Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells.

BACKGROUND & AIMS: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). METHODS: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. RESULTS: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. CONCLUSIONS: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Self-consistent field theory of chiral nematic worm-like chains.

Many macromolecules of biological and technological interest are both chiral and semi-flexible. DNA and collagen are good examples. Such molecules often form chiral nematic (or cholesteric) phases, as is well-documented in collagen and chitin. This work presents a method for studying cholesteric phases in the highly successful self-consistent field theory of worm-like chains, offering a new way of studying many biologically relevant molecules. The method involves an effective Hamiltonian with a chiral term inspired by the Oseen-Frank (OF) model of liquid crystals. This method is then used to examine the formation of cholesteric phases in chiral-nematic worm-like chains as a function of polymer flexibility, as well as the optimal cholesteric pitch and distribution of polymer segment orientations. Our approach not only allows for the determination of the isotropic-cholesteric transition and segment distributions, beyond what the OF model promises, but also explicitly incorporates polymer flexibility into the study of the cholesteric phase, offering a more complete understanding of the behavior of semiflexible chiral-nematic polymers.

A theoretical model of collective cell polarization and alignment.

Collective cell polarization and alignment play important roles in tissue morphogenesis, wound healing and cancer metastasis. How cells sense the direction and position in these processes, however, has not been fully understood. Here we construct a theoretical model based on describing cell layer as a nemato-elastic medium, by which the cell polarization, cell alignment and cell active contraction are explicitly expressed as functions of components of the nematic order parameter. To determine the order parameter we derive two sets of governing equations, one for the force equilibrium of the system, and the other for the minimization of the system's free energy including the energy of cell polarization and alignment. By solving these coupled governing equations, we can predict the effects of substrate stiffness, geometries of cell layers, external forces and myosin activity on the direction- and position-dependent cell aspect ratio and cell orientation. Moreover, the axisymmetric problem with cells on a ring-like pattern is solved analytically, and the analytical solution for cell aspect ratio are governed by parameter groups which include the stiffness of the cell and the substrate, the strength of myosin activity and the external forces. Our predictions of the cell aspect ratio and orientation are generally comparable to experimental observations. These results show that the pattern of cell polarization is determined by the anisotropic degree of active contractile stress, and suggest a stress-driven polarization mechanism that enables cells to sense their spatial positions to develop direction- and position-dependent behavior. This, in turn, sheds light on the ways to control pattern formation in tissue engineering for potential biomedical applications.

Nematic ordering of worm-like polymers near an interface.

The phase behavior of semi-flexible polymers is integral to various contexts, from materials science to biophysics, many of which utilize or require specific confinement geometries as well as the orientational behavior of the polymers. Inspired by collagen assembly, we study the orientational ordering of semi-flexible polymers, modeled as Maier-Saupe worm-like chains, using self-consistent field theory. We first examine the bulk behavior of these polymers, locating the isotropic-nematic transition and delineating the limit of stability of the isotropic and nematic phases. This effort explains how nematic ordering emerges from the isotropic phase and offers insight into how different (e.g., mono-domain vs multi-domain) nematic phases form. We then clarify the influence of planar confinement on the nematic ordering of the polymers. We find that while the presence of a single confining wall does not shift the location of nematic transition, it aligns the polymers in parallel to the wall; above the onset of nematic transition, this preference tends to propagate into the bulk phase. Introducing a second, perpendicular, wall leads to a nematic phase that is parallel to both walls, allowing the ordering direction to be uniquely set by the geometry of the experimental setup. The advantage of wall-confinement is that it can be used to propagate mono-domain nematic phases into the bulk phase.

Nonalcoholic fatty liver disease and gastric bypass surgery regulate serum and hepatic levels of pyruvate kinase isoenzyme M2.

Treatment of nonalcoholic fatty liver disease (NAFLD) focuses on the underlying metabolic syndrome, and Roux-en-Y gastric bypass surgery (RYGB) remains one of the most effective options. In rodents and human patients, RYGB induces an increase in the gene and protein expression levels of the M2 isoenzyme of pyruvate kinase (PKM2) in the jejunum. Since PKM2 can be secreted in the circulation, our hypothesis was that the circulating levels of PKM2 increase after RYGB. Our data, however, revealed an unexpected finding and a potential new role of PKM2 for the natural history of metabolic syndrome and NAFLD. Contrary to our initial hypothesis, RYGB-treated patients had decreased PKM2 blood levels compared with a well-matched group of patients with severe obesity before RYGB. Interestingly, PKM2 serum concentration correlated with body mass index before but not after the surgery. This prompted us to evaluate other potential mechanisms and sites of PKM2 regulation by the metabolic syndrome and RYGB. We found that in patients with NAFLD and nonalcoholic steatohepatitis (NASH), the liver had increased PKM2 expression levels, and the enzyme appears to be specifically localized in Kupffer cells. The study of murine models of metabolic syndrome and NASH replicated this pattern of expression, further suggesting a metabolic link between hepatic PKM2 and NAFLD. Therefore, we conclude that PKM2 serum and hepatic levels increase in both metabolic syndrome and NAFLD and decrease after RYGB. Thus, PKM2 may represent a new target for monitoring and treatment of NAFLD.

Regulation of Energy Homeostasis After Gastric Bypass Surgery.

The obesity epidemic continues to escalate each year in the United States more than anywhere else in the world. The existing pharmaceutical and other nonsurgical treatments for morbid obesity produce suboptimal physiologic outcomes compared with those of Roux-en-Y gastric bypass (RYGB) surgery. RYGB has been the gold standard of bariatric surgery because the beneficial long-term outcomes, which include sustainable weight loss and type 2 diabetes mellitus (T2DM) resolution, are far superior to those obtained with other bariatric surgeries. However, the current understanding of RYGB's mechanisms of actions remains limited and incomplete. There is an urgent need to understand these mechanisms as gaining this knowledge may lead to the development of innovative and less invasive procedures and/or medical devices, which can mirror the favorable outcomes of RYGB surgery. In this review, we highlight current observations of the metabolic and physiologic events following RYGB, with a particular focus on the role of the anatomical reconfiguration of the gastrointestinal tract after RYGB.

Physical properties of food or bile redirection do not contribute to the intestinal adaptations after Roux-en-Y Gastric Bypass in rats.

Objective: Metabolic and morphological adaptations of the intestine have been suggested to play a role in the various therapeutic benefits of Roux-en-Y Gastric Bypass (RYGB) surgery. However, the precise underlying mechanisms remain unclear. In this study, the effects of physical properties of ingested food and redirection of biliopancreatic secretions on intestinal remodeling were investigated in RYGB operated rats. Methods: RYGB employing two different Roux Limb (RL) lengths was performed on high fat diet induced obese rats. Post-operatively, rats were fed either Solid or isocaloric Liquid diets. Metabolic and morphological remodeling of intestine was compared across both diet forms (Solid and Liquid diets) and surgical models (Short RL and Long RL). Results: RYGB surgery in rats induced weight loss and improved glucose tolerance which was independent of physical properties of ingested food and biliopancreatic secretions. Intestinal glucose utilization after RYGB was not determined by either food form or biliopancreatic secretions. The GLUT-1 expression in RL was not influenced by physical properties of food. Furthermore, both physical properties of food and biliopancreatic secretions showed no effects on intestinal morphological adaptations after RYGB. Conclusion: Results of this study demonstrate that physical properties of food and bile redirection are not major determinants of intestinal remodeling after RYGB in rats.

PhaseFIT: live-organoid phase-fluorescent image transformation via generative AI.

Organoid models have provided a powerful platform for mechanistic investigations into fundamental biological processes involved in the development and function of organs. Despite the potential for image-based phenotypic quantification of organoids, their complex 3D structure, and the time-consuming and labor-intensive nature of immunofluorescent staining present significant challenges. In this work, we developed a virtual painting system, PhaseFIT (phase-fluorescent image transformation) utilizing customized and morphologically rich 2.5D intestinal organoids, which generate virtual fluorescent images for phenotypic quantification via accessible and low-cost organoid phase images. This system is driven by a novel segmentation-informed deep generative model that specializes in segmenting overlap and proximity between objects. The model enables an annotation-free digital transformation from phase-contrast to multi-channel fluorescent images. The virtual painting results of nuclei, secretory cell markers, and stem cells demonstrate that PhaseFIT outperforms the existing deep learning-based stain transformation models by generating fine-grained visual content. We further validated the efficiency and accuracy of PhaseFIT to quantify the impacts of three compounds on crypt formation, cell population, and cell stemness. PhaseFIT is the first deep learning-enabled virtual painting system focused on live organoids, enabling large-scale, informative, and efficient organoid phenotypic quantification. PhaseFIT would enable the use of organoids in high-throughput drug screening applications.

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro.

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold-based and scaffold-free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS--five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long-term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick-freeze/deep-etch electron microscopy. Both the scaffold-free and the collagen-based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell-derived matrix in the scaffold-based constructs was significantly lower than that produced by scaffold-free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell-derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response.

Interplay between nematic and cholesteric interactions in self-consistent field theory.

Chirality is a design feature of a number of biomolecules (e.g., collagen). In these molecules, cholesteric (chiral-nematic) behavior emerges from a combination of the tendency for the biopolymers to align (nematic interactions) and for the alignment direction to change with position, rotating around an axis normal to the alignment direction. This paper presents self-consistent field theory (SCFT) of chiral-nematic polymers, which takes into account polymer flexibility and the orientational degrees of freedom of polymer segments. Using the resulting SCFT, we construct a phase diagram showing regions of stability for isotropic, nematic, and cholesteric phases. Furthermore, we find that nematic interactions can stabilize the cholesteric phase, pushing the isotropic-cholesteric phase transition to lower cholesteric interaction strength, until the isotropic-nematic-cholesteric triple point is reached.

Rewiring of the Liver Transcriptome across Multiple Time-Scales Is Associated with the Weight Loss-Independent Resolution of NAFLD Following RYGB.

Roux-en-Y gastric bypass (RYGB) surgery potently improves obesity and a myriad of obesity-associated co-morbidities including type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Time-series omics data are increasingly being utilized to provide insight into the mechanistic underpinnings that correspond to metabolic adaptations in RYGB. However, the conventional computational biology methods used to interpret these temporal multi-dimensional datasets have been generally limited to pathway enrichment analysis (PEA) of isolated pair-wise comparisons based on either experimental condition or time point, neither of which adequately capture responses to perturbations that span multiple time scales. To address this, we have developed a novel graph network-based analysis workflow designed to identify modules enriched with biomolecules that share common dynamic profiles, where the network is constructed from all known biological interactions available through the Kyoto Encyclopedia of Genes and Genomes (KEGG) resource. This methodology was applied to time-series RNAseq transcriptomics data collected on rodent liver samples following RYGB, and those of sham-operated and weight-matched control groups, to elucidate the molecular pathways involved in the improvement of as NAFLD. We report several network modules exhibiting a statistically significant enrichment of genes whose expression trends capture acute-phase as well as long term physiological responses to RYGB in a single analysis. Of note, we found the HIF1 and P53 signaling cascades to be associated with the immediate and the long-term response to RYGB, respectively. The discovery of less intuitive network modules that may have gone overlooked with conventional PEA techniques provides a framework for identifying novel drug targets for NAFLD and other metabolic syndrome co-morbidities.

Mechanoscopy: A Novel Device and Procedure for in vivo Detection of Chronic Colitis in Mice.

BACKGROUND: Gut stiffening caused by fibrosis plays a critical role in the progression of inflammatory bowel disease (IBD) and colon cancer. Previous studies have characterized the biomechanical response of healthy and pathological gut, with most measurements obtained ex vivo. METHODS: Here, we developed a device and accompanying procedure for in vivo quantification of gut stiffness, termed mechanoscopy. Mechanoscopy includes a flexible balloon catheter, pressure sensor, syringe pump, and control system. The control system activates the balloon catheter and performs automated measurements of the gut stress-strain biomechanical response. RESULTS: A gut stiffness index (GSI) is identified based on the slope of the obtained stress-strain response. Using a colitis mouse model, we demonstrated that GSI positively correlates with the extent of gut fibrosis, the severity of mucosal damage, and the infiltration of immune cells. Furthermore, a critical strain value is suggested, and GSI efficiently detects pathological gut fibrotic stiffening when the strain exceeds this value. CONCLUSIONS: Based on these results, we envision that mechanoscopy and GSI will facilitate the clinical diagnosis of IBD.

Here, we present a novel procedure/device, termed mechanoscopy, which we have demonstrated to accurately detect and differentiate between fibrosis and inflammation in rodent models of colitis. Thus, mechanoscopy offers a translationally relevant approach for ultrasensitive and minimally invasive IBD diagnosis.

Molecular mechanochemistry: low force switch slows enzymatic cleavage of human type I collagen monomer.

In vertebrate animals, fibrillar collagen accumulates, organizes, and persists in structures which resist mechanical force. This antidissipative behavior is possibly due to a mechanochemical force-switch which converts collagen from enzyme-susceptible to enzyme-resistant. Degradation experiments on native tissue and reconstituted fibrils suggest that collagen/enzyme kinetics favor the retention of loaded collagen. We used a massively parallel, single molecule, mechanochemical reaction assay to demonstrate that the effect is derivative of molecular mechanics. Tensile loads higher than 3 pN dramatically reduced (10×) the enzymatic degradation rate of recombinant human type I collagen monomers by Clostridium histolyticum compared to unloaded controls. Because bacterial collagenase accesses collagen at multiple sites and is an aggressive cleaver of the collagen triple helical domain, the results suggest that collagen molecular architecture is generally more stable when mechanically strained in tension. Thus the tensile mechanical state of collagen monomers is likely to be correlated to their longevity in tissues. Further, strain-actuated molecular stability of collagen may constitute the fundamental basis of a smart structural mechanism which enhances the ability of animals to place, retain, and load-optimize material in the path of mechanical forces.

In situ metabolic flux analysis to quantify the liver metabolic response to experimental burn injury.

Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post-burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady-state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham-burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic-like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose-dependent metabolic response, but rather leads to qualitatively different responses.

Probing collagen/enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep.

Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal and enzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance, and disease in vertebrate animals. However, extracting a quantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissue due to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay and diffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and the cutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing of enzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initial strain. The results, obtained on uniaxially loaded strips of bovine corneal tissue (0.1, 0.25, or 0.5 N), demonstrate that small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that a change in tissue elongation of only 1.5% (at approximately 5% strain) reduces the maximum cutting rate of the enzyme by more than half. Estimation of the average load per monomer in the tissue strips indicates that this protective "cutoff" occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime. The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep response indicates that the decrease in the cleavage rate of the BC is nonlinear (initially steep between 4.5 and 6.5% and then flattens out from 6.5 to 9.5%). The high sensitivity to strain at low strain implies that even lightly loaded collagenous tissue may exhibit significant strain protection. The dynamic, enzyme-induced creep assay described herein has the potential to permit the rapid characterization of collagen/enzyme mechanochemistry in many different tissue types.

Rejuvenation of aged rat skin with pulsed electric fields.

The demand for skin rejuvenation procedures has progressively increased in the past decade. Additionally, clinical trials have shown that current therapies might cause downtime and side effects in patients including prolonged erythema, scarring, and dyspigmentation. The goal of this study was to explore the effect of partial irreversible electroporation (pIRE) with pulsed electric fields in aged skin rejuvenation as a novel, non-invasive skin resurfacing technique. In this study, we used an experimental model of aged rats. We showed that treatment with pIRE promoted keratinocyte proliferation and blood flow in aged rat skin. We also found significant evidence indicating that pIRE reformed the dermal extracellular matrix (ECM). Both the collagen protein and fibre density in aged skin increased after pIRE administration. Furthermore, using an image-processing algorithm, we found that the collagen fibre orientation in the histological sections did not change, indicating a lack of scar formation in the treated areas. The results showed that pIRE approach could effectively stimulate keratinocyte proliferation, ECM synthesis, and angiogenesis in an aged rat model.

Metabolomic Modularity Analysis (MMA) to Quantify Human Liver Perfusion Dynamics.

Large-scale -omics data are now ubiquitously utilized to capture and interpret global responses to perturbations in biological systems, such as the impact of disease states on cells, tissues, and whole organs. Metabolomics data, in particular, are difficult to interpret for providing physiological insight because predefined biochemical pathways used for analysis are inherently biased and fail to capture more complex network interactions that span multiple canonical pathways. In this study, we introduce a nov-el approach coined Metabolomic Modularity Analysis (MMA) as a graph-based algorithm to systematically identify metabolic modules of reactions enriched with metabolites flagged to be statistically significant. A defining feature of the algorithm is its ability to determine modularity that highlights interactions between reactions mediated by the production and consumption of cofactors and other hub metabolites. As a case study, we evaluated the metabolic dynamics of discarded human livers using time-course metabolomics data and MMA to identify modules that explain the observed physiological changes leading to liver recovery during subnormothermic machine perfusion (SNMP). MMA was performed on a large scale liver-specific human metabolic network that was weighted based on metabolomics data and identified cofactor-mediated modules that would not have been discovered by traditional metabolic pathway analyses.

Multi-omic network-based interrogation of rat liver metabolism following gastric bypass surgery featuring SWATH proteomics.

Morbidly obese patients often elect for Roux-en-Y gastric bypass (RYGB), a form of bariatric surgery that triggers a remarkable 30% reduction in excess body weight and reversal of insulin resistance for those who are type II diabetic. A more complete understanding of the underlying molecular mechanisms that drive the complex metabolic reprogramming post-RYGB could lead to innovative non-invasive therapeutics that mimic the beneficial effects of the surgery, namely weight loss, achievement of glycemic control, or reversal of non-alcoholic steatohepatitis (NASH). To facilitate these discoveries, we hereby demonstrate the first multi-omic interrogation of a rodent RYGB model to reveal tissue-specific pathway modules implicated in the control of body weight regulation and energy homeostasis. In this study, we focus on and evaluate liver metabolism three months following RYGB in rats using both SWATH proteomics, a burgeoning label free approach using high resolution mass spectrometry to quantify protein levels in biological samples, as well as MRM metabolomics. The SWATH analysis enabled the quantification of 1378 proteins in liver tissue extracts, of which we report the significant down-regulation of Thrsp and Acot13 in RYGB as putative targets of lipid metabolism for weight loss. Furthermore, we develop a computational graph-based metabolic network module detection algorithm for the discovery of non-canonical pathways, or sub-networks, enriched with significantly elevated or depleted metabolites and proteins in RYGB-treated rat livers. The analysis revealed a network connection between the depleted protein Baat and the depleted metabolite taurine, corroborating the clinical observation that taurine-conjugated bile acid levels are perturbed post-RYGB.

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates.

In a fibroblast colony model of corneal stromal development, we asked how physiological tension influences the patterning dynamics of fibroblasts and the orientation of deposited extracellular matrix (ECM). Using long-term live-cell microscopy, enabled by an optically accessible mechanobioreactor, a primary human corneal fibroblast colony was cultured on three types of substrates: a mechanically biased, loaded, dense, disorganized collagen substrate (LDDCS), a glass coverslip, and an unloaded, dense, disorganized collagen substrate (UDDCS). On LDDCS, fibroblast orientation and migration along a preferred angle developed early, cell orientation was correlated over long distances, and the colony pattern was stable. On glass, fibroblast orientation was poorly correlated, developed more slowly, and colony patterns were metastable. On UDDCS, cell orientation was correlated over shorter distances compared with LDDCS specimens. On all substrates, the ECM pattern reflected the cell pattern. In summary, mechanically biasing the collagen substrate altered the early migration behavior of individual cells, leading to stable emergent cell patterning, which set the template for newly synthesized ECM.