Characterizing how cells in three-dimensional (3D) environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension.

The ways that fibroblasts remodel their environment is central to wound healing, development of musculoskeletal tissues, and progression of pathologies such as fibrosis. However, the changes that fibroblasts make to the material around them and the mechanical consequences of these changes have proven difficult to quantify, especially in a realistic, viscoelastic three dimensional culture environments, leaving a critical need for quantitative data. Here, we observed the mechanisms and quantified the mechanical effects of fibroblast remodeling in engineered tissue constructs (ETCs) comprised of reconstituted rat tail (type I) collagen and human fibroblast cells. To study the effects of remodeling on tissue mechanics, stress-relaxation tests were performed on ETCs cultured for 24, 48, and 72 hours. ETCs were treated with deoxycholate and tested again to assess the ECM response. Viscoelastic relaxation spectra were obtained using the generalized Maxwell model. Cells exhibited viscoelastic damping at two finite time constants over which the ECM showed little damping, approximately 0.2 s and 10-30 s. Different finite time constants in the range of 1-7000 s were attributed to ECM relaxation. Cells remodeled the ECM to produce a relaxation time constant on the order of 7000 s, and to merge relaxation finite time constants in the 0.5-2 s range into a single time content in the 1 s range. Results shed light on hierarchical deformation mechanisms in tissues, and on pathologies related to collagen relaxation such as diastolic dysfunction.

Myocardial function deteriorates over the course of fibrotic cardiomyopathy, due to electrophysiological and mechanical effects of myofibroblasts that are not completely understood. Although a range of experimental model systems and associated theoretical treatments exist at the levels of isolated cardiomyocytes and planar co-cultures of myofibroblasts and cardiomyocytes, interactions between these cell types at the tissue level are less clear. We studied these interactions through an engineered heart tissue (EHT) model of fibrotic myocardium and a mathematical model of the effects of cellular composition on EHT impulse conduction velocity. The EHT model allowed for modulation of cardiomyocyte and myofibroblast volume fractions, and observation of cell behavior in a three-dimensional environment that is more similar to native heart tissue than is planar cell culture. The cardiomyocyte and myofibroblast volume fractions determined the retardation of impulse conduction (spread of the action potential) in EHTs as measured by changes of the fluorescence of the Ca2+ probe, Fluo-2. Interpretation through our model showed retardation far in excess of predictions by homogenization theory, with conduction ceasing far below the fibroblast volume fraction associated with steric percolation. Results point to an important multiscale structural role of myofibroblasts in attenuating impulse conduction in fibrotic cardiomyopathy.

Imaging of thick biological samples has been very challenging because of severe light scattering. The transparent cornea with the unique organization of stromal collagen makes it a good candidate for deep imaging and is responsible for mechanical strength and optical clarity of the eye. However, limitation on traditional histology method provides incomplete spatial information and details on the structural organization of corneal tissue is still not sound. Second harmonic generation (SHG) microscopy is a noninvansive and nonstained technique to characterize the macromolecular organization of collagen in biological tissues. Through the combination of SHG microcopy and optimized Fourier-transform analysis, adult and embryonic chick corneas are investigated. Our results show that the anterior stroma demonstrates a fanlike distribution of rotated fibrous lamellae. In comparison with the anterior structure, the posterior stroma maintains a nonrotating pattern while increasing the depth of corneal tissue. In particular, the rotational pattern in anterior stroma exhibits a potential role of corneal maturation. Moreover, SHG microscopy in combination with the Fourier-transform-based analysis exhibits a useful tool in determination of collagen alignment in biological tissues and discrimination of diseases.