Biaxial stretch effects on fibroblast-mediated remodeling of fibrin gel equivalents

Balestrini, Jenna Leigh

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Biaxial stretch effects on fibroblast-mediated remodeling of fibrin gel equivalents

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Mechanical loads play a pivotal role in the growth, maintenance, remodeling, and disease onset in connective tissues. Harnessing the relationship between mechanical signals and how cells remodel their surrounding extracellular matrix would provide new insights into the fundamental processes of wound healing and fibrosis and also assist in the creation of custom-tailored tissue equivalents for use in regenerative medicine. In 3D tissue models, uniaxial cyclic stretch has been shown to stimulate the synthesis and crosslinking of collagen while increasing the matrix density, fiber alignment, stiffness, and tensile strength in the direction of principal stretch. Unfortunately, the profound fiber realignment in these systems render it difficult to differentiate between passive effects and cell-mediated remodeling. Further, these previous studies generally focus on a single level of stretch magnitude and duration, and they also investigate matrix remodeling under a homogeneous strain conditions. Therefore, these studies are not sufficient to establish key information regarding stretch-dependent remodeling for use in tissue engineering and also do not simulate the complex mechanical environment of connective tissue. We first developed a novel in vitro model system using equibiaxial stretch on fibrin gels (early models of wound healing) that enabled the isolation of mechanical effects on cell-mediated matrix remodeling. Using this system we demonstrated that in the absence of in-plane alignment, stretch stimulates fibroblasts to produce a stronger tissue by synthesizing collagen and condensing their surrounding matrix. We then developed dose-response curves for multiple aspects of tissue remodeling as a function of stretch magnitude and duration (intermittent versus continuous stretch). Our results indicate that both the magnitude and the duration per day of stretch are important factors in mechanically induced cell activity, as evidenced by dose-dependent responses of several remodeling metrics in response to these two parameters (UTS, matrix stiffness, collagen content, cell number). In addition, we found that cellularity, collagen content, and resistance to tension increased when the tissues were mechanically loaded intermittently as opposed to continuously. Finally, we developed a novel model system that produces non-homogeneous strain distribution, allowing for the simultaneous study of strain gradients, strain anisotropy, and strain magnitude in 2D and 3D. Establishing a system that produces complex strain distributions provides a more accurate model of the mechanical conditions found in connective tissue, and also allows for the investigation of cellular adaptations to a changing mechanical environment.

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