Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties

Abstract

To successfully induce tissue repair or regeneration in vivo, bioengineered constructs must possess both optimal bioactivity and mechanical strength. This is because cell interaction with the extracellular matrix (ECM) produces two different but concurrent signaling mechanisms: ligation-induced signaling, which depends on ECM biological stimuli, and traction-induced signaling, which depends on ECM mechanical stimuli. In this report, we provide a fundamental understanding of how alterations in mechanical stimuli alone, produced by varying the viscoelastic properties of our bioengineered construct, modulate phenotypic behavior at the whole-cell level. Using a physiologically relevant ECM mimic composed of hyaluronan and fibronectin, we found that adult human dermal fibroblasts modify their mechanical response in order to match substrate stiffness. More specifically, the cells on stiffer substrates had higher modulus and a more stretched and organized actin cytoskeleton (and vice versa), which translated into larger traction forces exerted on the substrate. This modulation of cellular mechanics had contrasting effects on migration and proliferation, where cells migrated faster on softer substrates while proliferating preferentially on the stiffer ones. These findings implicate substrate rigidity as a critical design parameter in the development of bioengineered constructs aimed at eliciting maximal cell and tissue function.

Figure 1. Cell stiffness/compliance as a function of hydrogel mechanics

(A) The AFM response amplitude generated from cell’s resistance to cantilever indentation increases linearly with the cantilever drive amplitude, which confirms the absence of any slip between the AFM tip and cell surface. The AFM was calibrated such that the smaller response amplitude corresponded to a stiffer surface and vice versa. (B) Response amplitude at drive amplitude of 120 mV is plotted against hydrogel mechanics, where cell stiffness increases with increasing substrate mechanics. * indicates p<0.05.

Figure 2. Actin cytoskeleton organization on different hydrogels

Immunofluorescent staining with Alexa Flour 488-Phalloidin shows that actin fibers become more stretched and organized with increasing substrate mechanics. Arrows indicate areas of filament discontinuity and kinks/bucklings. Scale bar = 16 μm.

Figure 3. DISC technique

A typical displacement field produced by a cell on a hydrogel substrate (4270 Pa). The actual displacement is very small; the magnitudes of all vectors have been amplified in this image for easier comprehension.

Figure 4. Shear stress and strain energy using FEA

Cellular shear stresses (Pa) on the different hydrogels shown (A) on different scales, and (B) on one fixed scale. These stress maps were obtained by applying a FEA model on the displacement field that was obtained using DISC (Fig. 3). (C) Strain energy stored in the hydrogels as a result of the cellular mechanical work is plotted against hydrogel stiffness (absolute value for 4270 Pa hydrogels = 7.6 ± 0.82 pJ). * indicates p<0.05.

Figure 5. (A) Single-cell migration

Migration speed decreases with increasing hydrogel stiffness; (B) Cell proliferation. Contrary to migration, cell proliferation increases with increasing hydrogel stiffness. Cell counting was performed using MetaMorph software. Day 3 cell count showed significant difference. * indicates p<0.05.