title: 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.
authors.affiliation: Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.
authors.forename: Janna V
authors.initials: JV
authors.lastname: Serbo
authors.affiliation: Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.|Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.
authors.forename: Scot
authors.initials: S
authors.lastname: Kuo
authors.affiliation: Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.
authors.forename: Shawna
authors.initials: S
authors.lastname: Lewis
authors.affiliation: Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Lehmann
authors.affiliation: Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.
authors.forename: Jiuru
authors.initials: J
authors.lastname: Li
authors.affiliation: Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.|Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Gracias
authors.affiliation: Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.|Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.|Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.|Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.|Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.
authors.forename: Lewis H
authors.initials: LH
authors.lastname: Romer
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D016880:Anisotropy; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004129:Dimethylpolysiloxanes / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D053778:Thiazolidines / Q000494:pharmacology
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D000577:Amides; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004129:Dimethylpolysiloxanes; D005353:Fibronectins; D011725:Pyridines; D034741:RNA, Small Interfering; D053778:Thiazolidines; C108830:Y 27632; D015739:Nocodazole; C037067:latrunculin A
keywords: 3D confinement; anisotropy; cytoskeleton; fibronectin; tissue engineering