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Research Papers

Multiscale Characterization of Engineered Cardiac Tissue Architecture

[+] Author and Article Information
Nancy K. Drew

Department of Biomedical Engineering,
Center for Complex Biological Systems,
The Edwards Lifesciences Center for
Advanced Cardiovascular Technology,
University of California, Irvine,
Irvine, CA 92697
e-mail: ndrew@uci.edu

Nicholas E. Johnsen

Department of Biomedical Engineering,
The Edwards Lifesciences Center for
Advanced Cardiovascular Technology,
University of California, Irvine,
Irvine, CA 92697
e-mail: nejohnsen@gmail.com

Jason Q. Core

Department of Biomedical Engineering,
The Edwards Lifesciences Center for
Advanced Cardiovascular Technology,
University of California, Irvine,
Irvine, CA 92697
e-mail: corej@uci.edu

Anna Grosberg

Department of Biomedical Engineering,
Center for Complex Biological Systems,
The Edwards Lifesciences Center for
Advanced Cardiovascular Technology,
Department of Chemical Engineering
and Material Science,
University of California, Irvine,
Irvine, CA 92697
e-mail: grosberg@uci.edu

1N. K. Drew and N. E. Johnsen contributed equally to this work.

2Corresponding author.

Manuscript received May 15, 2016; final manuscript received August 26, 2016; published online October 21, 2016. Assoc. Editor: Jessica E. Wagenseil.

J Biomech Eng 138(11), 111003 (Oct 21, 2016) (8 pages) Paper No: BIO-16-1202; doi: 10.1115/1.4034656 History: Received May 15, 2016; Revised August 26, 2016

In a properly contracting cardiac muscle, many different subcellular structures are organized into an intricate architecture. While it has been observed that this organization is altered in pathological conditions, the relationship between length-scales and architecture has not been properly explored. In this work, we utilize a variety of architecture metrics to quantify organization and consistency of single structures over multiple scales, from subcellular to tissue scale as well as correlation of organization of multiple structures. Specifically, as the best way to characterize cardiac tissues, we chose the orientational and co-orientational order parameters (COOPs). Similarly, neonatal rat ventricular myocytes were selected for their consistent architectural behavior. The engineered cells and tissues were stained for four architectural structures: actin, tubulin, sarcomeric z-lines, and nuclei. We applied the orientational metrics to cardiac cells of various shapes, isotropic cardiac tissues, and anisotropic globally aligned tissues. With these novel tools, we discovered: (1) the relationship between cellular shape and consistency of self-assembly; (2) the length-scales at which unguided tissues self-organize; and (3) the correlation or lack thereof between organization of actin fibrils, sarcomeric z-lines, tubulin fibrils, and nuclei. All of these together elucidate some of the current mysteries in the relationship between force production and architecture, while raising more questions about the effect of guidance cues on self-assembly function. These types of metrics are the future of quantitative tissue engineering in cardiovascular biomechanics.

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References

Figures

Grahic Jump Location
Fig. 1

Example of actin fibril consistency in NRVM. (a) Images of stained NRVMS cultured on various shaped FN islands. Thestains in the images are green for actin, red for α-actinin, and blue for nuclei. (b) The average COOP for consistency of actin fibril organization at different areas and length-scales in rectangular-shaped NRVMs with an aspect ratio ≈ 11. Scale bars = 10 μm.

Grahic Jump Location
Fig. 2

Actin fibril consistency for various aspect ratios. The average COOP for consistency of actin fibril organization for different aspect ratios at the small length-scale of 1 μm (a) and at the large length-scale 21 μm (b). Inset of (b) shows aspect ratios for the isosceles triangle shapes.

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Fig. 3

Orientational order parameter (OOP) analysis over multiple spatial scales. (a) and (b) Immunostain images of anisotropic (i) and isotropic (ii) tissues for nuclei (a), sarcomeric z-lines (b), actin (c), and tubulin (d). (e) and (f) OOP data for anisotropic N = 4 (e) and isotropic N = 8 (f) tissues over multiple spatial scales. (g) The area at which the isotropic OOP is half way between minimum and maximum for each structure (legend at figure top); *indicates statistical significance with p < 0.001. Error bars represent the standard deviation of the data. Scale bar = 10 μm.

Grahic Jump Location
Fig. 4

Co-orientational order parameter analysis between tissue structure pairs. (a) and (b) Immunostain images of sarcomeric z-lines (red—(a)), actin (green (a) and (b)-(i)), nuclei (blue (a) and (b)), and tubulin of microtubules (yellow (b)-(ii)). (c) COOP data representing correlation between actin and sarcomeric z-lines ((c)-(i)) (N = 4), actin and tubulin ((c)-(ii)) (N = 8), actin and nuclei ((c)-(iii)) (N = 11), and tubulin and nuclei ((c)-(iv)) (N = 6). Error bars represent the standard deviation of the data. Significance was tested within each pair of structures between the COOP, uncorrelated COOP, and correlated COOP, and labeled with an asterisk where significant. (d) Immunostain image of nuclei with Voronoi diagram (purple mesh) and individual nuclei organization vectors (blue arrows). (e) and (f) Immunostain image of actin with detailed nuclei and actin organization vectors—blue and green arrows, respectively; (f) is the enlarged section of (e) outlined in dashed rectangle. Scale bar = 10 μm.

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