (right) for all three ECM sheet types. The asterisk denotes a significant difference between elastase-digested and control values within a group (p < 0.05). The pound sign denotes a significant difference between group values at a given treatment (p < 0.05).

Figure 6

Electron microscopic images of EC6 sheets at 0% (A) and 30% (B) strain. Images were taken at 12,500×. Arrows denote collagen fibers and e denotes elastin. Scale bar represents 0.5 μm.

Figure 7

Examples of (A) an organized network with stiffer springs comprising ∼14% of the network but organized into a single fiber, and (B) a network in which stiff springs make up 40% of the network but are randomly distributed. Both networks are strained to 40% strain. Note that darker shading represents higher force and lighter shading represents lower force.

Figure 8

Stress-strain curves from network model simulations of varying amounts of collagen content by randomly dispersing stiffer springs in a homogeneous network. Note that the solid line with square symbols represents a simulation of a network with 6:1 normal to stiff spring ratio in which the stiff springs have been organized into fibers that transverse the network. Stress is given in arbitrary units.

Figure 9

Correlation between failure stress and the stress-strain amplitude parameter, A, for all three groups at control conditions. Note that A is a reasonable predictor of failure stress as evidenced by the regression coefficient (r² = 0.40).