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Research Papers

Investigation of Cellular Confinement in Three-Dimensional Microscale Fibrous Substrates: Fabrication and Metrology

[+] Author and Article Information
Filippos Tourlomousis

Department of Mechanical Engineering,
Highly Filled Materials Institute,
Stevens Institute of Technology,
Hoboken, NJ 07030
e-mail: ftourlom@stevens.edu

William Boettcher

Department of Electrical and
Computer Engineering,
Stevens Institute of Technology,
Hoboken, NJ 07030

Houzhu Ding

Department of Mechanical Engineering,
Stevens Institute of Technology,
Hoboken, NJ 07030
e-mail: hding4@stevens.edu

Robert C. Chang

Department of Mechanical Engineering,
Stevens Institute of Technology,
Hoboken, NJ 07030
e-mail: rchang6@stevens.edu

1Corresponding author.

Contributed by the Manufacturing Engineering Division of ASME for publication in the JOURNAL OF MICRO- AND NANO-MANUFACTURING. Manuscript received September 12, 2017; final manuscript received November 29, 2017; published online January 18, 2018. Editor: Jian Cao.

J. Micro Nano-Manuf 6(2), 021003 (Jan 18, 2018) (7 pages) Paper No: JMNM-17-1055; doi: 10.1115/1.4038803 History: Received September 12, 2017; Revised November 29, 2017

Engineered microenvironments along with robust quantitative models of cell shape metrology that can decouple the effect of various well-defined cues on a stem cell's phenotypic response would serve as an illuminating tool for testing mechanistic hypotheses on how stem cell fate is fundamentally regulated. As an experimental testbed to probe the effect of geometrical confinement on cell morphology, three-dimensional (3D) poly(ε-caprolactone) (PCL) layered fibrous meshes are fabricated with an in-house melt electrospinning writing system (MEW). Gradual confinement states of fibroblasts are demonstrated by seeding primary fibroblasts on defined substrates, including a classical two-dimensional (2D) petri dish and porous 3D fibrous substrates with microarchitectures tunable within a tight cellular dimensional scale window (1–50 μm). To characterize fibroblast confinement, a quantitative 3D confocal fluorescence imaging workflow for 3D cell shape representation is presented. The methodology advanced allows the extraction of cellular and subcellular morphometric features including the number, location, and 3D distance distribution metrics of the shape-bearing focal adhesion (FA) proteins.

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Figures

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Fig. 1

Workflow followed in the present study for fibrous substrate fabrication using an in-house melt electrospinning writing and subsequent substrate functionalization with poly-L-lysine, cell seeding, and cell morphology observation after 24 h

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Fig. 2

Gradual confinement of NHDF primary fibroblast cells: (a) Classical 2D petri dish substrate used as control, (b) 3D fibrous mesh with “0–90 deg” pore microarchitecture, and (c) 3D fibrous mesh with “0–45–135–90 deg” pore microarchitecture. Cell morphology of representative NHDFs 1 day after seeding stained for vinculin (green), actin microfilament (red) and nucleus (blue) on the different substrate dimensionalities ((a)−(c)).

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Fig. 3

Image processing workflow for the detection of FA proteins in fluorescent images. Initially, the cell body is segmented using thresholding and filtering techniques from a raw grayscale image. Then, the individual FAs are detected and accurately segmented within the detected ROI.

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Fig. 4

(a) Demonstration of an optimized algorithm for accurate FA detection in z-section image data obtained from fluorescent confocal microscopy. The cell is confined within the porous microarchitecture of the printed substrate. Detected FAs are overlaid with red color. The overlay image is inverted into black and white for subsequent 3D volume rendering. (b) 3D volume renderings of cytoskeleton (red), FAs (green), and nucleus (blue) yield the composite image used for FA metrology directly from the 3D reconstructed cell shape.

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Fig. 5

FA metrology using reconstructed cell shapes from the 2D substrates. (a) Representative 3D reconstructed cell shapes from the control groups (cell 1, cell 2, and cell 3) (axes units: μm) and (b) 3D radial Euclidean distance metric plotted as function of individual FA for each representative reconstructed cell shape shown in Figure A. The 3D radial Euclidean distance is computed based on the distance between the centroids of individual FAs and the centroid of the nucleus, (c) histograms of CFD of 3Dradial Euclidean distance for each reconstructed cell shape. Straight lines are fitted to the data using linear regression to determine the slope of the curve.

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Fig. 6

FA metrology using reconstructed cell shapes from the 3D fibrous substrates: (a) various 3D reconstructed shapes of cells confined within the porous microarchitecture of the 3D fibrous substrates with average interfiber distance 75 μm (axes units: μm) and (b) 3D radial Euclidean distance metric plotted as function of individual FA for each representative reconstructed cell shape shown in Figure A. (c) Histograms of CFD of 3D radial Euclidean distance for each reconstructed cell shape. Straight lines fitted with linear regression to determine the slope of the curve.

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