Automated spatially targeted optical micro proteomics identifies fibroblast- and macrophage-specific regulation of myocardial infarction scar maturation in rats.

Myocardial infarction (MI) results from occlusion of blood supply to the heart muscle causing death of cardiac muscle cells. Following myocardial infarction (MI), extracellular matrix deposition and scar formation mechanically stabilize the injured heart as damaged myocytes undergo necrosis and removal. Fibroblasts and macrophages are key drivers of post-MI scar formation, maturation, and ongoing long-term remodelling; however, their individual contributions are difficult to assess from bulk analyses of infarct scar. Here, we employ state-of-the-art automated spatially targeted optical micro proteomics (autoSTOMP) to photochemically tag and isolate proteomes associated with subpopulations of fibroblasts (SMA+) and macrophages (CD68+) in the context of the native, MI tissue environment. Over a time course of 6-weeks post-MI, we captured dynamic changes in the whole-infarct proteome and determined that some of these protein composition signatures were differentially localized near SMA+ fibroblasts or CD68+ macrophages within the scar region. These results link specific cell populations to within-infarct protein remodelling and illustrate the distinct metabolic and structural processes underlying the observed physiology of each cell type.

Individual variability in animal-specific hemodynamic compensation following myocardial infarction.

Ventricular enlargement and heart failure are common in patients who survive a myocardial infarction (MI). There is striking variability in the degree of post-infarction ventricular remodeling, however, and no one factor or set of factors have been identified that predicts heart failure risk well. Sympathetic activation directly and indirectly modulates hypertrophic stimuli by altering both neurohormonal milieu and ventricular loading. In a recent study, we developed a method to identify the balance of reflex compensatory mechanisms employed by individual animals following MI based on measured hemodynamics. Here, we conducted prospective studies of acute myocardial infarction in rats to test the degree of variability in reflex compensation as well as whether responses to pharmacologic agents targeted at those reflex mechanisms could be anticipated in individual animals. We found that individual animals use very different mixtures of reflex compensation in response to experimental coronary ligation. Some of these mechanisms were related - animals that compensated strongly with venoconstriction tended to exhibit a decrease in the contractility of the surviving myocardium and those that increased contractility tended to exhibit venodilation. Furthermore, some compensatory mechanisms - such as venoconstriction - increased the extent of predicted ventricular enlargement. Unfortunately, initial reflex responses to infarction were a poor predictor of subsequent responses to pharmacologic agents, suggesting that customizing pharmacologic therapy to individuals based on an initial response will be challenging.

The connexin 43 carboxyl terminal mimetic peptide αCT1 prompts differentiation of a collagen scar matrix in humans resembling unwounded skin.

Phase II clinical trials have reported that acute treatment of surgical skin wounds with the therapeutic peptide alpha Connexin Carboxy-Terminus 1 (αCT1) improves cutaneous scar appearance by 47% 9-month postsurgery. While Cx43 and ZO-1 have been identified as molecular targets of αCT1, the mode-of-action of the peptide in scar mitigation at cellular and tissue levels remains to be further characterized. Scar histoarchitecture in αCT1 and vehicle-control treated skin wounds within the same patient were compared using biopsies from a Phase I clinical trial at 29-day postwounding. The sole effect on scar structure of a range of epidermal and dermal variables examined was that αCT1-treated scars had less alignment of collagen fibers relative to control wounds-a characteristic that resembles unwounded skin. The with-in subject effect of αCT1 on scar collagen order observed in Phase I testing in humans was recapitulated in Sprague-Dawley rats and the IAF hairless guinea pig. Transient increase in histologic collagen density in response to αCT1 was also observed in both animal models. Mouse NIH 3T3 fibroblasts and primary human dermal fibroblasts treated with αCT1 in vitro showed more rapid closure in scratch wound assays, with individual cells showing decreased directionality in movement. An agent-based computational model parameterized with fibroblast motility data predicted collagen alignments in simulated scars consistent with that observed experimentally in human and the animal models. In conclusion, αCT1 prompts decreased directionality of fibroblast movement and the generation of a 3D collagen matrix postwounding that is similar to unwounded skin-changes that correlate with long-term improvement in scar appearance.

Automated Spatially Targeted Optical Microproteomics Investigates Inflammatory Lesions In Situ.

Tissue microenvironment properties like blood flow, extracellular matrix, or proximity to immune-infiltrate are important regulators of cell biology. However, methods to study regional protein expression in the native tissue environment are limited. To address this need, we developed a novel approach to visualize, purify, and measure proteins in situ using automated spatially targeted optical microproteomics (AutoSTOMP). Here, we report custom codes to specify regions of heterogeneity in a tissue section and UV-biotinylate proteins within those regions. We have developed liquid chromatography-mass spectrometry (LC-MS)/MS-compatible biochemistry to purify those proteins and label-free quantification methodology to determine protein enrichment in target cell types or structures relative to nontarget regions in the same sample. These tools were applied to (a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and (b) characterize inflammatory proteins enriched in IgG4+ lesions in human esophageal tissues. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools and sample availability are limited.

Role of boundary conditions in determining cell alignment in response to stretch.

The ability of cells to orient in response to mechanical stimuli is essential to embryonic development, cell migration, mechanotransduction, and other critical physiologic functions in a range of organs. Endothelial cells, fibroblasts, mesenchymal stem cells, and osteoblasts all orient perpendicular to an applied cyclic stretch when plated on stretchable elastic substrates, suggesting a common underlying mechanism. However, many of these same cells orient parallel to stretch in vivo and in 3D culture, and a compelling explanation for the different orientation responses in 2D and 3D has remained elusive. Here, we conducted a series of experiments designed specifically to test the hypothesis that differences in strains transverse to the primary loading direction give rise to the different alignment patterns observed in 2D and 3D cyclic stretch experiments ("strain avoidance"). We found that, in static or low-frequency stretch conditions, cell alignment in fibroblast-populated collagen gels correlated with the presence or absence of a restraining boundary condition rather than with compaction strains. Cyclic stretch could induce perpendicular alignment in 3D culture but only at frequencies an order of magnitude greater than reported to induce perpendicular alignment in 2D. We modified a published model of stress fiber dynamics and were able to reproduce our experimental findings across all conditions tested as well as published data from 2D cyclic stretch experiments. These experimental and model results suggest an explanation for the apparently contradictory alignment responses of cells subjected to cyclic stretch on 2D membranes and in 3D gels.

Implications of scar structure and mechanics for post-infarction cardiac repair and regeneration.

Regenerating cardiac muscle lost during a heart attack is a topic of broad interest and enormous potential impact. One promising approach is to regenerate or re-engineer new myocardium in situ, at the site of damage, by injecting cells, growth factors, and other materials, or by reprogramming aspects of the normal wound healing process. A wide variety of strategies have been explored, from promoting angiogenesis to injection of a variety of different progenitor cell types, to re-engineering resident cells to produce key growth factors or even transdifferentiate into myocytes. Despite substantial progress and continued promise, clinical impact of this work has fallen short of expectations. One contributing factor may be that many efforts focus primarily on generating cardiomyocytes, with less attention to re-engineering the extracellular matrix (ECM). Yet the role of the ECM is particularly crucial to consider following myocardial infarction, which leads to rapid formation of a collagen-rich scar. This review combines a brief summary of current efforts to regenerate cardiomyocytes with what is currently known about the structure and mechanics of post-infarction scar, with the goal of identifying principles that can guide efforts to produce new myocytes embedded in an extracellular environment that facilitates their differentiation, maintenance, and function.

Longitudinal Reinforcement of Acute Myocardial Infarcts Improves Function by Transmurally Redistributing Stretch and Stress.

A wide range of emerging therapies, from surgical restraint to biomaterial injection to tissue engineering, aim to improve heart function and limit adverse remodeling following myocardial infarction (MI). We previously showed that longitudinal surgical reinforcement of large anterior infarcts in dogs could significantly enhance systolic function without restricting diastolic function, but the underlying mechanisms for this improvement are poorly understood. The goal of this study was to construct a finite element model that could match our previously published data on changes in regional strains and left ventricular function following longitudinal surgical reinforcement, then use the model to explore potential mechanisms for the improvement in systolic function we observed. The model presented here, implemented in febio, matches all the key features of our experiments, including diastolic remodeling strains in the ischemic region, small shifts in the end-diastolic pressure-volume relationship (EDPVR), and large changes in the end-systolic pressure-volume relationship (ESPVR) in response to ischemia and to patch application. Detailed examination of model strains and stresses suggests that longitudinal reinforcement reduces peak diastolic fiber stretch and systolic fiber stress in the remote myocardium and shifts those peaks away from the endocardial surface by reshaping the left ventricle (LV). These findings could help to guide the development of novel therapies to improve post-MI function by providing specific design objectives.

Open-Source Routines for Building Personalized Left Ventricular Models From Cardiac Magnetic Resonance Imaging Data.

Creating patient-specific models of the heart is a promising approach for predicting outcomes in response to congenital malformations, injury, or disease, as well as an important tool for developing and customizing therapies. However, integrating multimodal imaging data to construct patient-specific models is a nontrivial task. Here, we propose an approach that employs a prolate spheroidal coordinate system to interpolate information from multiple imaging datasets and map those data onto a single geometric model of the left ventricle (LV). We demonstrate the mapping of the location and transmural extent of postinfarction scar segmented from late gadolinium enhancement (LGE) magnetic resonance imaging (MRI), as well as mechanical activation calculated from displacement encoding with stimulated echoes (DENSE) MRI. As a supplement to this paper, we provide MATLAB and Python versions of the routines employed here for download from SimTK.

Multiscale computational model of Achilles tendon wound healing: Untangling the effects of repair and loading.

Mechanical stimulation of the healing tendon is thought to regulate scar anisotropy and strength and is relatively easy to modulate through physical therapy. However, in vivo studies of various loading protocols in animal models have produced mixed results. To integrate and better understand the available data, we developed a multiscale model of rat Achilles tendon healing that incorporates the effect of changes in the mechanical environment on fibroblast behavior, collagen deposition, and scar formation. We modified an OpenSim model of the rat right hindlimb to estimate physiologic strains in the lateral/medial gastrocnemius and soleus musculo-tendon units during loading and unloading conditions. We used the tendon strains as inputs to a thermodynamic model of stress fiber dynamics that predicts fibroblast alignment, and to determine local collagen synthesis rates according to a response curve derived from in vitro studies. We then used an agent-based model (ABM) of scar formation to integrate these cell-level responses and predict tissue-level collagen alignment and content. We compared our model predictions to experimental data from ten different studies. We found that a single set of cellular response curves can explain features of observed tendon healing across a wide array of reported experiments in rats-including the paradoxical finding that repairing transected tendon reverses the effect of loading on alignment-without fitting model parameters to any data from those experiments. The key to these successful predictions was simulating the specific loading and surgical protocols to predict tissue-level strains, which then guided cellular behaviors according to response curves based on in vitro experiments. Our model results provide a potential explanation for the highly variable responses to mechanical loading reported in the tendon healing literature and may be useful in guiding the design of future experiments and interventions.

Model First and Ask Questions Later: Confessions of a Reformed Experimentalist.

This paper is an invited perspective written in association with the awarding of the 2018 American Society of Mechanical Engineers Van C. Mow Medal. Inspired by Professor Mow's collaboration with Professor Michael Lai and the role mathematical modeling played in their work on cartilage biomechanics, this article uses our group's work on myocardial infarct healing as an example of the potential value of models in modern experimental biomechanics. Focusing more on the thought process and lessons learned from our studies on infarct mechanics than on the details of the science, this article argues that the complexity of current research questions and the wealth of information already available about almost any cell, tissue, or organ should change how we approach problems and design experiments. In particular, this paper proposes that constructing a mathematical or computational model is now in many cases a critical prerequisite to designing scientifically useful, informative experiments.

The Impact of Hemodynamic Reflex Compensation Following Myocardial Infarction on Subsequent Ventricular Remodeling.

Patients who survive a myocardial infarction (MI) are at high risk for ventricular dilation and heart failure. While infarct size is an important determinant of post-MI remodeling, different patients with the same size infarct often display different levels of left ventricular (LV) dilation. The acute physiologic response to MI involves reflex compensation, whereby increases in heart rate (HR), arterial resistance, venoconstriction, and contractility of the surviving myocardium act to maintain mean arterial pressure (MAP). We hypothesized that variability in reflex compensation might underlie some of the reported variability in post-MI remodeling, a hypothesis that is difficult to test using experimental data alone because some reflex responses are difficult or impossible to measure directly. We, therefore, employed a computational model to estimate the balance of compensatory mechanisms from experimentally reported hemodynamic data. We found a strikingly wide range of compensatory reflex profiles in response to MI in dogs and verified that pharmacologic blockade of sympathetic and parasympathetic reflexes nearly abolished this variability. Then, using a previously published model of postinfarction remodeling, we showed that observed variability in compensation translated to variability in predicted LV dilation consistent with published data. Treatment with a vasodilator shifted the compensatory response away from arterial and venous vasoconstriction and toward increased HR and myocardial contractility. Importantly, this shift reduced predicted dilation, a prediction that matched prior experimental studies. Thus, postinfarction reflex compensation could represent both a source of individual variability in the extent of LV remodeling and a target for therapies aimed at reducing that remodeling.

Surgical reinforcement alters collagen alignment and turnover in healing myocardial infarcts.

Previous studies have suggested that the composition and global mechanical properties of the scar tissue that forms after a myocardial infarction (MI) are key determinants of long-term survival, and emerging therapies such as biomaterial injection are designed in part to alter those mechanical properties. However, recent evidence suggests that local mechanics regulate scar formation post-MI, so that perturbing infarct mechanics could have unexpected consequences. We therefore tested the effect of changes in local mechanical environment on scar collagen turnover, accumulation, and alignment in 77 Sprague-Dawley rats at 1, 2, 3 and 6 wk post-MI by sewing a Dacron patch to the epicardium to eliminate circumferential strain while permitting continued longitudinal stretching with each heart beat. We found that collagen in healing infarcts aligned parallel to regional strain and perpendicular to the preinfarction muscle and collagen fiber direction, strongly supporting our hypothesis that mechanical environment is the primary determinant of scar collagen alignment. Mechanical reinforcement reduced levels of carboxy-terminal propeptide of type I procollagen (PICP; a biomarker for collagen synthesis) in samples collected by microdialysis significantly, particularly in the first 2 wk. Reinforcement also reduced carboxy-terminal telopeptide of type I collagen (ICTP; a biomarker for collagen degradation), particularly at later time points. These alterations in collagen turnover produced no change in collagen area fraction as measured by histology but significantly reduced wall thickness in the reinforced scars compared with untreated controls. Our findings confirm the importance of regional mechanics in regulating scar formation after infarction and highlight the potential for therapies that reduce stretch to also reduce wall thickness in healing infarcts. NEW & NOTEWORTHY This study shows that therapies such as surgical reinforcement, which reduce stretch in healing infarcts, can also reduce collagen synthesis and wall thickness and modify collagen alignment in postinfarction scars.

Guidelines for experimental models of myocardial ischemia and infarction.

Myocardial infarction is a prevalent major cardiovascular event that arises from myocardial ischemia with or without reperfusion, and basic and translational research is needed to better understand its underlying mechanisms and consequences for cardiac structure and function. Ischemia underlies a broad range of clinical scenarios ranging from angina to hibernation to permanent occlusion, and while reperfusion is mandatory for salvage from ischemic injury, reperfusion also inflicts injury on its own. In this consensus statement, we present recommendations for animal models of myocardial ischemia and infarction. With increasing awareness of the need for rigor and reproducibility in designing and performing scientific research to ensure validation of results, the goal of this review is to provide best practice information regarding myocardial ischemia-reperfusion and infarction models. Listen to this article's corresponding podcast at ajpheart.podbean.com/e/guidelines-for-experimental-models-of-myocardial-ischemia-and-infarction/.

Imaging left-ventricular mechanical activation in heart failure patients using cine DENSE MRI: Validation and implications for cardiac resynchronization therapy.

PURPOSE: To image late mechanical activation and identify effective left-ventricular (LV) pacing sites for cardiac resynchronization therapy (CRT). There is variability in defining mechanical activation time, with some studies using the time to peak strain (TPS) and some using the time to the onset of circumferential shortening (TOS). We developed improved methods for imaging mechanical activation and evaluated them in heart failure (HF) patients undergoing CRT. MATERIALS AND METHODS: We applied active contours to cine displacement encoding with stimulated echoes (DENSE) strain images to detect TOS. Six healthy volunteers underwent magnetic resonance imaging (MRI) at 1.5T, and 50 patients underwent pre-CRT MRI (strain, scar, volumes) and echocardiography, assessment of the electrical activation time (Q-LV) at the LV pacing site, and echocardiography assessment of LV reverse remodeling 6 months after CRT. TPS at the LV pacing site was also measured by DENSE. RESULTS: The latest TOS was greater in HF patients vs. healthy subjects (112 ± 28 msec vs. 61 ± 7 msec, P < 0.01). The correlation between TOS and Q-LV was strong (r > 0.75; P < 0.001) and better than between TPS and Q-LV (r < 0.62; P ≥ 0.006). Twenty-three of 50 patients had the latest activating segment in a region other than the mid-ventricular lateral wall, the most common site for the CRT LV lead. Using a multivariable model, TOS/QRS was significantly associated with LV reverse remodeling even after adjustment for overall dyssynchrony and scar (P < 0.05), whereas TPS was not (P = 0.49). CONCLUSION: Late activation by cine DENSE TOS analysis is associated with improved LV reverse remodeling with CRT and deserves further study as a tool to achieve optimal LV lead placement in CRT. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;46:887-896.

Effect of ablation pattern on mechanical function in the atrium.

BACKGROUND: Atrial fibrillation (AF) is often treated with catheter ablation, which induces scar formation to isolate misfiring electrical signals in the left atrium. Successful ablation restores sinus rhythm at the cost of replacing viable myocardium with scar. The impact of ablation scar on mechanical function of the left atrium is poorly understood. OBJECTIVE: We used a computational model to simulate various ablation patterns and determine their effect on atrial global and regional mechanical function. METHODS: A coupled finite-element and hemodynamic circuit model of the left atrium that represents the regional and global mechanics in paroxysmal AF patients was modified to simulate different ablation patterns: step-wise pulmonary vein isolation (PVI), wide area circumferential ablation (WACA), and a posterior ablation developed by nContact, Inc (Morrisville, NC, USA). Atrial pressure-volume relationships and regional wall motion were compared among the models. RESULTS: Ablation increased passive stiffness and decreased active work performed by the atrium. Active emptying volume decreased with increasing scar by up to 44% (11 mL) at a scar volume of 31%. At matched scar volumes, WACA decreased active emptying more severely than PVI and nContact. Similarly, wall motion was depressed most in the WACA model because WACA involved portions of the lateral wall with higher baseline motion. CONCLUSION: Simulated ablation depressed atrial mechanical function to an extent that depended on both scar volume and location, primarily through reducing active emptying. Placing ablation scar in regions with high baseline motion resulted in greater depression of active function, while ablation of the posterior wall was less disruptive.

Emergence of Collagen Orientation Heterogeneity in Healing Infarcts and an Agent-Based Model.

Spatial heterogeneity of matrix structure can be an important determinant of tissue function. Although bulk properties of collagen structure in healing myocardial infarcts have been characterized previously, regional heterogeneity in infarct structure has received minimal attention. Herein, we quantified regional variations of collagen and nuclear orientations over the initial weeks of healing after infarction in rats, and employed a computational model of infarct remodeling to test potential explanations for the heterogeneity we observed in vivo. Fiber and cell orientation maps were generated from infarct samples acquired previously at 1, 2, 3, and 6 weeks postinfarction in a rat ligation model. We analyzed heterogeneity by calculating the dot product of each fiber or cell orientation vector with every other fiber or cell orientation vector, and plotting that dot product versus distance between the fibers or cells. This analysis revealed prominent regional heterogeneity, with alignment of both fibers and cell nuclei in local pockets far exceeding the global average. Using an agent-based model of fibroblast-mediated collagen remodeling, we found that similar levels of heterogeneity can spontaneously emerge from initially isotropic matrix via locally reinforcing cell-matrix interactions. Specifically, cells that sensed fiber orientation at a distance or remodeled fibers at a distance by traction-mediated reorientation or aligned deposition gave rise to regionally heterogeneous structures. However, only the simulations in which cells deposited collagen fibers aligned with their own orientation reproduced experimentally measured patterns of heterogeneity across all time points. These predictions warrant experimental follow-up to test the role of such mechanisms in vivo and identify opportunities to control heterogeneity for therapeutic benefit.

Modifying the mechanics of healing infarcts: Is better the enemy of good?

Myocardial infarction (MI) is a major source of morbidity and mortality worldwide, with over 7 million people suffering infarctions each year. Heart muscle damaged during MI is replaced by a collagenous scar over a period of several weeks, and the mechanical properties of that scar tissue are a key determinant of serious post-MI complications such as infarct rupture, depression of heart function, and progression to heart failure. Thus, there is increasing interest in developing therapies that modify the structure and mechanics of healing infarct scar. Yet most prior attempts at therapeutic scar modification have failed, some catastrophically. This article reviews available information about the mechanics of healing infarct scar and the functional impact of scar mechanical properties, and attempts to infer principles that can better guide future attempts to modify scar. One important conclusion is that collagen structure, mechanics, and remodeling of healing infarct scar vary so widely among experimental models that any novel therapy should be tested across a range of species, infarct locations, and reperfusion protocols. Another lesson from past work is that the biology and mechanics of healing infarcts are sufficiently complex that the effects of interventions are often counterintuitive; for example, increasing infarct stiffness has little effect on heart function, and inhibition of matrix metalloproteases (MMPs) has little effect on scar collagen content. Computational models can help explain such counterintuitive results, and are becoming an increasingly important tool for integrating known information to better identify promising therapies and design experiments to test them. Moving forward, potentially exciting new opportunities for therapeutic modification of infarct mechanics include modulating anisotropy and promoting scar compaction.

Computational modeling of cardiac fibroblasts and fibrosis.

Altered fibroblast behavior can lead to pathologic changes in the heart such as arrhythmia, diastolic dysfunction, and systolic dysfunction. Computational models are increasingly used as a tool to identify potential mechanisms driving a phenotype or potential therapeutic targets against an unwanted phenotype. Here we review how computational models incorporating cardiac fibroblasts have clarified the role for these cells in electrical conduction and tissue remodeling in the heart. Models of fibroblast signaling networks have primarily focused on fibroblast cell lines or fibroblasts from other tissues rather than cardiac fibroblasts, specifically, but they are useful for understanding how fundamental signaling pathways control fibroblast phenotype. In the future, modeling cardiac fibroblast signaling, incorporating -omics and drug-interaction data into signaling network models, and utilizing multi-scale models will improve the ability of in silico studies to predict potential therapeutic targets against adverse cardiac fibroblast activity.

Comparison of quantitative wall-motion analysis and strain for detection of coronary stenosis with three-dimensional dobutamine stress echocardiography.

BACKGROUND: Quantitative analysis of wall motion from three-dimensional (3D) dobutamine stress echocardiography (DSE) could provide additional diagnostic information not available from qualitative analysis. In this study, we compare the effectiveness of 3D fractional shortening (3DFS), a measure of wall motion computed from 3D echocardiography (3DE), to strain and strain rate measured with sonomicrometry for detecting critical stenoses during DSE. METHODS: Eleven open-chest dogs underwent DSE both with and without a critical stenosis. 3DFS was measured from 3DE images acquired at peak stress. 3DFS was normalized by subtracting average 3DFS during control peak stress (∆3DFS). Strains in the perfusion defect (PD) were measured from sonomicrometry, and PD size and location were measured with microspheres. RESULTS: A ∆3DFS abnormality indicated the presence of a critical stenosis with high sensitivity and specificity (88% and 100%, respectively), and ∆3DFS abnormality size correlated with PD size (R(2) = 0.54). The sensitivity and specificity for ∆3DFS were similar to that for area strain (88%, 100%) and circumferential strain and strain rate (88%, 92% and 88%, 86%, respectively), while longitudinal strain and strain rate were less specific. ∆3DFS correlated significantly with both coronary flow reserve (R(2) = 0.71) and PD size (R(2) = 0.97), while area strain correlated with PD size only (R(2) = 0.67), and other measures were not significantly correlated with flow reserve or PD size. CONCLUSION: Quantitative wall-motion analysis using ∆3DFS is effective for detecting critical stenoses during DSE, performing similar to 3D strain, and provides potentially useful information on the size and location of a perfusion defect.

Mechanical boundary conditions bias fibroblast invasion in a collagen-fibrin wound model.

Because fibroblasts deposit the collagen matrix that determines the mechanical integrity of scar tissue, altering fibroblast invasion could alter wound healing outcomes. Anisotropic mechanical boundary conditions (restraint, stretch, or tension) could affect the rate of fibroblast invasion, but their importance relative to the prototypical drivers of fibroblast infiltration during wound healing--cell and chemokine concentration gradients--is unknown. We tested whether anisotropic mechanical boundary conditions affected the directionality and speed of fibroblasts migrating into a three-dimensional model wound, which could simultaneously expose fibroblasts to mechanical, structural, steric, and chemical guidance cues. We created fibrin-filled slits in fibroblast-populated collagen gels and applied uniaxial mechanical restraint along the short or long axis of the fibrin wounds. Anisotropic mechanical conditions increased the efficiency of fibroblast invasion by guiding fibroblasts without increasing their migration speed. The migration behavior could be modeled as a biased random walk, where the bias due to multiple guidance cues was accounted for in the shape of a displacement orientation probability distribution. Taken together, modeling and experiments suggested an effect of strain anisotropy, rather than strain-induced fiber alignment, on fibroblast invasion.