The micromechanics of three-dimensional collagen-I gels

We study the micromechanics of collagen-I gel with the goal of bridging the gap between theory and experiment in the study of biopolymer networks. Three-dimensional images of fluorescently labeled collagen are obtained by confocal microscopy, and the network geometry is extracted using a 3D network skeletonization algorithm. Each fiber is modeled as an elastic beam that resists stretching and bending, and each crosslink is modeled as torsional spring. The stress–strain curves of networks at three different densities are compared with rheology measurements. The model shows good agreement with experiment, confirming that strain stiffening of collagen can be explained entirely by geometric realignment of the network, as opposed to entropic stiffening of individual fibers. The model also suggests that at small strains, crosslink deformation is the main contributer to network stiffness, whereas at large strains, fiber stretching dominates. As this modeling effort uses networks with realistic geometries, this analysis can ultimately serve as a tool for understanding how the mechanics of fibers and crosslinks at the microscopic level produce the macroscopic properties of the network. © 2010 Wiley Periodicals, Inc. Complexity 16: 22-28, 2011Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83463/1/20332_ftp.pd

Remodeling of Fibrous Extracellular Matrices by Contractile Cells: Predictions from Discrete Fiber Network Simulations

Contractile forces exerted on the surrounding extracellular matrix (ECM) lead to the alignment and stretching of constituent fibers within the vicinity of cells. As a consequence, the matrix reorganizes to form thick bundles of aligned fibers that enable force transmission over distances larger than the size of the cells. Contractile force-mediated remodeling of ECM fibers has bearing on a number of physiologic and pathophysiologic phenomena. In this work, we present a computational model to capture cell-mediated remodeling within fibrous matrices using finite element based discrete fiber network simulations. The model is shown to accurately capture collagen alignment, heterogeneous deformations, and long-range force transmission observed experimentally. The zone of mechanical influence surrounding a single contractile cell and the interaction between two cells are predicted from the strain-induced alignment of fibers. Through parametric studies, the effect of cell contractility and cell shape anisotropy on matrix remodeling and force transmission are quantified and summarized in a phase diagram. For highly contractile and elongated cells, we find a sensing distance that is ten times the cell size, in agreement with experimental observations.Comment: Accepted for publication in the Biophysical Journa

Cell contraction induces long-ranged stress stiffening in the extracellular matrix

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing Nonlinear Stress Inference Microscopy (NSIM), a novel technique to infer stress fields in a 3D matrix from nonlinear microrheology measurement with optical tweezers. Using NSIM and simulations, we reveal a long-ranged propagation of cell-generated stresses resulting from local filament buckling. This slow decay of stress gives rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which could form a mechanism for mechanical communication between cells

Concentration Independent Modulation of Local Micromechanics in a Fibrin Gel

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues

Computational biomechanics of acute myocardial infarction and its treatment

The intramyocardial injection of biomaterials is an emerging therapy for myocardial infarction. Computational methods can help to study the mechanical effect s of biomaterial injectates on the infarcted heart s and can contribute to advance and optimise the concept of this therapy. The distribution of polyethylene glycol hydrogel injectate delivered immediately after the infarct induction was studied using rat infarct model. A micro-structural three-dimensional geometrical model of the entire injectate was reconstructed from histological micro graphs. The model provides a realistic representation of biomaterial injectates in computational models at macroscopic and microscopic level. Biaxial and compression mechanical testing was conducted for healing rat myocardial infarcted tissue at immediate (0 day), 7, 14 and 28 days after infarction onset. Infarcts were found to be mechanically anisotropic with the tissue being stiffer in circumferential direction than in longitudinal direction. The 0, 7, 14 and 28 days infarcts showed 443, 670, 857 and 1218 kPa circumferential tensile moduli. The 28 day infarct group showed a significantly higher compressive modulus compared to the other infarct groups (p= 0.0055, 0.028, and 0.018 for 0, 7 and 14 days groups). The biaxial mechanical data were utilized to establish material constitutive models of rat healing infarcts. Finite element model s and genetic algorithms were employed to identify the parameters of Fung orthotropic hyperelastic strain energy function for the healing infarcts. The provided infarct mechanical data and the identified constitutive parameters offer a platform for investigations of mechanical aspects of myocardial infarction and therapies in the rat, an experimental model extensively used in the development of infarct therapies. Micro-structurally detailed finite element model of a hydrogel injectate in an infarct was developed to provide an insight into the micromechanics of a hydrogel injectate and infarct during the diastolic filling. The injectate caused the end-diastolic fibre stresses in the infarct zone to decrease from 22.1 to 7.7 kPa in the 7 day infarct and from 35.7 to 9.7 kPa in the 28 day infarct. This stress reduction effect declined as the stiffness of the biomaterial increased. It is suggested that the gel works as a force attenuating system through micromechanical mechanisms reducing the force acting on tissue layers during the passive diastolic dilation of the left ventricle and thus reducing the stress induced in these tissue layers

Biomaterial-Mediated Reprogramming of the Wound Interface to Enhance Meniscal Repair

Endogenous repair of fibrous connective tissues is limited, and there exist few successful strategies to improve healing after injury. As such, new methods that advance repair by enhancing cell migration to the wound interface, extracellular matrix (ECM) production, and tissue integration would represent a marked clinical advance. Using the adult meniscus as a test platform, we hypothesized that ECM density and stiffness increase throughout tissue maturation, and that these age-related changes present biophysical barriers to interstitial cell migration during wound healing. We further posited that modulating the matrix could remove these impediments, enabling endogenous cells to reach the injury site. To test our hypotheses, we compared the microenvironment of fetal and adult meniscal ECM via atomic force microscopy (AFM) indentation and second harmonic generation (SHG) imaging of the collagenous matrix. We also explored interstitial cell mobility through fetal and adult native tissue environments using a three-dimensional ex vivo system. We further investigated strategies that might expedite cell migration, including enzymatic degradation of the ECM with collagenase to reduce matrix stiffness and increase porosity. To restrict these biological manipulations to the wound interface, we fabricated a delivery system in which selected biofactors were stored inside composite electrospun nanofibrous scaffolds and released upon hydration. The ability for bioactive scaffolds to enhance the cellularity and integration of meniscal injuries was evaluated in vivo using tissue explants in a subcutaneous implantation model, as well as an orthotopic meniscal injury model. Our findings suggest that matrix stiffness, density, and organization increase with meniscal development at the expense of cell mobility. Our results also indicate that partial digestion of the wound interface with collagenase improves repair by creating a more compliant and porous microenvironment that facilitates cell migration. Furthermore, when scaffolds containing collagenase-releasing fibers were placed inside meniscal defects, enzymatic digestion was localized and resulted in improved cellular colonization and closure of the wound site, similar to treatment with aqueous collagenase. This innovative approach of targeted delivery may aid the many patients that exhibit meniscal tears by promoting integrative repair, thereby circumventing the pathologic consequences of partial meniscus removal, and may find widespread application in the treatment of injuries to a variety of dense connective tissues

Collagen-hyaluronic acid scaffolds for adipose tissue engineering.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.This was supported by the Biotechnology and Biological Sciences Research Council