A Combined In Vitro Imaging and Multi-Scale Modeling System for Studying the Role of Cell Matrix Interactions in Cutaneous Wound Healing.

Many cell types remodel the extracellular matrix of the tissues they inhabit in response to a wide range of environmental stimuli, including mechanical cues. Such is the case in dermal wound healing, where fibroblast migrate into and remodel the provisional fibrin matrix in a complex manner that depends in part on the local mechanical environment and the evolving multi-scale mechanical interactions of the system. In this study, we report on the development of an image-based multi-scale mechanical model that predicts the short-term (24 hours), structural reorganization of a fibrin gel by fibroblasts. These predictive models are based on an in vitro experimental system where clusters of fibroblasts (i.e., explants) were spatially arranged into a triangular geometry onto the surface of fibrin gels that were subjected to either Fixed or Free in-plane mechanical constraints. Experimentally, regional differences in short-term structural remodeling and cell migration were observed for the two gel boundary conditions. A pilot experiment indicated that these small differences in the short-term remodeling of the fibrin gel translate into substantial differences in long-term (4 weeks) remodeling, particularly in terms of collagen production. The multi-scale models were able to predict some regional differences in remodeling and qualitatively similar reorganization patterns for the two boundary conditions. However, other aspects of the model, such as the magnitudes and rates of deformation of gel, did not match the experiments. These discrepancies between model and experiment provide fertile ground for challenging model assumptions and devising new experiments to enhance our understanding of how this multi-scale system functions. These efforts will ultimately improve the predictions of the remodeling process, particularly as it relates to dermal wound healing and the reduction of patient scarring. Such models could be used to recommend patient-specific mechanical-based treatment dependent on parameters such as wound geometry, location, age, and health

Comparison of Regional Differences in Microsphere Displacements.

<p>In both (A) <i>Fixed</i> and (B) <i>Free</i> gels the cumulative displacements of images associated with Region 1 (yellow box) and Region 4 (red box) at each hour are represented with box plots, where the box edges show the 25^th and 75^th percentiles, the median is depicted with a red line, and the whiskers indicate the full range of the data points. Color-coded arrows show the direction and magnitudes of the cumulative displacements at t = 24 hours. Microsphere displacements are directed towards either the explants or the axial regions between explants. Larger microsphere displacements occurred in the <i>Free</i> gel compared to the <i>Fixed</i> gel.</p

Comparison of regional average magnitude and period average rate of microsphere displacement for each boundary condition and between the experiment and model.

<p>(A) Average cumulative microsphere displacement and (B) period average rate of microsphere displacement in the experiment for all four regions and for each boundary condition plotted every six hours. Error bars represent the standard error of the mean. Equivalent plots for the model show the (C) average cumulative FE nodal displacement, and (D) average rates of nodal displacement for all four regions in the model for each boundary condition. The model predicts larger displacements and rates of displacement in the <i>Free</i> gel compared to the <i>Fixed</i> gel, as was observed experimentally. However, instead of the experimentally observed decrease in the displacement rates with time, the model predicts a gradually increasing rate of displacement with time.</p

Pilot Study of Compositional Remodeling after 4 weeks of culture.

<p>Mason’s trichrome stain for a (A) <i>Fixed</i> and (B) <i>Free</i> gel after 4 weeks of culture supplemented with TGF-β1 and ascorbic acid. More collagen (blue) was produced in <i>Fixed</i> gels than in <i>Free</i> gels.</p

Microsphere displacements in a <i>Free</i> gel.

<p>Cumulative microsphere displacements at (A,B) t = 12 hours and (C,D) t = 24 are depicted spatially with color coded arrows that indicate the direction and magnitudes. Also shown are the corresponding histograms of microsphere displacement.</p

Model prediction of fiber realignment.

<p>Fiber network realignment at t = 24 hours for a (A) <i>Fixed</i> and (B) <i>Free</i> gel. The color map indicates the change in the strength of fiber alignment (Δ<i>α</i>) from the initial network configuration. Also shown as white arrows are the principal directions of fiber alignment for those elements where Δ<i>α</i> > 0.05. Network reorganization and fiber forces for a representative network in an axial (C,D) and non-axial region (E,F) in roughly equivalent locations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148254#pone.0148254.g007" target="_blank">Fig 7</a>.</p

Microsphere displacements in a <i>Fixed</i> gel.

<p>Cumulative microsphere displacements at (A,B) t = 12 hours and (C,D) t = 24 are depicted spatially with color coded arrows that indicate the direction and magnitudes. Also shown are the corresponding histograms of microsphere displacement.</p

In vitro data collection and image-based computational model.

<p>(A) Three explants were placed at a distance of approximately 2 mm from each other to form the vertices of a triangle. A tiled image consisting of 36 individual 10x DIC images was acquired every 15 minutes in order to observe and quantify structural and morphological changes in the fibrin gel. (B) Over the course of the experiment the instantaneous and cumulative displacements of a subset of embedded microspheres was analyzed at each location (the depicted image corresponds to the black box in (A)). (C) The image-based computational model consists of finite elements containing fiber networks that are representative of the fibrin fibers in the gel. (D) The model is partitioned into cellular networks (white elements) and surrounding fibrin networks (yellow elements) that are configured to approximate the geometric configuration of the explants and gel in the experiments.</p

Overall Average Magnitude and Rate of Microsphere Displacement.

<p>Overall Average Magnitude and Rate of Microsphere Displacement.</p