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

Abstract

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 72 h 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.

A thermodynamically motivated model for stress-fiber reorganization

“…Associated with the tension-dependent stress-fiber assembly is the development of structural anisotropy. For example, uniaxially constrained, fibroblast-populated collagen gels develop a high degree of fiber alignment and mechanical anisotropy, while gels constrained biaxially remain isotropic (Thomopoulos et al 2005). (ii) Cells "sense" the stiffness of their substrates and exert smaller tractions on more compliant substrates (Discher et al 2005).…”

Potential-based and non-potential-based cohesive zone formulations under mixed-mode separation and over-closure. Part I: Theoretical analysis

“…Cross-bridge cycling between the myosin motor proteins and actin filaments generates active contractility in the actin cytoskeleton. Several experimental studies have demonstrated that a reduction in cell tension leads to the dissociation of stress fibres (Franke et al, 1984;Kolega, 1986;Burridge and Chrzanowska-Wodnicka, 1996;Tan et al, 2003;Thomopoulos et al, 2005).…”

Remodeling of Engineered Tissue Anisotropy in Response to Altered Loading Conditions

“…Images of the tissue constructs (1024 X 768 pixels) were transferred to a personal computer, and the titanium oxide marker centroids were detected semi-automatically by threshholding the binary image (Scion Image, Frederick, MD). As described elsewhere 32 , homogeneous, two-dimensional finite Lagrangian strains (i.e., normal strains E xx and E yy , and shear strain E xy ) were computed from the digitized marker coordinates at each time point after t* referenced to the pre-switch configuration immediately prior to t*. In the steady state, the final deformation relative to the pre-switch reference configuration (i.e.…”

“…Therefore, deformation of the tissue was monitored throughout the loading experiment for a representative case of t* = 24 hours (n = 4). Before culture media was added to float the gel, a 3 × 3 array of nine titanium oxide markers was carefully painted with a fine sterile brush tip on the central third of the exposed tissue surface, where the strain field has been shown to be relatively homogeneous 32 . Throughout the course of the experiment, the construct and dish were intermittently moved to a photography copy stand for imaging with a digital camera (PowerShot G2, Canon Inc.).…”