Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Tel-Aviv University

In vivo, eukaryotic cells are embedded in a matrix environment, where they grow and develop. Generally, this extracellular matrix (ECM) is an anisotropic fibrous structure, through which macromolecules and biochemical signaling molecules at the nanometer scale diffuse. The ECM is continuously remodeled by cells, via mechanical interactions, which lead to a potential link between biomechanical and biochemical cell-cell interactions. Here, it is studied how cell-induced forces applied on the ECM impact the biochemical transport of molecules between distant cells. It is experimentally observed that cells remodel the ECM by increasing fiber alignment and density of the matrix between them over time. Using random walk simulations on a 3D lattice, elongated fixed obstacles are implemented that mimic the fibrous ECM structure. Both diffusion of a tracer molecule and the mean first-passage time a molecule secreted from one cell takes to reach another cell are measured. The model predicts that cell-induced remodeling can lead to a dramatic speedup in the transport of molecules between cells. Fiber alignment and densification cause reduction of the transport dimensionality from a 3D to a much more rapid 1D process. Thus, a novel mechanism of mechano-biochemical feedback in the regulation of long-range cell-cell communication is suggested.

Mechanical Interaction between Cells Facilitates Molecular Transport.

It is well recognized that isolated cardiac muscle cells beat in a periodic manner. Recently, evidence indicates that other, non-muscle cells, also perform periodic motions that are either imperceptible under conventional lab microscope lens or practically not easily amenable for analysis of oscillation amplitude, frequency, phase of movement and its direction. Here, we create a real-time video analysis tool to visually magnify and explore sub-micron rhythmic movements performed by biological cells and the induced movements in their surroundings. Using this tool, we suggest that fibroblast cells perform small fluctuating movements with a dominant frequency that is dependent on their surrounding substrate and its stiffness.

Motion magnification analysis of microscopy videos of biological cells.

Generation of vessel networks within engineered tissues is critical for integration and perfusion of the implanted tissue in vivo. The effect of mechanical cues in guiding and stabilizing the vessels has begun to attract marked interest. This review surveys the impact of mechanical cues on formation of vascular networks in 2D and 3D gel matrices. We give less emphasis to regulation of endothelial monolayers and single endothelial cells. Several vascularization models have consistently found that the stress generated in the gel, and encountered by embedded cells, control various aspects of vascular network formation, including sprouting, branching, alignment, and vessel maturation. This internal stress is generated by cell contractile forces, and is balanced by gel stiffness and boundary constrains imposed on the gel. Actin and myosin II are key molecular players in controlling initiation of vessel sprouting and branching morphogenesis. Additionally, the impact of external mechanical cues on tissue vascularization, and studies supporting the notion that mechanical forces regulate vascularization in the live animal are reviewed.

Mechanical regulation of vascular network formation in engineered matrices.

Biological cells embedded in fibrous matrices have been observed to form intercellular bands of dense and aligned fibers through which they mechanically interact over long distances. Such matrix-mediated cellular interactions have been shown to regulate various biological processes. This study aimed to explore the effects of elastic nonlinearity of the fibers contained in the extracellular matrix (ECM) on the transmission of mechanical loads between contracting cells. Based on our biological experiments, we developed a finite-element model of two contracting cells embedded within a fibrous network. The individual fibers were modeled as showing linear elasticity, compression microbuckling, tension stiffening, or both of the latter two. Fiber compression buckling resulted in smaller loads in the ECM, which were primarily directed toward the neighboring cell. These loads decreased with increasing cell-to-cell distance; when cells were >9 cell diameters apart, no such intercellular interaction was observed. Tension stiffening further contributed to directing the loads toward the neighboring cell, though to a smaller extent. The contraction of two neighboring cells resulted in mutual attraction forces, which were considerably increased by tension stiffening and decayed with increasing cell-to-cell distances. Nonlinear elasticity contributed also to the onset of force polarity on the cell boundaries, manifested by larger contractile forces pointing toward the neighboring cell. The density and alignment of the fibers within the intercellular band were greater when fibers buckled under compression, with tension stiffening further contributing to this structural remodeling. Although previous studies have established the role of the ECM nonlinear mechanical behavior in increasing the range of force transmission, our model demonstrates the contribution of nonlinear elasticity of biological gels to directional and efficient mechanical signal transfer between distant cells, and rehighlights the importance of using fibrous gels in experimental settings for facilitating intercellular communication. VIDEO ABSTRACT.

Nonlinear Elasticity of the ECM Fibers Facilitates Efficient Intercellular Communication.

Force chains (FCs) are a key determinant of the micromechanical properties and behaviour of heterogeneous materials, such as granular systems. However, less is known about FCs in fibrous materials, such as the networks composing the extracellular matrix (ECM) of biological systems. Using a finite-element computational model, we simulated the contraction of a single cell and two nearby cells embedded in two-dimensional fibrous elastic networks and analysed the tensile FCs that developed in the ECM. The role of ECM nonlinear elasticity on FC formation was evaluated by considering linear and nonlinear, i.e. exhibiting 'buckling' and/or 'strain-stiffening', stress-strain curves. The effect of the degree of cell contraction and network coordination value was assessed. We found that nonlinear elasticity of the ECM fibres influenced the structure of the FCs, facilitating the transition towards more distinct chains that were less branched and more radially oriented than the chains formed in linear elastic networks. When two neighbouring cells contract, a larger number of FCs bridged between the cells in nonlinear networks, and these chains had a larger effective rigidity than the chains that did not reach a neighbouring cell. These results suggest that FCs function as a route for mechanical communication between distant cells and highlight the contribution of ECM fibre nonlinear elasticity to the formation of FCs.

Force chains in cell-cell mechanical communication.

External forces play an important role in the development and regulation of many tissues. Such effects are often studied using specialized stretchers-standardized commercial and novel laboratory-designed. While designs for 2D stretchers are abundant, the range of available 3D stretcher designs is more limited, especially when live imaging is required. This work presents a novel method and a stretching device that allow straining of 3D hydrogels from their circumference, using a punctured elastic silicone strip as the sample carrier. The system was primarily constructed from 3D-printed parts and low-cost electronics, rendering it simple and cost-efficient to reproduce in other labs. To demonstrate the system functionality, > 100 μm thick soft fibrin gels (< 1 KPa) were stretched, while performing live confocal imaging. The subsequent strains and fiber alignment were analyzed and found to be relatively homogenous throughout the gel's thickness (Z axis). The uniform Z-response enabled by our approach was found to be in contrast to a previously reported approach that utilizes an underlying elastic substrate to convey strain to a 3D thick sample. This work advances the ability to study the role of external forces on biological processes under more physiological 3D conditions, and can contribute to the field of tissue engineering.

Straining 3D Hydrogels with Uniform Z-Axis Strains While Enabling Live Microscopy Imaging.

The unique nonlinear mechanics of the fibrous extracellular matrix (ECM) facilitates long-range cell-cell mechanical communications that would be impossible for linear elastic substrates. Past research has described the contribution of two separated effects on the range of force transmission, including ECM elastic nonlinearity and fiber alignment. However, the relation between these different effects is unclear, and how they combine to dictate force transmission range is still elusive. Here, we combine discrete fiber simulations with continuum modeling to study the decay of displacements induced by a contractile cell in fibrous networks. We demonstrate that fiber nonlinearity and fiber reorientation both contribute to the strain-induced elastic anisotropy of the cell's local environment. This elastic anisotropy is a "lumped" parameter that governs the slow decay of displacements, and it depends on the magnitude of applied strain, either an external tension or an internal contraction, as a model of the cell. Furthermore, we show that accounting for artificially prescribed elastic anisotropy dictates the decay of displacements induced by a contracting cell. Our findings unify previous single effects into a mechanical theory that explains force transmission in fibrous networks. This work may provide insights into biological processes that involve communication of distant cells mediated by the ECM, such as those occurring in morphogenesis, wound healing, angiogenesis, and cancer metastasis. It may also provide design parameters for biomaterials to control force transmission between cells as a way to guide morphogenesis in tissue engineering.

Elastic Anisotropy Governs the Range of Cell-Induced Displacements.

When seeded in fibrous gels, pairs of cells or cell aggregates can induce bands of deformed gel, extending to surprisingly long distances in the intercellular medium. The formation of bands has been previously shown and studied in collagen systems. In this study, we strive to further our understanding of this fundamental mechanical mechanism in fibrin, a key element in wound healing and angiogenesis processes. We embedded fibroblast cells in 3D fibrin gels, and monitored band formation by real-time confocal microscopy. Quantitative dynamic analysis of band formation revealed a gradual increase in fiber density and alignment between pairs of cells. Such intercellular bands extended into a large-scale network of mechanically connected cells, in which the connected cells exhibited a more spread morphology than the isolated cells. Moreover, computational modeling demonstrated that the direction of cell-induced force triggering band formation can be applied in a wide range of angles relative to a neighboring cell. Our findings indicate that long-range mechanical coupling between cells is an important mechanism in regulating multicellular processes in reconstituted fibrin gels. As such, it should motivate exploration of this mechanism in studies in vivo, in wound healing or angiogenesis, in which fibrin is contracted by fibroblast cells.

Long-range mechanical coupling of cells in 3D fibrin gels.

There is a growing need for biomaterial scaffolds that support engineering of soft tissue substitutes featuring structure and mechanical properties similar to those of the native tissue. This work introduces a new biomaterial system that is based on centimeter-long collagen fibers extracted from  soft corals, wrapped around frames to create aligned fiber arrays. The collagen arrays displayed hyperelastic and viscoelastic mechanical properties that resembled those of collagenous-rich tissues. Cytotoxicity tests demonstrated that the collagen arrays were nontoxic to fibroblast cells. In addition, fibroblast cells seeded on the collagen arrays demonstrated spreading and increased growth for up to 40 days, and their orientation followed that of the aligned fibers. The possibility to combine the collagen cellular arrays with poly(ethylene glycol) diacrylate (PEG-DA) hydrogel, to create integrated biocomposites, was also demonstrated. This study showed that coral collagen fibers in combination with a hydrogel can support biological tissue-like growth, with predefined orientation over a long period of time in culture. As such, it is an attractive scaffold for the construction of various engineered tissues to match their native oriented morphology.

Ayelet Lesman

School of Mechanical Engineering,
Faculty of Engineering,
Center for the Physics and Chemistry of Living Systems,
School of Mechanical Engineering, Faculty of Engineering

Tel-Aviv University

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Ayelet Lesman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Mechanical Engineering,Faculty of Engineering,Center for the Physics and Chemistry of Living Systems,School of Mechanical Engineering, Faculty of Engineering

Tel-Aviv University

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

School of Mechanical Engineering,
Faculty of Engineering,
Center for the Physics and Chemistry of Living Systems,
School of Mechanical Engineering, Faculty of Engineering