Research Papers

Magnitude and Duration of Stretch Modulate Fibroblast Remodeling

[+] Author and Article Information
Jenna L. Balestrini, Kristen L. Billiar

Departments of Biomedical Engineering and Mechanical Engineering, Worcester Polytechnic University, Worcester, MA 01609-2280; Department of Mechanical Engineering, Department of Surgery, University of Massachusetts Medical School, Worcester, MA 01655

J Biomech Eng 131(5), 051005 (Mar 24, 2009) (9 pages) doi:10.1115/1.3049527 History: Received April 14, 2008; Revised September 03, 2008; Published March 24, 2009

Mechanical cues modulate fibroblast tractional forces and remodeling of extracellular matrix in healthy tissue, healing wounds, and engineered matrices. The goal of the present study is to establish dose-response relationships between stretch parameters (magnitude and duration per day) and matrix remodeling metrics (compaction, strength, extensibility, collagen content, contraction, and cellularity). Cyclic equibiaxial stretch of 2–16% was applied to fibroblast-populated fibrin gels for either 6 h or 24 h/day for 8 days. Trends in matrix remodeling metrics as a function of stretch magnitude and duration were analyzed using regression analysis. The compaction and ultimate tensile strength of the tissues increased in a dose-dependent manner with increasing stretch magnitude, yet remained unaffected by the duration in which they were cycled (6 h/day versus 24 h/day). Collagen density increased exponentially as a function of both the magnitude and duration of stretch, with samples stretched for the reduced duration per day having the highest levels of collagen accumulation. Cell number and failure tension were also dependent on both the magnitude and duration of stretch, although stretch-induced increases in these metrics were only present in the samples loaded for 6 h/day. Our results indicate that both the magnitude and the duration per day of stretch are critical parameters in modulating fibroblast remodeling of the extracellular matrix, and that these two factors regulate different aspects of this remodeling. These findings move us one step closer to fully characterizing culture conditions for tissue equivalents, developing improved wound healing treatments and understanding tissue responses to changes in mechanical environments during growth, repair, and disease states.

Copyright © 2009 by American Society of Mechanical Engineers
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Figure 1

Schematics representing (a) fibrin gel with foam anchor attached prior to and (b) after loading onto the biaxial device. The dotted lines represent areas to be sectioned, and the holes represent areas the hooks will be placed. The four arrows in the x and y axis represent force at each of the tethering points. Note that the force is distributed equally at each loading point.

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Figure 2

Representative brightfield images of hematoxylin and eosin stained sections of fibrin gels stretched intermittently for 8 days at 0%, 2%, 4%, 8%, and 16% stretch. These images demonstrate the dose-dependent decrease in thickness with stretch magnitude and the corresponding increase in protein and cell density as seen in both continuous and intermittently stretched groups. The indentations in the 0% and 2% stretch group are artifact of histological sectioning, and not present during culture. Original magnification 200×; scale bar=100 μm.

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Figure 3

(a) Tissue thickness, (b) UTS, (c) collagen density, (d) extensibility, (e) failure tension, (f) stiffness, (g) active retraction, (h) passive retraction, and (i) cell number of CS (24 h/day), and IS (6 h/day) fibrin gels cycled at 2%, 4%, 8%, and 16% stretch for 8 days at 0.2 Hz, and normalized to statically cultured controls from each experiment. Note that UTS, stiffness, and collagen density are, by definition, directly dependent on the thickness, whereas the other parameters are independent of the degree of compaction. For clarity, statistical models are provided in Table 1.

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Figure 4

(a) Representative fibroblast-populated fibrin gel at 40 s and 7 min post release from its substrate. The gels are placed in a 35 mm tissue culture dish and are free floating in culture media. The dashed line represents the initial area of the fibrin gel (25 mm in diameter) that was dynamically cultured for 8 days and then cut away from its circumferential anchors. Note the rapid decrease in projected-sectional area. (b) Representative data of total matrix retraction as a function of time. The data are fitted to an exponential increase equation as represented by the dotted line with three parameters; RT (total retraction), RA (active retraction), R0 (passive retraction), and τ (time constant), shown schematically in the figure. The bulk of the retraction occurs in less than 10 min (τ∼9 min), and tensional homeostasis is reached by approximately 30 min.

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Figure 5

(a) Representative engineering stress-strain plot of equibiaxial loading along orthogonal “1” and “2” directions demonstrating isotropy of the matrix in a gel cycled intermittently at 4% strain for 8 days. Note that the 1 and 2 directions result in identical stress-strain profiles; this similarity is a result of the in-plane isotropy of the material. (b) Representative stress-strain data from gels stretched at 0%, 2%, 4%, 8%, and 16% stretch for 6 h a day at 0.2 Hz. Stiffness generally increases with increasing culture stretch magnitude, with a large increase between the 4% and 8% strain-conditioned groups, but no significant change between the 8% and 16% strain-conditioned groups.




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