Patterning of Fibroblast and Matrix Anisotropy within 3D Confinement is Driven by the Cytoskeleton

Abstract

Effects of 3D confinement on cellular growth and matrix assembly are important in tissue engineering, developmental biology, and regenerative medicine. Polydimethylsiloxane wells with varying anisotropy are microfabicated using soft‐lithography. Microcontact printing of bovine serum albumin is used to block cell adhesion to surfaces between wells. The orientations of fibroblast stress fibers, microtubules, and fibronectin fibrils are examined 1 day after cell seeding using laser scanning confocal microscopy, and anisotropy is quantified using a custom autocorrelation analysis. Actin, microtubules, and fibronectin exhibit higher anisotropy coefficients for cells grown in rectangular wells with aspect ratios of 1:4 and 1:8, as compared to those in wells with lower aspect ratios or in square wells. The effects of disabling individual cytoskeletal components on fibroblast responses to anisotropy are then tested by applying actin or microtubule polymerization inhibitors, Rho kinase inhibitor, or by siRNA‐mediated knockdown of AXL or cofilin‐1. Latrunculin A decreases cytoskeletal and matrix anisotropy, nocodazole ablates both, and Y27632 mutes cellular polarity while decreasing matrix anisotropy. AXL siRNA knockdown has little effect, as does siRNA knockdown of cofilin‐1. These data identify several specific cytoskeletal strategies as targets for the manipulation of anisotropy in 3D tissue constructs.

Mechanochemical Patternable ECM‐Mimetic Hydrogels for Programmed Cell Orientation

“…[ 90–94 ] These materials are mostly based on synthetic elastomers (e.g., PDMS) and polymers (i.e., polycaprolactone, polylactide, and polyethylene glycol) or polysaccharide‐based hydrogels, thus yielding geometrically confined cell distribution. [ 95–100 ] Due to the lack of mechanically adaptable features in such systems, geometrical topography is typically decoupled from ECM‐like materials presenting biofunctional cell‐active motifs. Such strategies, although initially useful for studying singular events of cell response and dynamics in controlled environments, do not accurately recapitulate native tissues which comprise entirely cell adhesive 3D topographical landscapes.…”

Engineered Tissue Development in Biofabricated 3D Geometrical Confinement–A Review

“…However, when the aspect ratio is small (e.g., less than 4:1), local anisotropy induced by the curvature and corner of the microwell is likely to dominate (Figure 2E). 72 2.3.1. Regulating the Development of Fibrous Tissue and Muscle.…”

“…Microwells of the same size but different aspect ratios ranging from 1:4 to 1:8 were found to induce high level of cellular anisotropic alignment and such alignment can be disrupted by cytoskeleton inhibiting drugs (Figure 2E). 72 In contrast, cardiac microtissues with fixed aspect ratio of 1:4 but different sizes and cellular compositions have been developed to study the effect of these factors on cardiac tissue maturation. Enhanced cytoskeletal organization, cardiac-specific protein expression and synchronous contraction were achieved in cocultured system with cardiomyocytes to cardiac fibroblasts ratio of 2:1, as compared to the monoculture regardless of the tissue geometry.…”

3D Hybrid Small Scale Devices

“…Developmental biology represents an important frontier for advancing 3D hybrid small scale devices. In this venue, imperatives of growth, adaptation, and the dynamic turnover of biological components will dictate mechanical properties, microenvironment topology and geometry, and spatial configurations of cues that integrate cells with synthetic scaffolds . Work with stem cells can shed light on microenvironmental determinants of cell fate determination, and such insights have already been used to successfully differentiate them into numerous tissue types such as blood vessels, bones, and neurons .…”