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

Janna V. Serbo, Scot Kuo, Shawna Lewis, Matthew Lehmann, Jiuru Li, David H. Gracias, Lewis H. Romer

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)146-158
Number of pages13
JournalAdvanced Healthcare Materials
Volume5
Issue number1
DOIs
StatePublished - Jan 1 2016

    Fingerprint

Keywords

  • 3D confinement
  • Anisotropy
  • Cytoskeleton
  • Fibronectin
  • Tissue engineering

ASJC Scopus subject areas

  • Biomaterials
  • Biomedical Engineering
  • Pharmaceutical Science

Cite this