Patterning of Fibroblast and Matrix Anisotropy within 3D Confinement is Driven by the Cytoskeleton.
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.
['Serbo|Janna V|JV|', 'Kuo|Scot|S|', 'Lewis|Shawna|S|', 'Lehmann|Matthew|M|', 'Li|Jiuru|J|', 'Gracias|David H|DH|', 'Romer|Lewis H|LH|']
D051339:Actin Depolymerizing Factors D000199:Actins D000577:Amides D016880:Anisotropy D019086:Bridged Bicyclo Compounds, Heterocyclic D002478:Cells, Cultured D003599:Cytoskeleton D004129:Dimethylpolysiloxanes D005109:Extracellular Matrix D005347:Fibroblasts D005353:Fibronectins D055785:Gene Knockdown Techniques D006801:Humans D008870:Microtubules D015739:Nocodazole D011725:Pyridines D034741:RNA, Small Interfering D053778:Thiazolidines
anisotropy 3D confinement cytoskeleton tissue engineering fibronectin