The influence of contact guidance on chemotaxis of human neutrophil leukocytes

Chemotaxis of human neutrophil leukocytes moving on or in aligned 3D fibrin gels is more efficient if the cells are moving along the axis of fibre alignment than if they have to cross the fibres. This was shown by using two assays, one in which the cells were responding to a distant (600 micrometers) gradient source diffusing from a filter paper impregnated with formyl-Met-Leu-Phe and incorporated into the gel, the other in which the cells were responding to nearby (20--30 micrometers) Candida albicans spores in serum. In the former assay, impairment of chemotaxis across the axis of fibre alignment was highly significant. In the latter, cells showed efficient chemotaxis to the spores, but took more irregular paths when crossing the aligned fibres than when running along them. Neutrophils show contact guidance in aligned collagen or fibrin gels (Wilkinson et al., Exp cell res 140 (1982) 55) [1], thus the cells were subjected simultaneously to two directional cues in these experiments, one the chemotactic gradient and the other a contact guidance field. These cues may reinforce or interfere with each other depending on their relative orientation. Since many tissues in vivo show alignment or more complex forms of patterning, tissue architecture is likely to be an important determinant of the efficiency of cellular mobilization in inflamed or infected sites.     

Macrophages influence a competition of contact guidance and chemotaxis for fibroblast alignment in a fibrin gel coculture assay

Rat dermal fibroblasts were dispersed initially in the outer shell of a fibrin gel sphere, while the inner core either was devoid of cells or contained peritoneal exudate cells (primarily macrophages), thereby mimicking the inflammatory phase of wound healing. The fibroblasts compacted floating fibrin microspheres over time. In the absence of macrophages, the initial distribution of fibroblasts (only in the shell) induced circumferential alignment of fibrin fibrils via compaction of the shell relative to the core. The aligned fibrils created a contact guidance field, which was manifested by strong circumferential alignment of the fibroblasts. However, in the presence of macrophages, the fibroblasts exhibited more radial alignment despite the simultaneous contact guidance field in the circumferential direction associated with compaction. This was attributed to a chemotactic gradient emanating from the core due to a putative factor(s) released by the macrophages. The presence of a radial chemotactic stimulus was supported by the finding of even greater radial alignment when fibrin microspheres were embedded in an agarose-fibrin gel that abolished compaction and consequently the contact guidance field. Our assay permits the simulation of tissue morphogenetic processes that involve cell guidance phenomena and tractional restructuring of the extracellular matrix.     

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.     

Fabrication of skeletal muscle constructs by topographic activation of cell alignment

Skeletal muscle fiber construction for tissue-engineered grafts requires assembly of unidirectionally aligned juxtaposed myotubes. To construct such a tissue, a polymer microchip with linearly aligned microgrooves was fabricated that could direct myoblast adaptation under stringent conditions. The closely spaced microgrooves fabricated by a modified replica molding process guided linear cellular alignment. Examination of the myoblasts by immunofluorescence microscopy demonstrated that the microgrooves with subcellular widths and appropriate height-to-width ratios were required for practically complete linear alignment of myoblasts. The topology-dependent cell alignment encouraged differentiation of myoblasts into multinucleate, myosin heavy chain positive myotubes. The monolayer of myotubes formed on the microstructured chips allowed attachment, growth and differentiation of subsequent layers of linearly arranged myoblasts, parallel to the primary monolayer of myotubes. The consequent deposition of additional myoblasts on the previous layer of myotubes resulted in three-dimensional multi-layered structures of myotubes, typical of differentiated skeletal muscle tissue. The findings demonstrate that the on-chip device holds promise for providing an efficient means for guided muscle tissue construction.     

Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation

Most tissue cells grown in sparse cultures on linearly elastic substrates typically display a small, round phenotype on soft substrates and become increasingly spread as the modulus of the substrate increases until their spread area reaches a maximum value. As cell density increases, individual cells retain the same stiffness-dependent differences unless they are very close or in molecular contact. On nonlinear strain-stiffening fibrin gels, the same cell types become maximally spread even when the low strain elastic modulus would predict a round morphology, and cells are influenced by the presence of neighbors hundreds of microns away. Time lapse microscopy reveals that fibroblasts and human mesenchymal stem cells on fibrin deform the substrate by several microns up to five cell lengths away from their plasma membrane through a force limited mechanism. Atomic force microscopy and rheology confirm that these strains locally and globally stiffen the gel, depending on cell density, and this effect leads to long distance cell-cell communication and alignment. Thus cells are acutely responsive to the nonlinear elasticity of their substrates and can manipulate this rheological property to induce patterning.     

Aligned electrospun fibers for neural patterning

Objectives

To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation.

Results

A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy.

Conclusions

The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.

     

Elastic characterization of transversely isotropic soft materials by dynamic shear and asymmetric indentation

The mechanical characterization of soft anisotropic materials is a fundamental challenge because of difficulties in applying mechanical loads to soft matter and the need to combine information from multiple tests. A method to characterize the linear elastic properties of transversely isotropic soft materials is proposed, based on the combination of dynamic shear testing (DST) and asymmetric indentation. The procedure was demonstrated by characterizing a nearly incompressible transversely isotropic soft material. A soft gel with controlled anisotropy was obtained by polymerizing a mixture of fibrinogen and thrombin solutions in a high field magnet (B?=?11.7 T); fibrils in the resulting gel were predominantly aligned parallel to the magnetic field. Aligned fibrin gels were subject to dynamic (20-40 Hz) shear deformation in two orthogonal directions. The shear storage modulus was 1.08?±?0. 42 kPa (mean?±?std. dev.) for shear in a plane parallel to the dominant fiber direction, and 0.58?±?0.21 kPa for shear in the plane of isotropy. Gels were indented by a rectangular tip of a large aspect ratio, aligned either parallel or perpendicular to the normal to the plane of transverse isotropy. Aligned fibrin gels appeared stiffer when indented with the long axis of a rectangular tip perpendicular to the dominant fiber direction. Three-dimensional numerical simulations of asymmetric indentation were used to determine the relationship between direction-dependent differences in indentation stiffness and material parameters. This approach enables the estimation of a complete set of parameters for an incompressible, transversely isotropic, linear elastic material.     

Cell orientation determines the alignment of cell-produced collagenous matrix

In healing ligaments and tendons, the cells are not aligned and collagen matrix is not organized as in normal tissues. In addition, the mechanical properties of the tissues are abnormal. We hypothesized that the lack of alignment of the collagen matrix results from random orientation of the cells seen in the healing area. To test this hypothesis, a novel in vitro model was used in which the orientation of cells could be controlled via microgrooves, and alignment of the collagen matrix formed by these cells could be easily observed. It is known that cells align uniformly along the direction of microgrooves; therefore MC3T3-E1 cells, which produce large amounts of collagen, were grown on silicone membranes with parallel microgrooves (10 microm wide x 3 microm deep) in the surface. As a control, the same cells were also grown on smooth silicone membranes. Cells on both the microgrooved and smooth silicone surfaces produced a layer of readily visible collagen matrix. Immunohistochemical staining showed that the matrix consisted of abundant type I collagen. Polarized light microscopy of the collagen matrix revealed the collagen fibers to be parallel to the direction of the microgrooves, whereas the collagen matrix produced by the randomly oriented cells on the smooth membranes was disorganized. Thus, the results of this study suggest that the orientation of cells affects the organization of the collagenous matrix produced by the cells. The results also suggest that orienting cells along the longitudinal direction of healing ligaments and tendons may lead to the production of aligned collagenous matrix that more closely represents the uninjured state. This may enhance the mechanical properties of healing ligaments and tendons.     

Electrosensitive polyacrylic acid/fibrin hydrogel facilitates cell seeding and alignment

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure.     

Dependence of alignment direction on magnitude of strain in esophageal smooth muscle cells

The response of cells in vitro to mechanical forces has been the subject of much research using devices to exert controlled mechanical stimulation on cultured cells or isolated tissue. In this study, esophageal smooth muscle cells were seeded on flexible polyurethane membranes to form a confluent cell layer. The cells were then subjected to uniform cyclic stretch of varying magnitudes at a frequency of approximately five cycles per minute in a custom made mechatronic bioreactor, providing similar strains experienced in the in vivo mechanical environment of the esophagus. The results show that the orientation response is dependent on the magnitude of cyclic stretch applied. Smooth muscle cells showed parallel alignment to the force direction at low cyclic strains (2%) compared to the hill‐valley morphology of static controls. At higher strains (5% and 10% magnitude), the cells exhibited a consistent alignment perpendicular to the strain. To our knowledge, this is the first time that the alignment direction's dependence on strain magnitude has been demonstrated. MTS analysis indicated that cell metabolism was reduced when mechanical strain was applied, and proliferation was inhibited by mechanical strain. Protein expression indicates a decrease in smooth muscle α‐actin, indicative of changes in cell phenotype, an increase in vimentin, which is associated with increased cell motility, and an increase in desmin, indicating differentiation in stimulated cells. Biotechnol. Bioeng. 2009;102: 1703–1711. © 2008 Wiley Periodicals, Inc.     

Orientation of neurites influences severity of mechanically induced tau pathology

Chronic traumatic encephalopathy is a neurodegenerative disease associated with repeated traumatic brain injury (TBI). Chronic traumatic encephalopathy is a tauopathy, in which cognitive decline is accompanied by the accumulation of neurofibrillary tangles of the protein tau in patients’ brains. We recently found that mechanical force alone can induce tau mislocalization to dendritic spines and loss of synaptic function in in vitro neuronal cultures with random cell organization. However, in the brain, neurons are highly aligned, so here we aimed to determine how neuronal organization influences early-stage tauopathy caused by mechanical injury. Using microfabricated cell culture constructs to control the growth of neurites and an in vitro simulated TBI device to apply controlled mechanical deformation, we found that neuronal orientation with respect to the direction of a uniaxial high-strain-rate stretch injury influences the degree of tau pathology in injured neurons. We found that a mechanical stretch applied parallel to the neurite alignment induces greater mislocalization of tau proteins to dendritic spines than does a stretch with the same strain applied perpendicular to the neurites. Synaptic function, characterized by the amplitude of miniature excitatory postsynaptic currents, was similarly decreased in neurons with neurites aligned parallel to stretch, whereas in neurons aligned perpendicular to stretch, it had little to no functional loss. Experimental injury parameters (strain, strain rate, direction of stretch) were combined with a standard viscoelastic solid model to show that in our in vitro model, neurite work density during stretch correlates with tau mislocalization. These findings suggest that in a TBI, the magnitude of brain deformation is not wholly predictive of neurodegenerative consequences of TBI but that deformation relative to local neuronal architecture and the neurite mechanical energy during injury are better metrics for predicting trauma-induced tauopathy.     

Fluid shear stress-induced alignment of cultured vascular smooth muscle cells

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering.     

Alignment of skeletal muscle myoblasts and myotubes using linear micropatterned surfaces ground with abrasives

Alignment of cells plays a significant key role in skeletal muscle tissue engineering because skeletal muscle tissue in vivo has a highly organized structure consisting of long parallel multinucleated myotubes formed through differentiation and fusion of myoblasts. In the present study, we developed an easy, simple, and low‐cost method for aligning skeletal muscle cells by using surfaces with linear microscale features fabricated by grinding. Iron blocks were ground in one direction with three kinds of abrasives (9 µm diamond suspension, #400 sandpaper, and #150 sandpaper) and then used as molds to make micropatterned polydimethylsiloxane (PDMS) substrates (type I, type II, and type III). Observation of the surface topography revealed that the PDMS substrates exhibited different degree of mean roughness (Ra), 0.03 µm for type I, 0.16 µm for type II, and 0.56 µm for type III, respectively. Murine skeletal muscle cell line C2C12 myoblasts were cultured and differentiated on the patterned PDMS substrates, and it was examined whether the alignment of C2C12 myoblasts and myotubes was possible. Although the cell growth and differentiation on the three types of patterned substrates were similar to those on the flat PDMS substrate as a control, the alignment of both C2C12 myoblasts and myotubes was obviously observed on types II and III, but not on type I or the control substrate. These results indicate that surfaces ground with abrasives will be useful for fabricating aligned skeletal muscle tissues. Biotechnol. Bioeng. 2009;103: 631–638. © 2009 Wiley Periodicals, Inc.     

Combined effects of microtopography and cyclic strain on vascular smooth muscle cell orientation

Cellular alignment studies have shown that cell orientation has a large effect on the expression and behavior of cells. Cyclic strain and substrate microtopography have each been shown to regulate cellular alignment. This study examined the combined effects of these two stimuli on the alignment of bovine vascular smooth muscle cells (VSMCs). Cells were cultured on substrates with microgrooves of varying widths oriented either parallel or perpendicular to the direction of an applied cyclic tensile strain. We found that microgrooves oriented parallel to the direction of the applied strain limited the orientation response of VSMCs to the mechanical stimulus, while grooves perpendicular to the applied strain enhanced cellular alignment. Further, the extent to which parallel grooves limited cell alignment was found to be dependent on the groove width. It was found that for both a small (15microm) and a large (70microm) groove width, cells were better able to reorient in response to the applied strain than for an intermediate groove width (40microm). This study indicates that microtopographical cues modulate the orientation response of VSMCs to cyclic strain. The results suggest that there is a range of microgroove dimensions that is most effective at maintaining the orientation of the cells in the presence of an opposing stimulus induced by cyclic strain.     

Three-dimensional simulation of anisotropic cell-driven collagen gel compaction

Tissue equivalents (TEs), formed by entrapping cells in a collagen gel, are an important model system for studying cell behavior. We have previously (Barocas and Tranquillo in J Biomech Eng 117:161–170, 1997a) developed an anisotropic biphasic theory of TE mechanics, which comprises five coupled partial differential equations describing interaction among cells and collagen fibers in the TE. The model equations, previously solved in one or two dimensions, were solved in three dimensions using an adaptive finite-element platform. The model was applied to three systems: a rectangular isometric cell traction assay, an otherwise- acellular gel containing two islands of cells, and an idealized tissue-engineered cardiac valve leaflet. In the first two cases, published experimental data were available for comparison, and the model results were consistent with the experimental observations. Fibers and cells aligned in the fixed direction in the isometric assay, and a region of strong fiber alignment arose between the two cell islands. For the valve problem, the alignment predicted by the model was generally similar to that observed experimentally, but an asymmetry in the experiment was not captured by the model.     

3D Collagen Alignment Limits Protrusions to Enhance Breast Cancer Cell Persistence

Patients with mammographically dense breast tissue have a greatly increased risk of developing breast cancer. Dense breast tissue contains more stromal collagen, which contributes to increased matrix stiffness and alters normal cellular responses. Stromal collagen within and surrounding mammary tumors is frequently aligned and reoriented perpendicular to the tumor boundary. We have shown that aligned collagen predicts poor outcome in breast cancer patients, and postulate this is because it facilitates invasion by providing tracks on which cells migrate out of the tumor. However, the mechanisms by which alignment may promote migration are not understood. Here, we investigated the contribution of matrix stiffness and alignment to cell migration speed and persistence. Mechanical measurements of the stiffness of collagen matrices with varying density and alignment were compared with the results of a 3D microchannel alignment assay to quantify cell migration. We further interpreted the experimental results using a computational model of cell migration. We find that collagen alignment confers an increase in stiffness, but does not increase the speed of migrating cells. Instead, alignment enhances the efficiency of migration by increasing directional persistence and restricting protrusions along aligned fibers, resulting in a greater distance traveled. These results suggest that matrix topography, rather than stiffness, is the dominant feature by which an aligned matrix can enhance invasion through 3D collagen matrices.     

Light transmission through blood in oscillatory flow

Measurements were made of the intensity of light transmitted through various thicknesses of normal human blood confined between two parallel plane surfaces, one fixed and the other oscillating in its own plane. When the light propagation direction is perpendicular to the direction of shear flow the transmitted intensity contains a steady component and a dominant second harmonic of the oscillation frequency. For a thin layer of blood, the steady intensity is a minimum value when the oscillation amplitude produces unit strain. The second harmonic is very small at small strains, but increases rapidly near unit strain where it is approximately in phase with the strain. For thicker layers, the effects of viscoelastic shear waves reduce the size of the second harmonic and modify its phase. Changes in light transmission are interpreted by relating the optical density of the blood to the total amount of contact between red cells. In oscillatory flow at low strains (less than 1) cell-to-cell contact is reduced by disaggregation of cell groups, and light transmission decreases. Near unit strain, disaggregation becomes complete, cell alignment occurs, and light transmission is minimized. At higher strains cell-to-cell contact is increased by formation of aligned layers of compacted cells separated by parallel plasma layers, and light transmission increases.     

Stress fibers of the aortic smooth muscle cells in tissues do not align with the principal strain direction during intraluminal pressurization

Stress fibers (SFs) in cells transmit external forces to cell nuclei, altering the DNA structure, gene expression, and cell activity. To determine whether SFs are involved in mechanosignal transduction upon intraluminal pressure, this study investigated the SF direction in smooth muscle cells (SMCs) in aortic tissue and strain in the SF direction. Aortic tissues were fixed under physiological pressure of 120 mmHg. First, we observed fluorescently labeled SFs using two-photon microscopy. It was revealed that SFs in the same smooth muscle layers were aligned in almost the same direction, and the absolute value of the alignment angle from the circumferential direction was 16.8° ± 5.2° (n = 96, mean ± SD). Second, we quantified the strain field in the aortic tissue in reference to photo-bleached markers. It was found in the radial-circumferential plane that the largest strain direction was − 21.3° ± 11.1°, and the zero normal strain direction was 28.1° ± 10.2°. Thus, the SFs in aortic SMCs were not in line with neither the largest strain direction nor the zero strain direction, although their orientation was relatively close to the zero strain direction. These results suggest that SFs in aortic SMCs undergo stretch, but not maximal and transmit the force to nuclei under intraluminal pressure.

     

Anisotropic stretch-induced hypertrophy in neonatal ventricular myocytes micropatterned on deformable elastomers

Because cell shape and alignment, cell-matrix adhesion, and cell-cell contact can all affect growth, and because mechanical strains in vivo are multiaxial and anisotropic, we developed an in vitro system for engineering aligned, rod-shaped, neonatal cardiac myocyte cultures. Photolithographic and microfluidic techniques were used to micropattern extracellular matrices in parallel lines on deformable silicone elastomers. Confluent, elongated, aligned myocytes were produced by varying the micropattern line width and collagen density. An elliptical cell stretcher applied 2:1 anisotropic strain statically to the elastic substrate, with the axis of greatest stretch (10%) either parallel or transverse to the myofibrils. After 24 h, the principal strain parallel to myocytes did not significantly alter myofibril accumulation or expression of atrial natriuretic factor (ANF), connexin-43 (Cx-43), or N-cadherin (by indirect immunofluorescent antibody labeling and immunoblotting) compared with unstretched controls. In contrast, 10% transverse principal strain resulted in continuous staining of actin filaments (rhodamine phalloidin); increased immunofluorescent labeling of ANF, Cx-43, and N-cadherin; and upregulation of protein signal intensity by western blotting. By using microfabrication and microfluidics to control cell shape and alignment on an elastic substrate, we found greater effects for transverse than for longitudinal stretch in regulating sarcomere organization, hypertrophy, and cell-to-cell junctions.     

Probing Cell Structure Responses Through a Shear and Stretching Mechanical Stimulation Technique

Cells are complex, dynamic systems that respond to various in vivo stimuli including chemical, mechanical, and scaffolding alterations. The influence of mechanics on cells is especially important in physiological areas that dictate what modes of mechanics exist. Complex, multivariate physiological responses can result from multi-factorial, multi-mode mechanics, including tension, compression, or shear stresses. In this study, we present a novel device based on elastomeric materials that allowed us to stimulate NIH 3T3 fibroblasts through uniaxial strip stretching or shear fluid flow. Cell shape and structural response was observed using conventional approaches such as fluorescent microscopy. Cell orientation and actin cytoskeleton alignment along the direction of applied force were observed to occur after an initial 3 h time period for shear fluid flow and static uniaxial strip stretching experiments although these two directions of alignment were oriented orthogonal relative to each other. This response was then followed by an increasingly pronounced cell and actin cytoskeleton alignment parallel to the direction of force after 6, 12, and 24 h, with 85% of the cells aligned along the direction of force after 24 h. These results indicate that our novel device could be implemented to study the effects of multiple modes of mechanical stimulation on living cells while probing their structural response especially with respect to competing directions of alignment and orientation under these different modes of mechanical stimulation. We believe that this will be important in a diversity of fields including cell mechanotransduction, cell–material interactions, biophysics, and tissue engineering.