The Development of Structural and Mechanical Anisotropy in Fibroblast Populated Collagen Gels

[+] Author and Article Information
Stavros Thomopoulos1

Department of Orthopaedic Surgery,  Washington University School of Medicine, 1 Barnes-Jewish Hospital Plaza, Suite 11300, Campus Box 8233, St. Louis, MO 63110ThomopoulosS@msnotes.wustl.edu

Gregory M. Fomovsky, Jeffrey W. Holmes

Department of Biomedical Engineering,  Columbia University, New York, NY 10027


Corresponding author.

J Biomech Eng 127(5), 742-750 (May 19, 2005) (9 pages) doi:10.1115/1.1992525 History: Received January 13, 2004; Revised May 19, 2005

An in vitro model system was developed to study structure-function relationships and the development of structural and mechanical anisotropy in collagenous tissues. Fibroblast-populated collagen gels were constrained either biaxially or uniaxially. Gel remodeling, biaxial mechanical properties, and collagen orientation were determined after 72h of culture. Collagen gels contracted spontaneously in the unconstrained direction, uniaxial mechanical constraints produced structural anisotropy, and this structural anisotropy was associated with mechanical anisotropy. Cardiac and tendon fibroblasts were compared to test the hypothesis that tendon fibroblasts should generate greater anisotropy in vitro. However, no differences were seen in either structure or mechanics of collagen gels populated with these two cell types, or between fibroblast populated gels and acellular gels. This study demonstrates our ability to control and measure the development of structural and mechanical anisotropy due to imposed mechanical constraints in a fibroblast-populated collagen gel model system. While imposed constraints were required for the development of anisotropy in this system, active remodeling of the gel by fibroblasts was not. This model system will provide a basis for investigating structure-function relationships in engineered constructs and for studying mechanisms underlying the development of anisotropy in collagenous tissues.

Copyright © 2005 by American Society of Mechanical Engineers
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Figure 1

Collagen gels were constrained either biaxially (BIAX) or uniaxially (UNIAX) for 72h of culture. Gel remodeling was defined by the deformation gradient FR, describing deformation of the central region of the gel from the beginning of culture (reference state) to immediately prior to testing at 72h (remodeled state). At 72h, gels underwent planar biaxial load controlled mechanical testing (where the applied load is defined by the traction tensor T) followed by measurement of collagen fiber orientation.

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Figure 2

Spatial variation in strain component E22 during mechanical loading of a UNIAX collagen gel. Strain was tracked using over 900 small markers scattered over the entire gel surface rather than the 9 marker chips we normally place in the central region of the gel. (a) Gray scale map of E22 at strain magnitudes similar to those reported in this study. Edge effects are prominent near loading bars (indicated by white rectangles) but strains are reasonably uniform in the central region where marker chips (indicated by white circles) are normally placed. (b) Fitted profiles showing variation of E22 with horizontal position across the center of the gel. Each curve indicates a different applied load, while the vertical dotted lines indicate the edges of the “central region” within which marker chips are normally placed. Edge effects are prominent and some variation exists within the central region at strain levels similar to those reported in this study (lower 2 curves); nonuniformities become increasingly pronounced at higher strain levels. (c) Profiles showing variation of E22 with vertical position across the center of the gel. Relative standard deviation (SD∕mean) of E22 in the central region was 0.24, 0.16, 0.17, and 0.19 (lowest load to highest) at the four loads for which profiles are shown.

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Figure 3

(a) Collagen fibers were visualized using confocal reflectance microscopy. (b) A gradient detection algorithm was used to determine the orientation of fibers. (c) The distribution of collagen fiber orientations was represented as a histogram. Fiber orientation data were corrected for bias introduced by polarization of the confocal laser by imaging samples at multiple orientations and fitting the resulting distributions as described in the text.

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Figure 4

Gel remodeling, collagen fiber orientations, and biaxial mechanical properties for gels 10.15.02biax and 10.15.02uniax. For the BIAX gel, remodeling strains were near zero, collagen fibers had no apparent preferred orientation, and mechanical testing revealed isotropic behavior. The UNIAX gel showed substantial contraction in the x2 direction, preferred orientation of collagen fibers toward the x1 direction, and clear mechanical anisotropy.

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Figure 5

Average collagen fiber distributions for collagen gels. Collagen fiber distributions in UNIAX gels were biased towards zero degrees (defined as the x1, or constrained, direction). Collagen fibers in BIAX gels were distributed randomly. There were no differences between cell types.

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Figure 6

Average biaxial mechanical properties for BIAX and UNIAX gels. BIAX gels displayed isotropic mechanical behavior under equibiaxial loading. UNIAX gels displayed anisotropic mechanical behavior. There were no differences between ACF and ATF gels. Error bars represent standard deviations about the mean strain for a given stress (N=9 for each group).

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Figure 7

Predicted collagen fiber orientations for ACF and ATF based on experimentally measured remodeling FR. It was assumed that fiber orientations were randomly distributed at t=0. Affine transformation predicted an increase in organization due to the remodeling strain at t=72h. Experimentally measured collagen orientations for ACF and ATF gels demonstrate similar alignment compared to the affine transformation prediction at t=72h.




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