Fabrication of Aligned Microstructures with a Single Elastomeric Stamp

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2002  |  Pubmed ID: 11842197

The fabrication of complex patterns of aligned microstructures has required the use of multiple applications of lithography. Here we describe an approach for microfabrication that encodes the two-dimensional spatial information of several photomasks onto a single elastomeric stamp by mapping each photomask onto distinct heights on the surface of the stamp. Pressing the stamp against a surface collapses the topography of the stamp such that each recessed layer contacts the surface in stepwise sequence; the greater the applied pressure, the larger the area of the stamp that contacts the surface. After contact of each new layer with the surface, we use techniques of soft lithography (microcontact printing, microfluidics, and patterning through membranes) to pattern the surfaces that contact the stamp and those that do not with inorganic, organic, or living materials. Microfabrication through the use of multilevel stamps provides a promising alternative to conventional lithography for the construction of multicomponent, aligned surfaces; these structures may find use as components of microfluidic devices or biological patterns.

Patterning the Cellular Microenvironment

IEEE Engineering in Medicine and Biology Magazine : the Quarterly Magazine of the Engineering in Medicine & Biology Society. Jan-Feb, 2002  |  Pubmed ID: 11935995

Cell-cell Signaling by Direct Contact Increases Cell Proliferation Via a PI3K-dependent Signal

FEBS Letters. Mar, 2002  |  Pubmed ID: 11943158

We report a novel mechanism of cellular growth control. Increasing the density of endothelial or smooth muscle cells in culture increased cell-cell contact and decreased cell spreading, leading to growth arrest. Using a new method to independently control cell-cell contact and cell spreading, we found that introducing cell-cell contact positively regulates proliferation, but that contact-mediated proliferation can be masked by changes in cell spreading: Round cells with many contacts proliferated less than spread cells with none. Physically blocking cell-cell contact or inhibiting PI3K signaling abrogated cell-cell induced proliferation, but inhibiting diffusible paracrine signaling did not. Thus, direct cell-cell contact induces proliferation in these cells.

Engineering Cellular Microenvironments to Improve Cell-based Drug Testing

Drug Discovery Today. Jun, 2002  |  Pubmed ID: 12047872

Recent progress in the biology of cell adhesion is enabling cell culture models to better reproduce in vivo functions. Cues from adhesion to extracellular matrix and neighboring cells are important regulators of cell behaviors. The recent adaptation of semiconductor tools to spatially organize cells and their adhesions has enhanced our ability to engineer cell functions ex vivo. By using these tools to create more in vivo-like cultures, cell-based drug discovery and target validation could be improved. This review explores the biological advances made by these microfabrication tools and discusses how they could enable high-throughput cell-based assays.

Cells Lying on a Bed of Microneedles: an Approach to Isolate Mechanical Force

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2003  |  Pubmed ID: 12552122

We describe an approach to manipulate and measure mechanical interactions between cells and their underlying substrates by using microfabricated arrays of elastomeric, microneedle-like posts. By controlling the geometry of the posts, we varied the compliance of the substrate while holding other surface properties constant. Cells attached to, spread across, and deflected multiple posts. The deflections of the posts occurred independently of neighboring posts and, therefore, directly reported the subcellular distribution of traction forces. We report two classes of force-supporting adhesions that exhibit distinct force-size relationships. Force increased with size of adhesions for adhesions larger than 1 microm(2), whereas no such correlation existed for smaller adhesions. By controlling cell adhesion on these micromechanical sensors, we showed that cell morphology regulates the magnitude of traction force generated by cells. Cells that were prevented from spreading and flattening against the substrate did not contract in response to stimulation by serum or lysophosphatidic acid, whereas spread cells did. Contractility in the unspread cells was rescued by expression of constitutively active RhoA. Together, these findings demonstrate a coordination of biochemical and mechanical signals to regulate cell adhesion and mechanics, and they introduce the use of arrays of mechanically isolated sensors to manipulate and measure the mechanical interactions of cells.

Cell Shape Provides Global Control of Focal Adhesion Assembly

Biochemical and Biophysical Research Communications. Jul, 2003  |  Pubmed ID: 12859964

Cell spreading was controlled independently of the amount and density of immobilized integrin ligand by culturing cells on single adhesive islands of different sizes (100-2500 microm(2)) and shapes (squares, circles, and lines) or on many smaller (3-5 microm diameter) circular islands that were coated with a saturating density of fibronectin and separated by non-adhesive regions. The amount of focal adhesions (FAs) containing vinculin and phosphotyrosine increased in direct proportion to cell spreading under all conditions. FAs localized asymmetrically along the periphery of the small islands that experienced highest tensional stress, and FA staining increased when cytoskeletal tension was stimulated with thrombin, whereas inhibitors of contractility promoted FA disassembly. Thus, these findings demonstrate the existence of an "inside-out" mechanism whereby global cell distortion produces increases in cytoskeletal tension that feed back to drive local changes in FA assembly. This complex interplay between cell morphology, mechanics, and adhesion may be critical to how cells integrate from and function in living tissues.

VE-cadherin Simultaneously Stimulates and Inhibits Cell Proliferation by Altering Cytoskeletal Structure and Tension

Journal of Cell Science. Sep, 2003  |  Pubmed ID: 12876221

Engagement of vascular endothelial (VE)-cadherin leads to the cessation of proliferation commonly known as 'contact inhibition'. We show that VE-cadherin inhibits growth by mediating changes in cell adhesion to the extracellular matrix. Increasing cell-cell contact decreased cell spreading and proliferation, which was reversed by blocking engagement of VE-cadherin. Using a new system to prevent the cadherin-induced changes in cell spreading, we revealed that VE-cadherin paradoxically increased proliferation. Treating cells with inhibitors of PKC and MEK abrogated the stimulatory signal at concentrations that disrupted the formation of actin fibers across the cell-cell contact. Directly disrupting actin fibers, blocking actin-myosin-generated tension, or inhibiting signaling through Rho specifically inhibited the cadherin-induced proliferative signal. By progressively altering the degree to which cell-cell contact inhibited cell spreading, we show that cell-cell contact ultimately increased or decreased the overall proliferation rate of the population by differentially shifting the balance between the two opposing proliferative cues. The existence of opposing growth signals induced by VE-cadherin that are both mediated through crosstalk with cytoskeletal structure highlights the complex interplay of mechanical and chemical signals with which cells navigate in their physical microenvironment.

Repositioning of Cells by Mechanotaxis on Surfaces with Micropatterned Young's Modulus

Journal of Biomedical Materials Research. Part A. Sep, 2003  |  Pubmed ID: 12918044

Adherent cells are strongly influenced by the mechanical aspects of biomaterials, but little is known about the cellular effects of spatial variations in these properties. This work describes a novel method to produce polymeric cell culture surfaces containing micrometer-scale regions of variable stiffness. Substrates made of acrylamide or poly(dimethylsiloxane) were patterned with 100- or 10-microm resolution, respectively. Cells were cultured on fibronectin-coated acrylamide having Young's moduli of 34 kPa and 1.8 kPa, or fibronectin-coated PDMS having moduli of 2.5 MPa and 12 kPa. Over several days, NIH/3T3 cells and bovine pulmonary arterial endothelial cells accumulated preferentially on stiffer regions of substrates. The migration, not proliferation, of cells in response to mechanical patterning (mechanotaxis) was responsible for the accumulation of cells on stiffer regions. Differential remodeling of extracellular matrix protein on stiff versus compliant regions was observed by immunofluorescence staining, and may have been responsible for the observed mechanotaxis. These results suggest that mechanically patterned substrates might provide a general means to study mechanotaxis, and a new approach to patterning cells.

Dielectrophoretic Registration of Living Cells to a Microelectrode Array

Biosensors & Bioelectronics. Feb, 2004  |  Pubmed ID: 14709396

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 micromdiameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20000 cells onto a 1cm(2) grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70+/-1% fidelity versus 17+/-1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.

Cell Shape, Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment

Developmental Cell. Apr, 2004  |  Pubmed ID: 15068789

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.

Vascular Endothelial-cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

Molecular Biology of the Cell. Jun, 2004  |  Pubmed ID: 15075376

Changes in vascular endothelial (VE)-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin-mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.

Dielectrophoretic Registration of Living Cells to a Microelectrode Array

Biosensors & Bioelectronics. Jul, 2004  |  Pubmed ID: 15198083

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 Ipm diameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20,000 cells onto aI cm2 grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70 +/- 1% fidelity versus 17 +/- 1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.

Mechanotransduction at Cell-matrix and Cell-cell Contacts

Annual Review of Biomedical Engineering. 2004  |  Pubmed ID: 15255771

Mechanical forces play an important role in the organization, growth, maturation, and function of living tissues. At the cellular level, many of the biological responses to external forces originate at two types of specialized microscale structures: focal adhesions that link cells to their surrounding extracellular matrix and adherens junctions that link adjacent cells. Transmission of forces from outside the cell through cell-matrix and cell-cell contacts appears to control the maturation or disassembly of these adhesions and initiates intracellular signaling cascades that ultimately alter many cellular behaviors. In response to externally applied forces, cells actively rearrange the organization and contractile activity of the cytoskeleton and redistribute their intracellular forces. Recent studies suggest that the localized concentration of these cytoskeletal tensions at adhesions is also a major mediator of mechanical signaling. This review summarizes the role of mechanical forces in the formation, stabilization, and dissociation of focal adhesions and adherens junctions and outlines how integration of signals from these adhesions over the entire cell body affects how a cell responds to its mechanical environment. This review also describes advanced optical, lithographic, and computational techniques for the study of mechanotransduction.

Simple Approach to Micropattern Cells on Common Culture Substrates by Tuning Substrate Wettability

Tissue Engineering. May-Jun, 2004  |  Pubmed ID: 15265304

The ability to spatially control cell adhesion and multicellular organization is critical to many biomedical and tissue-engineering applications. This work describes a straightforward method to micropattern cells onto glass, silicone rubber, and polystyrene using commercially available reagents. An elastomeric polydimethylsiloxane stamp is used to contact-transfer extracellular matrix protein onto a surface followed by blocking cell adhesion in the surrounding regions by the physisorption of Pluronic surfactants. Using self-assembled monolayers of alkanethiols on gold as model surfaces to control surface wettability, we found that protein printing was most effective at intermediate to highly wetting surfaces whereas Pluronic adsorption occurred at intermediate to low wetting surfaces. Within a regimen of intermediate wettability both techniques were applied in conjunction to restrict cell adhesion to specified patterns. Adjusting the wettability of common tissue culture substrates to the same intermediate range again allowed the micropatterning of cells, suggesting that this approach is likely to be generally applicable to many types of materials. This technique therefore may allow for wider adoption of cell patterning.

Characterization of the Nuclear Deformation Caused by Changes in Endothelial Cell Shape

Journal of Biomechanical Engineering. Oct, 2004  |  Pubmed ID: 15648807

We investigated the mechanotransduction pathway in endothelial cells between their nucleus and adhesions to the extracellular matrix. First, we measured nuclear deformations in response to alterations of cell shape as cells detach from a flat surface. We found that the nuclear deformation appeared to be in direct and immediate response to alterations of the cell adhesion area. The nucleus was then treated as a neo-Hookean compressible material, and we estimated the stress associated with the cytoskeleton and acting on the nucleus during cell rounding. With the obtained stress field, we estimated the magnitude of the forces deforming the nucleus. Considering the initial and final components of this adhesion-cytoskeleton-nucleus force transmission pathway, we found our estimate for the internal forces acting on the nucleus to be on the same order of magnitude as previously measured traction forces, suggesting a direct mechanical link between adhesions and the nucleus.

Strategies for Engineering the Adhesive Microenvironment

Journal of Mammary Gland Biology and Neoplasia. Oct, 2004  |  Pubmed ID: 15838609

Cells exist within a complex tissue microenvironment, which includes soluble factors, extracellular matrix molecules, and neighboring cells. In the breast, the adhesive microenvironment plays a crucial role in driving both normal mammary gland development as well tumor initiation and progression. Researchers are designing increasingly more complex ways to mimic the in vivo microenvironment in an in vitro setting, so that cells in culture may serve as model systems for tissue structures. Here, we explore the use of microfabrication technologies to engineer the adhesive microenvironment of cells in culture. These new tools permit the culture of cells on well-defined surface chemistries, patterning of cells into defined geometries either alone or in coculture scenarios, and measurement of forces associated with cell-ECM interactions. When applied to questions in mammary gland development and neoplasia, these new tools will enable a better understanding of how adhesive, structural, and mechanical cues regulate mammary epithelial biology.

Using Microenvironment to Engineer Stem Cell Function

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2004  |  Pubmed ID: 17271427

Stem cells differentiate into different lineages depending on the local cues present in their microenvironment. While soluble cues have been studied in great depth, little is known about the role of insoluble cues such as cell adhesion and mechanical forces. We now show that human mesenchymal stem cells (MSCs) commit to different lineages depending on their physical microenvironment. Using microfabricated tools, we found that cell shape regulates MSCs commitment, and does so through a cytoskeletal tension-dependent pathway. We will discuss the novel tools that we are developing to study these mechanical signals, and how they have informed us about how cells probe and respond to their environment.

Assembly of Multicellular Constructs and Microarrays of Cells Using Magnetic Nanowires

Lab on a Chip. Jun, 2005  |  Pubmed ID: 15915251

An approach is described for controlling the spatial organization of mammalian cells using ferromagnetic nanowires in conjunction with patterned micromagnet arrays. The nanowires are fabricated by electrodeposition in nanoporous templates, which allows for precise control of their size and magnetic properties. The high aspect ratio and large remanent magnetization of the nanowires enable suspensions of cells bound to Ni nanowires to be controlled with low magnetic fields. This was used to produce one- and two-dimensional field-tuned patterning of suspended 3T3 mouse fibroblasts. Self-assembled one-dimensional chains of cells were obtained through manipulation of the wires' dipolar interactions. Ordered patterns of individual cells in two dimensions were formed through trapping onto magnetic microarrays of ellipsoidal permalloy micromagnets. Cell chains were formed on the arrays by varying the spacing between the micromagnets or the strength of fluid flow over the arrays. The positioning of cells on the array was further controlled by varying the direction of an external magnetic field. These results demonstrate the possibility of using magnetic nanowires to organize cells.

T Cell-to-T Cell Clustering Enhances NF-kappaB Activity by a PI3K Signal Mediated by Cbl-b and Rho

Biochemical and Biophysical Research Communications. Jul, 2005  |  Pubmed ID: 15922296

Full activation of T cells requires the binding of antigen to the T cell receptor and stimulation of the CD28 molecule, a process which typically occurs when T cells bind to an antigen presenting cell. The transcription factor, NF-kappaB, is an integration point for these two signals and its activation is critical for T cell function. Using antibodies to the TCR and CD28 molecules to activate Jurkat T cells, we show that cells that were permitted to aggregate into multi-cellular clusters increased NF-kappaB activity compared to unclustered cells. Inhibition of PI3K signaling with wortmannin decreased the clustering-mediated NF-kappaB signal. Over-expression of a dominant negative form of Cbl-b, an endogenous inhibitor of PI3K, in unclustered cells rescued NF-kappaB activation to the same levels caused by cell clustering. Inhibiting signaling through Rho with dominant negative RhoA abrogated both clustering-mediated and dominant negative Cbl-b-mediated NF-kappaB inactivation, but not TCR/CD28 mediated NF-kappaB activation. Taken together, these results suggest that in addition to pathways stimulated by classical T cell-APC interactions, another signal arising from T cell clustering can enhance activation.

Emergent Patterns of Growth Controlled by Multicellular Form and Mechanics

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2005  |  Pubmed ID: 16049098

Spatial patterns of cellular growth generate mechanical stresses that help to push, fold, expand, and deform tissues into their specific forms. Genetic factors are thought to specify patterns of growth and other behaviors to drive morphogenesis. Here, we show that tissue form itself can feed back to regulate patterns of proliferation. Using micro-fabrication to control the organization of sheets of cells, we demonstrated the emergence of stable patterns of proliferative foci. Regions of concentrated growth corresponded to regions of high tractional stress generated within the sheet, as predicted by a finite-element model of multicellular mechanics and measured directly by using a micromechanical force sensor array. Inhibiting actomyosin-based tension or cadherin-mediated connections between cells disrupted the spatial pattern of proliferation. These findings demonstrate the existence of patterns of mechanical forces that originate from the contraction of cells, emerge from their multicellular organization, and result in patterns of growth. Thus, tissue form is not only a consequence but also an active regulator of tissue growth.

Finite-element Analysis of the Adhesion-cytoskeleton-nucleus Mechanotransduction Pathway During Endothelial Cell Rounding: Axisymmetric Model

Journal of Biomechanical Engineering. Aug, 2005  |  Pubmed ID: 16121529

Endothelial cells possess a mechanical network connecting adhesions on the basal surface, the cytoskeleton, and the nucleus. Transmission of force at adhesions via this pathway can deform the nucleus, ultimately resulting in an alteration of gene expression and other cellular changes (mechanotransduction). Previously, we measured cell adhesion area and apparent nuclear stretch during endothelial cell rounding. Here, we reconstruct the stress map of the nucleus from the observed strains using finite-element modeling. To simulate the disruption of adhesions, we prescribe displacement boundary conditions at the basal surface of the axisymmetric model cell. We consider different scenarios of the cytoskeletal arrangement, and represent the cytoskeleton as either discrete fibers or as an effective homogeneous layer When the nucleus is in the initial (spread) state, cytoskeletal tension holds the nucleus in an elongated, ellipsoidal configuration. Loss of cytoskeletal tension during cell rounding is represented by reactive forces acting on the nucleus in the model. In our simulations of cell rounding, we found that, for both representations of the cytoskeleton, the loss of cytoskeletal tension contributed more to the observed nuclear deformation than passive properties. Since the simulations make no assumption about the heterogeneity of the nucleus, the stress components both within and on the surface of the nucleus were calculated. The nuclear stress map showed that the nucleus experiences stress on the order of magnitude that can be significant for the function of DNA molecules and chromatin fibers. This study of endothelial cell mechanobiology suggests the possibility that mechanotransduction could result, in part, from nuclear deformation, and may be relevant to angiogenesis, wound healing, and endothelial barrier dysfunction.

Shear Force at the Cell-matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors

Mechanics & Chemistry of Biosystems : MCB. 2005  |  Pubmed ID: 16708468

The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.

Facile Modification of Collagen Directed by Collagen Mimetic Peptides

Journal of the American Chemical Society. Mar, 2005  |  Pubmed ID: 15783169

Recent widespread interest in the development of engineered tissue and organ replacement therapies has prompted demand for new approaches to immobilize exogenous components to natural collagen. Chemical coupling of synthetic moieties to amino acid side chains has been commonly practiced for such purposes; however, such coupling reactions are difficult to control on large proteins and are generally not conducive to modifying integrated collagen scaffolds that contain live cells and tissues. As an alternative to the conventional "covalent" modification method, we have developed a novel "physical" modification technique that is based on collagen's native ability to associate into a triple-helical molecular architecture. Here, we present a finding that collagen mimetic peptides (CMPs) of sequence -(Pro-Hyp-Gly)x- exhibit strong affinity to both native and gelatinized type I collagen under controlled thermal conditions. We also show that the cell adhesion characteristics of collagen can be readily altered by applying a poly(ethylene glycol)-CMP conjugate to a prefabricated collagen film.

Optimization of Yield in Magnetic Cell Separations Using Nickel Nanowires of Different Lengths

Biotechnology Progress. Mar-Apr, 2005  |  Pubmed ID: 15801791

Ferromagnetic nanowires are shown to perform both high yield and high purity single-step cell separations on cultures of NIH-3T3 mouse fibroblast cells. The nanowires are made by electrochemical deposition in nanoporous templates, permitting detailed control of their chemical and physical properties. When added to fibroblast cell cultures, the nanowires are internalized by the cells via the integrin-mediated adhesion pathway. The effectiveness of magnetic cell separations using Ni nanowires 350 nm in diameter and 5-35 micrometers long in field gradients of 40 T/m was compared to commercially available superparamagnetic beads. The percent yield of the separated populations is found to be optimized when the length of the nanowire is matched to the diameter of the cells in the culture. Magnetic cell separations performed under these conditions achieve 80% purity and 85% yield, a 4-fold increase over the beads. This effect is shown to be robust when the diameter of the cell is changed within the same cell line using mitomycin-C.

Nanopattern-induced Changes in Morphology and Motility of Smooth Muscle Cells

Biomaterials. Sep, 2005  |  Pubmed ID: 15814139

Cells are known to be surrounded by nanoscale topography in their natural extracellular environment. The cell behavior, including morphology, proliferation, and motility of bovine pulmonary artery smooth muscle cells (SMC) were studied on poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces comprising nanopatterned gratings with 350 nm linewidth, 700 nm pitch, and 350 nm depth. More than 90% of the cells aligned to the gratings, and were significantly elongated compared to the SMC cultured on non-patterned surfaces. The nuclei were also elongated and aligned. Proliferation of the cells was significantly reduced on the nanopatterned surfaces. The polarization of microtubule organizing centers (MTOC), which are associated with cell migration, of SMC cultured on nanopatterned surfaces showed a preference towards the axis of cell alignment in an in vitro wound healing assay. In contrast, the MTOC of SMC on non-patterned surfaces preferentially polarized towards the wound edge. It is proposed that this nanoimprinting technology will provide a valuable platform for studies in cell-substrate interactions and for development of medical devices with nanoscale features.

Heparin Binding Directs Activation of T Cells Against Adeno-associated Virus Serotype 2 Capsid

Nature Medicine. Aug, 2006  |  Pubmed ID: 16845388

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8(+) T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.

An Inhibitory Role for FAK in Regulating Proliferation: a Link Between Limited Adhesion and RhoA-ROCK Signaling

The Journal of Cell Biology. Jul, 2006  |  Pubmed ID: 16847103

Focal adhesion kinase (FAK) transduces cell adhesion to the extracellular matrix into proliferative signals. We show that FAK overexpression induced proliferation in endothelial cells, which are normally growth arrested by limited adhesion. Interestingly, displacement of FAK from adhesions by using a FAK-/- cell line or by expressing the C-terminal fragment FRNK also caused an escape of adhesion-regulated growth arrest, suggesting dual positive and negative roles for FAK in growth regulation. Expressing kinase-dead FAK-Y397F in FAK-/- cells prevented uncontrolled growth, demonstrating the antiproliferative function of inactive FAK. Unlike FAK overexpression-induced growth, loss of growth control in FAK-/- or FRNK-expressing cells increased RhoA activity, cytoskeletal tension, and focal adhesion formation. ROCK inhibition rescued adhesion-dependent growth control in these cells, and expression of constitutively active RhoA or ROCK dysregulated growth. These findings demonstrate the ability of FAK to suppress and promote growth, and underscore the importance of multiple mechanisms, even from one molecule, to control cell proliferation.

Nanotechnology for Cell-substrate Interactions

Annals of Biomedical Engineering. Jan, 2006  |  Pubmed ID: 16525764

In the pursuit to understand the interaction between cells and their underlying substrates, the life sciences are beginning to incorporate micro- and nanotechnology-based tools to probe and measure cells. The development of these tools portends endless possibilities for new insights into the fundamental relationships between cells and their surrounding microenvironment that underlie the physiology of human tissue. Here, we review techniques and tools that have been used to study how a cell responds to the physical factors in its environment. We also discuss unanswered questions that could be addressed by these approaches to better elucidate the molecular processes and mechanical forces that dominate the interactions between cells and their physical scaffolds.

E-cadherin Engagement Stimulates Proliferation Via Rac1

The Journal of Cell Biology. May, 2006  |  Pubmed ID: 16682529

E-cadherin has been linked to the suppression of tumor growth and the inhibition of cell proliferation in culture. We observed that progressively decreasing the seeding density of normal rat kidney-52E (NRK-52E) or MCF-10A epithelial cells from confluence, indeed, released cells from growth arrest. Unexpectedly, a further decrease in seeding density so that cells were isolated from neighboring cells decreased proliferation. Experiments using microengineered substrates showed that E-cadherin engagement stimulated the peak in proliferation at intermediate seeding densities, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding-dependent decrease in cell spreading against the underlying substrate. Rac1 activity, which was induced by E-cadherin engagement specifically at intermediate seeding densities, was required for the cadherin-stimulated proliferation, and the control of Rac1 activation by E-cadherin was mediated by p120-catenin. Together, these findings demonstrate a stimulatory role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell-cell contact may trigger or inhibit epithelial cell proliferation in different settings.

Separate but Not Equal: Differential Mechanical Roles for Myosin Isoforms

Biophysical Journal. May, 2007  |  Pubmed ID: 17325018

Manipulation of Cell-cell Adhesion Using Bowtie-shaped Microwells

Methods in Molecular Biology (Clifton, N.J.). 2007  |  Pubmed ID: 17416983

Traditional methods to study cell-cell adhesion have been limited by their inability to manipulate cell-cell interactions without simultaneously affecting other microenvironmental factors. Here we describe a novel method that enables the culture of cells with precise simultaneous control of both cell-cell and cell-substratum adhesion. Using microfabricated stamps of poly(dimethylsiloxane), we construct bowtie-shaped agarose microwells into which cells can be cultured. The degree to which cells spread is controlled by the size of the microwell; cell-cell contacts form between neighboring cells within the microwell. This chapter describes the details of stamp fabrication, agarose microwell construction, and cell culture in micropatterned substrata.

Bait and Switch: Synthetic GEFs Divert an Input Signal to Diverse Cellular Responses

Developmental Cell. Jul, 2007  |  Pubmed ID: 17609105

The Rho family GTPases regulate many cellular functions including the organization, mechanics, and dynamics of the actin cytoskeleton. Re-engineering the highly conserved autoinhibitory structure of GEFs, Yeh et al. (2007) generated novel synthetic proteins that regulated Rho GTPase signaling and cell morphology, highlighting the power of protein module engineering.

Microfabricated Silicone Elastomeric Post Arrays for Measuring Traction Forces of Adherent Cells

Methods in Cell Biology. 2007  |  Pubmed ID: 17613314

Nonmuscle cells exert biomechanical forces known as traction forces on the extracellular matrix (ECM). Spatial coordination of these traction forces against the ECM is in part responsible for directing cell migration, for remodeling the surrounding tissue scaffold, and for the folds and rearrangements seen during morphogenesis. The traction forces are applied through a number of discrete adhesions between a cell and the ECM. We have developed a device consisting of an array of flexible, microfabricated posts capable of measuring these forces under an adherent cell. Functionalizing the top of each post with ECM protein allows cells to attach and spread across the tops of the posts. Deflection of the tips of the posts is proportional to cell-generated traction forces during cell migration or contraction. In this chapter, we describe the microfabrication, preparation, and experimental use of such microfabricated post array detector system (mPADs).

Activation of ROCK by RhoA is Regulated by Cell Adhesion, Shape, and Cytoskeletal Tension

Experimental Cell Research. Oct, 2007  |  Pubmed ID: 17673200

Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.

Cadherins, RhoA, and Rac1 Are Differentially Required for Stretch-mediated Proliferation in Endothelial Versus Smooth Muscle Cells

Circulation Research. Aug, 2007  |  Pubmed ID: 17712140

Abnormal mechanical forces can trigger aberrant proliferation of endothelial and smooth muscle cells, as observed in the progression of vascular diseases such as atherosclerosis. It has been previously shown that cells can sense physical forces such as stretch through adhesions to the extracellular matrix. Here, we set out to examine whether cell-cell adhesions are also involved in transducing mechanical stretch into a proliferative response. We found that both endothelial and smooth muscle cells exhibited an increase in proliferation in response to stretch. Using micropatterning to isolate the role of cell-cell adhesion from cell-extracellular matrix adhesion, we demonstrate that endothelial cells required cell-cell contact and vascular endothelial cadherin engagement to transduce stretch into proliferative signals. In contrast, smooth muscle cells responded to stretch without contact to neighboring cells. We further show that stretch stimulated Rac1 activity in endothelial cells, whereas RhoA was activated by stretch in smooth muscle cells. Blocking Rac1 signaling by pharmacological or adenoviral reagents abrogated the proliferative response to stretch in endothelial cells but not in smooth muscle cells. Conversely, blocking RhoA completely inhibited the proliferative response in smooth muscle cells but not in endothelial cells. Together, these data suggest that vascular endothelial cadherin has an important role in mechanotransduction and that endothelial and smooth muscle cells use different mechanisms to respond to stretch.

Magnetic Microposts As an Approach to Apply Forces to Living Cells

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2007  |  Pubmed ID: 17804810

Cells respond to mechanical forces whether applied externally or generated internally via the cytoskeleton. To study the cellular response to forces separately, we applied external forces to cells via microfabricated magnetic posts containing cobalt nanowires interspersed among an array of elastomeric posts, which acted as independent sensors to cellular traction forces. A magnetic field induced torque in the nanowires, which deflected the magnetic posts and imparted force to individual adhesions of cells attached to the array. Using this system, we examined the cellular reaction to applied forces and found that applying a step force led to an increase in local focal adhesion size at the site of application but not at nearby nonmagnetic posts. Focal adhesion recruitment was enhanced further when cells were subjected to multiple force actuations within the same time interval. Recording the traction forces in response to such force stimulation revealed two responses: a sudden loss in contractility that occurred within the first minute of stimulation or a gradual decay in contractility over several minutes. For both types of responses, the subcellular distribution of loss in traction forces was not confined to locations near the actuated micropost, nor uniformly across the whole cell, but instead occurred at discrete locations along the cell periphery. Together, these data reveal an important dynamic biological relationship between external and internal forces and demonstrate the utility of this microfabricated system to explore this interaction.

Cellular and Multicellular Form and Function

Advanced Drug Delivery Reviews. Nov, 2007  |  Pubmed ID: 17884241

Engineering artificial tissue constructs requires the appropriate spatial arrangement of cells within scaffolds. The introduction of microengineering tools to the biological community has provided a valuable set of techniques to manipulate the cellular environment, and to examine how cell structure affects cellular function. Using micropatterning techniques, investigators have found that the geometric presentation of cell-matrix adhesions are important regulators of various cell behaviors including cell growth, proliferation, differentiation, polarity and migration. Furthermore, the presence of neighboring cells in multicellular aggregates has a significant impact on the proliferative and differentiated state of cells. Using microengineering tools, it will now be possible to manipulate the various environmental factors for practical applications such as engineering tissue constructs with greater control over the physical structure and spatial arrangement of cells within their surrounding microenvironment.

Versatile, Fully Automated, Microfluidic Cell Culture System

Analytical Chemistry. Nov, 2007  |  Pubmed ID: 17953452

There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy. Using this device, we perform the first quantitative measurements of the influence of transient stimulation schedules on the proliferation, osteogenic differentiation, and motility of human primary mesenchymal stem cells.

Rac-dependent Cyclin D1 Gene Expression Regulated by Cadherin- and Integrin-mediated Adhesion

Journal of Cell Science. Jan, 2008  |  Pubmed ID: 18187454

Integrin-mediated adhesion to substratum is required for cyclin D1 induction in mesenchymal cells, but we show here that the induction of cyclin D1 persists despite blockade of ECM-integrin signaling in MCF10A mammary epithelial cells. E-cadherin-mediated cell-cell adhesion also supports cyclin D1 induction in these cells, and the combined inhibition of both E-cadherin and integrin adhesion is required to prevent the expression of cyclin D1 mRNA and protein. Our previous studies described a pro-proliferative effect of E-cadherin in MCF10A cells, mediated by Rac, and we now show that Rac is required for cyclin D1 mRNA induction by both E-cadherin and integrin engagement. The levels of p21Cip1 and p27Kip1, Cdk inhibitors that are also targets of integrin signaling, are not affected by E-cadherin-mediated cell-cell adhesion. Finally, we show that the increased expression of cyclin D1 mRNA associated with E-cadherin-dependent cell-cell adhesion is causally linked to an increased entry into S phase. Our results identify Rac signaling to cyclin D1 as a crucial pro-proliferative effect of E-cadherin-mediated cell-cell adhesion.

Spatial Patterning of Gene Expression Using Surface-immobilized Recombinant Adenovirus

Biomedical Microdevices. Aug, 2008  |  Pubmed ID: 18246428

Spatially patterned gene expression drives tissue organization and is a critical determinant of tissue function. Approaches in functional tissue engineering will require not only the spatial organization of cells but also control of their gene expression patterns. We report a method to generate patterns of gene expression within a monolayer of cells by using surface-immobilized recombinant adenovirus. This study represents a new approach to engineering tissues that relies on controlling spatial patterns of gene expression, and can be used independently or in combination with positioning of different cell types. This technique may have broad applications in biotechnology including tissue engineering and targeted gene delivery.

Magnetic Microposts for Mechanical Stimulation of Biological Cells: Fabrication, Characterization, and Analysis

The Review of Scientific Instruments. Apr, 2008  |  Pubmed ID: 18447536

Cells use force as a mechanical signal to sense and respond to their microenvironment. Understanding how mechanical forces affect living cells requires the development of tool sets that can apply nanoscale forces and also measure cellular traction forces. However, there has been a lack of techniques that integrate actuation and sensing components to study force as a mechanical signal. Here, we describe a system that uses an array of elastomeric microposts to apply external forces to cells through cobalt nanowires embedded inside the microposts. We first biochemically treat the posts' surfaces to restrict cell adhesion to the posts' tips. Then by applying a uniform magnetic field (B<0.3 T), we induce magnetic torque on the nanowires that is transmitted to a cell's adhesion site as an external force. We have achieved external forces of up to 45 nN, which is in the upper range of current nanoscale force-probing techniques. Nonmagnetic microposts, similarly prepared but without nanowires, surround the magnetic microposts and are used to measure the traction forces and changes in cell mechanics. We record the magnitude and direction of the external force and the traction forces by optically measuring the deflection of the microposts, which linearly deflect as cantilever springs. With this approach, we can measure traction forces before and after force stimulation in order to monitor cellular response to forces. We present the fabrication methods, magnetic force characterization, and image analysis techniques used to achieve the measurements.

Myofibrillar Architecture in Engineered Cardiac Myocytes

Circulation Research. Aug, 2008  |  Pubmed ID: 18635822

Morphogenesis is often considered a function of transcriptional synchrony and the spatial limits of diffusing mitogens; however, physical constrainment by the cell microenvironment represents an additional mechanism for regulating self-assembly of subcellular structures. We asked whether myocyte shape is a distinct signal that potentiates the organization of myofibrillar arrays in cardiac muscle myocytes. We engineered the shape of neonatal rat ventricular myocytes by culturing them on microfabricated fibronectin islands, where they spread and assumed the shape of the island. Myofibrillogenesis followed, both spatially and temporally, the assembly of unique actin networks whose architecture was predictable given the shape of the island. Subsequently, the z lines of the sarcomeres aligned and registered in distinct patterns in different regions of the myocytes in such a way that orthogonal axes of contraction could be distinctly engineered. These data suggest that physical constrainment of muscle cells by extracellular matrix may be an important regulator of myofibrillar organization.

Engineering Amount of Cell-cell Contact Demonstrates Biphasic Proliferative Regulation Through RhoA and the Actin Cytoskeleton

Experimental Cell Research. Sep, 2008  |  Pubmed ID: 18652824

Endothelial cell-cell contact via VE-cadherin plays an important role in regulating numerous cell functions, including proliferation. However, using different experimental approaches to manipulate cell-cell contact, investigators have observed both inhibition and stimulation of proliferation depending on the adhesive context. In this study, we used micropatterned wells combined with active positioning of cells by dielectrophoresis in order to investigate whether the number of contacting neighbors affected the proliferative response. Varying cell-cell contact resulted in a biphasic effect on proliferation; one contacting neighbor increased proliferation, while two or more neighboring cells partially inhibited this increase. We also observed that cell-cell contact increased the formation of actin stress fibers, and that expression of dominant negative RhoA (RhoN19) blocked the contact-mediated increase in stress fibers and proliferation. Furthermore, examination of heterotypic pairs of untreated cells in contact with RhoN19-expressing cells revealed that intracellular, but not intercellular, tension is required for the contact-mediated stimulation of proliferation. Moreover, engagement of VE-cadherin with cadherin-coated beads was sufficient to stimulate proliferation in the absence of actual cell-cell contact. In all, these results demonstrate that cell-cell contact signals through VE-cadherin, RhoA, and intracellular tension in the actin cytoskeleton to regulate proliferation.

Biotechnology: Remote Control of Living Cells

Nature Nanotechnology. Jan, 2008  |  Pubmed ID: 18654443

Emergence of Patterned Stem Cell Differentiation Within Multicellular Structures

Stem Cells (Dayton, Ohio). Nov, 2008  |  Pubmed ID: 18703661

The ability of stem cells to differentiate into specified lineages in the appropriate locations is vital to morphogenesis and adult tissue regeneration. Although soluble signals are important regulators of patterned differentiation, here we show that gradients of mechanical forces can also drive patterning of lineages. In the presence of soluble factors permitting osteogenic and adipogenic differentiation, human mesenchymal stem cells at the edge of multicellular islands differentiate into the osteogenic lineage, whereas those in the center became adipocytes. Interestingly, changing the shape of the multicellular sheet modulated the locations of osteogenic versus adipogenic differentiation. Measuring traction forces revealed gradients of stress that preceded and mirrored the patterns of differentiation, where regions of high stress resulted in osteogenesis, whereas stem cells in regions of low stress differentiated to adipocytes. Inhibiting cytoskeletal tension suppressed the relative degree of osteogenesis versus adipogenesis, and this spatial patterning of differentiation was also present in three-dimensional multicellular clusters. These findings demonstrate a role for mechanical forces in linking multicellular organization to spatial differentials of cell differentiation, and they represent an important guiding principle in tissue patterning that could be exploited in stem cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.

Immobilization of Growth Factors on Collagen Scaffolds Mediated by Polyanionic Collagen Mimetic Peptides and Its Effect on Endothelial Cell Morphogenesis

Biomacromolecules. Oct, 2008  |  Pubmed ID: 18816098

Angiogenesis, a morphogenic event endothelial cells (ECs) undergo in response to 3-D environmental triggers, is critical to the survival and ultimate functional capacity of engineered tissue constructs. Here we present a new collagen mimetic peptide (CMP) architecture consisting of multiple anionic charges at the peptide's N-terminus designed to attract growth factors by charge-charge interactions and bind to collagen by CMP-collagen interaction. The anionic CMPs exhibited specific binding affinity to type I collagen substrates while attracting vascular endothelial growth factors (VEGFs), which led to enhanced morphological features of ECs, indicative of tubulogenesis. The results show that these new CMPs could be used to direct proliferation and differentiation of cells in collagen scaffolds by localization and sustained delivery of growth factors and other morphogens.

Mechanotransduction - a Field Pulling Together?

Journal of Cell Science. Oct, 2008  |  Pubmed ID: 18843115

Mechanical stresses are ever present in the cellular environment, whether through external forces that are applied to tissues or endogenous forces that are generated within the active cytoskeleton. Despite the wide array of studies demonstrating that such forces affect cellular signaling and function, it remains unclear whether mechanotransduction in different contexts shares common mechanisms. Here, I discuss possible mechanisms by which applied forces, cell-generated forces and changes in substrate mechanics could exert changes in cell function through common mechanotransduction machinery. I draw from examples that are primarily focused on the role of adhesions in transducing mechanical forces. Based on this discussion, emerging themes arise that connect these different areas of inquiry and suggest multiple avenues for future studies.

Cell Traction Forces Direct Fibronectin Matrix Assembly

Biophysical Journal. Jan, 2009  |  Pubmed ID: 19167317

Interactions between cells and the surrounding matrix are critical to the development and engineering of tissues. We have investigated the role of cell-derived traction forces in the assembly of extracellular matrix using what we believe is a novel assay that allows for simultaneous measurement of traction forces and fibronectin fibril growth at discrete cell-matrix attachment sites. NIH3T3 cells were plated onto arrays of deformable cantilever posts for 2-24 h. Data indicate that developing fibril orientation is guided by the direction of the traction force applied to that fibril. In addition, cells initially establish a spatial distribution of traction forces that is largest at the cell edge and decreases toward the cell center. This distribution progressively shifts from a predominantly peripheral pattern to a more uniform pattern as compressive strain at the cell perimeter decreases with time. The impact of these changes on fibrillogenesis was tested by treating cells with blebbistatin or calyculin A to tonically block or augment, respectively, myosin II activity. Both treatments blocked the inward translation of traction forces, the dissipation of compressive strain, and fibronectin fibrillogenesis over time. These data indicate that dynamic spatial and temporal changes in traction force and local strain may contribute to successful matrix assembly.

Mechanotransduction in Development: a Growing Role for Contractility

Nature Reviews. Molecular Cell Biology. Jan, 2009  |  Pubmed ID: 19197330

Mechanotransduction research has focused historically on how externally applied forces can affect cell signalling and function. A growing body of evidence suggests that contractile forces that are generated internally by the actomyosin cytoskeleton are also important in regulating cell behaviour, and suggest a broader role for mechanotransduction in biology. Although the molecular basis for these cellular forces in mechanotransduction is being pursued in cell culture, researchers are also beginning to appreciate their contribution to in vivo developmental processes. Here, we examine the role for mechanical forces and contractility in regulating cell and tissue structure and function during development.

Using Self-assembled Monolayers to Pattern ECM Proteins and Cells on Substrates

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19247618

We present a method that uses microcontact printing of alkanethiols on gold to generate patterned substrates presenting "islands" of extracellular matrix (ECM) surrounded by nonadhesive regions such that single cells attach and spread only on the adhesive regions. We have used this micropatterning technology to demonstrate that mammalian cells can be switched between growth and apoptosis programs in the presence of saturating concentrations of growth factors by either promoting or preventing cell spreading (Science 276:1425-1428, 1997). From the perspective of fundamental cell biology, these results suggested that the local differentials in growth and viability that are critical for the formation of complex tissue patterns may be generated by local changes in cell-ECM interactions. In the context of cell culture technologies, such as bioreactors and cellular engineering applications, the regulation of cell function by cell shape indicates that the adhesive microenvironment around cells can be carefully optimized by patterning a substrate in addition to using soluble factors (Biotech. Prog. 14:356-363, 1998). Micropatterning technology is playing a central role both in our understanding how ECM and cell shape regulate cell physiology and in facilitating the development of cellular biosensor and tissue engineering applications (Science 264:696-698, 1994; J. Neurosci. Res. 13:213-20, 1985; Biotech. Bioeng. 43:792-800, 1994).

Cell Polarity Triggered by Cell-cell Adhesion Via E-cadherin

Journal of Cell Science. Apr, 2009  |  Pubmed ID: 19258396

Cell polarity is orchestrated by numerous extracellular cues, and guides events such as chemotaxis, mitosis and wound healing. In scrape-wound assays of cell monolayers, wound-edge cells orient their centrosomes towards the wound, a process that appears to depend on the formation of new cell-extracellular-matrix adhesions as cells spread into the wound. In direct contrast to scrape-wounded cells, isolated cells without cell-cell contacts failed to polarize, suggesting that asymmetry of cell-cell adhesions resulting from monolayer disruption might contribute to polarization. By using micropatterned substrates to engineer such asymmetries in kidney epithelial cells, we found that cell-cell contact induced displacement of the nucleus towards the contact, and also caused centrosomal reorientation and lamellipodial ruffling to the distal side of the nucleus. Upon release from micropatterned constraints, cells exhibited directed migration away from the cell-cell contact. Disrupting E-cadherin engagement randomized nuclear position and lamellipodial ruffling in patterned cultures, and abrogated scrape-wound-induced cell reorientation, but not migration rate. Polarity that was induced by cell-cell contact required an intact actin cytoskeleton and Cdc42 activity, but not RhoA or Rac signaling. Together, these findings demonstrate a novel role for cell-cell adhesion in polarization, and have implications for wound healing and developmental patterning.

Microfabricated Tissue Gauges to Measure and Manipulate Forces from 3D Microtissues

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2009  |  Pubmed ID: 19541627

Physical forces generated by cells drive morphologic changes during development and can feedback to regulate cellular phenotypes. Because these phenomena typically occur within a 3-dimensional (3D) matrix in vivo, we used microelectromechanical systems (MEMS) technology to generate arrays of microtissues consisting of cells encapsulated within 3D micropatterned matrices. Microcantilevers were used to simultaneously constrain the remodeling of a collagen gel and to report forces generated during this process. By concurrently measuring forces and observing matrix remodeling at cellular length scales, we report an initial correlation and later decoupling between cellular contractile forces and changes in tissue morphology. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that cellular forces increased with boundary or matrix rigidity whereas levels of cytoskeletal and extracellular matrix (ECM) proteins correlated with levels of mechanical stress. By mapping these relationships between cellular and matrix mechanics, cellular forces, and protein expression onto a bio-chemo-mechanical model of microtissue contractility, we demonstrate how intratissue gradients of mechanical stress can emerge from collective cellular contractility and finally, how such gradients can be used to engineer protein composition and organization within a 3D tissue. Together, these findings highlight a complex and dynamic relationship between cellular forces, ECM remodeling, and cellular phenotype and describe a system to study and apply this relationship within engineered 3D microtissues.

Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix

Cell Stem Cell. Jul, 2009  |  Pubmed ID: 19570510

A diverse array of environmental factors contributes to the overall control of stem cell activity. In particular, new data continue to mount on the influence of the extracellular matrix (ECM) on stem cell fate through physical interactions with cells, such as the control of cell geometry, ECM geometry/topography at the nanoscale, ECM mechanical properties, and the transmission of mechanical or other biophysical factors to the cell. Here, we review some of the physical processes by which cues from the ECM can influence stem cell fate, with particular relevance to the use of stem cells in tissue engineering and regenerative medicine.

Cytoskeleton-based Forecasting of Stem Cell Lineage Fates

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2010  |  Pubmed ID: 20080726

Stem cells that adopt distinct lineages cannot be distinguished based on traditional cell shape. This study reports that higher-order variations in cell shape and cytoskeletal organization that occur within hours of stimulation forecast the lineage commitment fates of human mesenchymal stem cells (hMSCs). The unique approach captures numerous early (24 h), quantitative features of actin fluororeporter shapes, intensities, textures, and spatial distributions (collectively termed morphometric descriptors). The large number of descriptors are reduced into "combinations" through which distinct subpopulations of cells featuring unique combinations are identified. We demonstrate that hMSCs cultured on fibronectin-treated glass substrates under environments permissive to bone lineage induction could be readily discerned within the first 24 h from those cultured in basal- or fat-inductive conditions by such cytoskeletal feature groupings. We extend the utility of this approach to forecast osteogenic stem cell lineage fates across a series of synthetic polymeric materials of diverse physicochemical properties. Within the first 24 h following stem cell seeding, we could successfully "profile" the substrate responsiveness prospectively in terms of the degree of bone versus nonbone predisposition. The morphometric methodology also provided insights into how substrates may modulate the pace of osteogenic lineage specification. Cells on glass substrates deficient in fibronectin showed a similar divergence of lineage fates, but delayed beyond 48 h. In summary, this high-content imaging and single cell modeling approach offers a framework to elucidate and manipulate determinants of stem cell behaviors, as well as to screen stem cell lineage modulating materials and environments.

Stem Cell Shape Regulates a Chondrogenic Versus Myogenic Fate Through Rac1 and N-cadherin

Stem Cells (Dayton, Ohio). Mar, 2010  |  Pubmed ID: 20082286

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into many cell types. Chondrogenesis is induced in hMSCs cultured as a micromass pellet to mimic cellular condensation during cartilage development, and exposed to transforming growth factor beta (TGFbeta). Interestingly, TGFbeta can also induce hMSC differentiation to smooth-muscle-like cell types, but it remains unclear what directs commitment between these two lineages. Our previous work revealed that cell shape regulates hMSC commitment between osteoblasts and adipocytes through RhoA signaling. Here we show that cell shape also confers a switch between chondrogenic and smooth muscle cell (SMC) fates. Adherent and well-spread hMSCs stimulated with TGF beta 3 upregulated SMC genes, whereas cells allowed to attach onto micropatterned substrates, but prevented from spreading and flattening, upregulated chondrogenic genes. Interestingly, cells undergoing SMC differentiation exhibited little change in RhoA, but significantly higher Rac1 activity than chondrogenic cells. Rac1 activation inhibited chondrogenesis and was necessary and sufficient for inducing SMC differentiation. Furthermore, TGF beta 3 and Rac1 signaling upregulated N-cadherin, which was required for SMC differentiation. These results demonstrate a chondrogenic-SMC fate decision mediated by cell shape, Rac1, and N-cadherin, and highlight the tight coupling between lineage commitment and the many changes in cell shape, cell-matrix adhesion, and cell-cell adhesion that occur during morphogenesis.

Bioactive Hydrogels Made from Step-growth Derived PEG-peptide Macromers

Biomaterials. May, 2010  |  Pubmed ID: 20138664

Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate-PEG-(peptide-PEG)(m)-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate-PEG-CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.

Geometrically Controlled Endothelial Tubulogenesis in Micropatterned Gels

Tissue Engineering. Part A. Jul, 2010  |  Pubmed ID: 20180698

We present a novel approach to control endothelial tubulogenesis by spatially patterning cells within micromolded collagen gels. Endothelial cells cultured within microscale channels that were filled with collagen gel organized into tubes with lumens within 24-48 h of seeding. These tubes extended up to 1 cm in length, and exhibited cell-cell junction formation characteristic of early stage capillary vessels. Tube diameter could be controlled by varying collagen concentrations or channel width. The geometry of the microfabricated template also could be used to guide the development of branches during tube formation, allowing for the generation of more complex capillary architectures. Time-lapse imaging of tube formation revealed a highly dynamic process involving coalescence of endothelial cells, reorganization and alignment of collagen fibers into a central core, and arrangement of cells into cords. This platform may be of use to generate geometrically defined vascular networks for tissue engineering applications as well as a means to better understand the process of endothelial tubulogenesis.

Mechanical Tugging Force Regulates the Size of Cell-cell Junctions

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010  |  Pubmed ID: 20463286

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell-matrix and cell-cell contact. While increased mechanical loading at cell-matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell-cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell-cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell-cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell-cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.

Measuring Mechanical Tension Across Vinculin Reveals Regulation of Focal Adhesion Dynamics

Nature. Jul, 2010  |  Pubmed ID: 20613844

Mechanical forces are central to developmental, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is approximately 2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

Mechanical Regulation of Cell Function with Geometrically Modulated Elastomeric Substrates

Nature Methods. Sep, 2010  |  Pubmed ID: 20676108

We report the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We demonstrated that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility predicted later stem cell fate decisions in single cells.

Decoupling Diffusional from Dimensional Control of Signaling in 3D Culture Reveals a Role for Myosin in Tubulogenesis

Journal of Cell Science. Sep, 2010  |  Pubmed ID: 20682635

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.

Mechanical Forces in Endothelial Cells During Firm Adhesion and Early Transmigration of Human Monocytes

Cellular and Molecular Bioengineering. Mar, 2010  |  Pubmed ID: 20862208

Transmigration of leukocytes across the endothelial barrier is a tightly controlled process involving multiple steps, including rolling adhesion, firm adhesion, and then penetration of leukocytes through the endothelial monolayer. While the key molecular signals have been described in great detail, we are only just beginning to unveil the mechanical forces involved in this process. Here, using a microfabricated system that reports traction forces generated by cells, we describe forces generated by endothelial cells during monocyte firm adhesion and transmigration. Average traction force across the endothelial monolayer increased dramatically when monocytes firmly adhered and transmigrated. Interestingly, the endothelial cell that was in direct contact with the monocyte exhibited much larger traction forces relative to its neighbors, and the direction of these traction forces aligned centripetally with respect to the monocyte. The increase in traction force occurred in the local subcellular zone of monocyte adhesion, and dissipated rapidly with distance. To begin to characterize the basis for this mechanical effect, we show that beads coated with anti-ICAM-1 or VCAM-1 antibodies bound to monolayers could reproduce this effect. Taken together, this study provides a new approach to examining the role of cellular mechanics in regulating leukocyte transmigration through the endothelium.

Micropatterned Dynamically Adhesive Substrates for Cell Migration

Langmuir : the ACS Journal of Surfaces and Colloids. Nov, 2010  |  Pubmed ID: 20886900

We present a novel approach to examine cell migration using dynamically adhesive substrates consisting of three spatially and functionally distinct regions: the first is permanently nonadhesive to cells, the second is permanently adhesive, and the final region is electrochemically switched from nonadhesive to adhesive. We applied a double microcontact printing approach to pattern gold surfaces with carboxylic acid-terminated self-assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that permit adsorption of a nonadhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically "switched" to permit cell migration from a prespecified pattern onto a new pattern. Using these substrates, we investigated the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to characterize how lead cells influence the direction of movement of followers. Time-lapse imaging revealed that, on average, five cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets. This platform may be of use in furthering our understanding of the mechanisms underlying cellular decision-making during migration in both individual and multicellular contexts.

Engineered Materials and the Cellular Microenvironment: a Strengthening Interface Between Cell Biology and Bioengineering

Trends in Cell Biology. Dec, 2010  |  Pubmed ID: 20965727

Cells constantly probe and respond to a myriad of cues that are present in their local surroundings. The effects of soluble cues are relatively straightforward to manipulate, yet teasing apart how cells transduce signals from the extracellular matrix and neighboring cells has proven to be challenging due to the spatially and mechanically complex adhesive interactions. Over the years, advances in the engineering of biocompatible materials have enabled innovative ways to study adhesion-mediated cell functions, and numerous insights have elucidated the significance of the cellular microenvironment. Here, we highlight some of the major approaches and discuss the potential for future advancement.

Measurement of Mechanical Tractions Exerted by Cells in Three-dimensional Matrices

Nature Methods. Dec, 2010  |  Pubmed ID: 21076420

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.

Assaying Stem Cell Mechanobiology on Microfabricated Elastomeric Substrates with Geometrically Modulated Rigidity

Nature Protocols. Feb, 2011  |  Pubmed ID: 21293460

We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different heights to yield substrates of different rigidities. The tips of the elastomeric microposts are functionalized with extracellular matrix through microcontact printing to promote cell adhesion. These substrates, therefore, present the same topographical cues to adherent cells while varying substrate rigidity only through manipulation of micropost height. This protocol describes how to fabricate the silicon micropost array masters (~2 weeks to complete) and elastomeric substrates (3 d), as well as how to perform cell culture experiments (1-14 d), immunofluorescence imaging (2 d), traction force analysis (2 d) and stem cell differentiation assays (1 d) on these substrates in order to examine the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization and differentiation.

Subcellular Spatial Segregation of Integrin Subtypes by Patterned Multicomponent Surfaces

Integrative Biology : Quantitative Biosciences from Nano to Macro. May, 2011  |  Pubmed ID: 21298148

While it is well known that individual integrins are critical mediators of cell behavior, recent work has shown that when multiple types of integrins simultaneously engage the ECM, cell functions are enhanced. However, it is not known how integrins spatially coordinate to regulate cell adhesion because no reliable method exists to segregate integrins on the cell membrane. Here, we use a microcontact printing-based strategy to pattern multiple ECMs that bind distinct integrins in order to study how integrins might interact. In our technique, proteins are first adsorbed uniformly to a poly(dimethyl siloxane) stamp, and then selectively "de-inked." Our strategy overcomes several inherent limitations of conventional microcontact printing, including stamp collapse and limited functionality of the surface patterns. We show that integrins spatially segregate on surfaces patterned with multiple ECMs, as expected. Interestingly, despite spatial segregation of distinct integrins, cells could form adhesions and migrate across multicomponent surfaces as well as they do on single component surfaces. Together, our data indicate that although cells can segregate individual integrins on the cell surface to mediate ECM-specific binding, integrins function cooperatively to guide cell adhesion and migration.

Cell Shape and Substrate Rigidity Both Regulate Cell Stiffness

Biophysical Journal. Mar, 2011  |  Pubmed ID: 21354386

Cells from many different tissues sense the stiffness and spatial patterning of their microenvironment to modulate their shape and cortical stiffness. It is currently unknown how substrate stiffness, cell shape, and cell stiffness modulate or interact with one another. Here, we use microcontact printing and microfabricated arrays of elastomeric posts to independently and simultaneously control cell shape and substrate stiffness. Our experiments show that cell cortical stiffness increases as a function of both substrate stiffness and spread area. For soft substrates, the influence of substrate stiffness on cell cortical stiffness is more prominent than that of cell shape, since increasing adherent area does not lead to cell stiffening. On the other hand, for cells constrained to a small area, cell shape effects are more dominant than substrate stiffness, since increasing substrate stiffness no longer affects cell stiffness. These results suggest that cell size and substrate stiffness can interact in a complex fashion to either enhance or antagonize each other's effect on cell morphology and mechanics.

The Cdc42 Guanine Nucleotide Exchange Factor FGD1 Regulates Osteogenesis in Human Mesenchymal Stem Cells

The American Journal of Pathology. Mar, 2011  |  Pubmed ID: 21356349

Loss of function mutations in FGD1 result in faciogenital dysplasia, an X-linked human developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor that specifically activates Cdc42, a Rho family small GTPase that regulates a variety of cellular behaviors. We have found that FGD1 is expressed in human mesenchymal stem cells (hMSCs) isolated from adult bone marrow. hMSCs are multipotent cells that can differentiate into many cell types, including fibroblasts, osteoblasts, adipocytes, and chondrocytes, and are thought to play a role in maintaining musculoskeletal tissues throughout life. We demonstrate an active role of FGD1 in osteogenic differentiation of hMSCs. During osteogenic differentiation of hMSCs in culture, we observed up-regulation of both FGD1 expression and Cdc42 activity. Activating FGD1/Cdc42 signaling by overexpression of either FGD1 or constitutively active Cdc42 promoted hMSC osteogenesis, while inhibiting Cdc42 signaling by either dominant negative mutants of FGD1 or Cdc42 suppressed osteogenesis. These results demonstrate an important role for FGD1/Cdc42 signaling in hMSC osteogenesis and suggest that the defects in bone remodeling in faciogenital dysplasia may persist throughout adult life and serve as a potential pathway that may be targeted for enhancing bone regeneration.

Measurement and Analysis of Traction Force Dynamics in Response to Vasoactive Agonists

Integrative Biology : Quantitative Biosciences from Nano to Macro. Jun, 2011  |  Pubmed ID: 21445393

Mechanical traction forces exerted by adherent cells on their surroundings serve an important role in a multitude of cellular and physiological processes including cell motility and multicellular rearrangements. For endothelial cells, contraction also provides a means to disrupt cell-cell junctions during inflammation to increase permeability between blood and interstitial tissue compartments. The degree of contractility exhibited by endothelial cells is influenced by numerous soluble factors, such as thrombin, histamine, lysophosphatidic acid, sphingosine-1-phosphate, and vascular endothelial growth factor (VEGF). Upon binding to cell surface receptors, these agents trigger changes in cytoskeletal organization, adhesion and myosin II activity to varying degrees. While conventional antibody-based biochemical assays are suitable for detecting relatively large changes in biomarkers of contractility in an end-point format, they cannot resolve subtle or rapid changes in contractility and cannot do so noninvasively. To overcome these limitations, we developed an approach to measure the contractile response of single cells exposed to contractility agonists with high spatiotemporal resolution. A previously developed traction force sensor, comprised of dense arrays of elastomeric microposts on which cells are cultured, was combined with custom, semi-automated software developed here to extract strain energy measurements from thousands of time-lapse images of micropost arrays deformed by adherent cells. Using this approach we corroborated the differential effects of known agonists of contractility and characterized the dynamics of their effects. All of these agonists produced a characteristic first-order rise and plateau in forces, except VEGF, which stimulated an early transient spike in strain energy followed by a sustained increase. This novel, two-phase contractile response was present in a subpopulation of cells, was mediated through both VEGFR2 and ROCK activation, and its magnitude was modulated by receptor internalization. Interestingly, the concentration of VEGF could shift the proportion of cells that responded with a spike versus only a gradual increase in forces. Furthermore, cells repeatedly exposed to VEGF were found to contract with different dynamics after pretreatment, suggesting that exposure history can impact the mechanical response. These studies highlight the importance of direct measurements of traction force dynamics as a tool for studies of mechanotransduction.

Decreased Cell Adhesion Promotes Angiogenesis in a Pyk2-dependent Manner

Experimental Cell Research. Aug, 2011  |  Pubmed ID: 21640103

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.

A Hitchhiker's Guide to Mechanobiology

Developmental Cell. Jul, 2011  |  Pubmed ID: 21763607

More than a century ago, it was proposed that mechanical forces could drive tissue formation. However, only recently with the advent of enabling biophysical and molecular technologies are we beginning to understand how individual cells transduce mechanical force into biochemical signals. In turn, this knowledge of mechanotransduction at the cellular level is beginning to clarify the role of mechanics in patterning processes during embryonic development. In this perspective, we will discuss current mechanotransduction paradigms, along with the technologies that have shaped the field of mechanobiology.

Repressor Transcription Factor 7-like 1 Promotes Adipogenic Competency in Precursor Cells

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2011  |  Pubmed ID: 21914845

The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit non-adipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell-cell contact with the transcriptional repression required for adipogenic competency.

Bone Morphogenetic Protein-2-Induced Signaling and Osteogenesis Is Regulated by Cell Shape, RhoA/ROCK, and Cytoskeletal Tension

Stem Cells and Development. Oct, 2011  |  Pubmed ID: 21967638

Abstract Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.

Bioresponsive Mesoporous Silica Nanoparticles for Triggered Drug Release

Journal of the American Chemical Society. Dec, 2011  |  Pubmed ID: 21981330

Mesoporous silica nanoparticles (MSNPs) have garnered a great deal of attention as potential carriers for therapeutic payloads. However, achieving triggered drug release from MSNPs in vivo has been challenging. Here, we describe the synthesis of stimulus-responsive polymer-coated MSNPs and the loading of therapeutics into both the core and shell domains. We characterize MSNP drug-eluting properties in vitro and demonstrate that the polymer-coated MSNPs release doxorubicin in response to proteases present at a tumor site in vivo, resulting in cellular apoptosis. These results demonstrate the utility of polymer-coated nanoparticles in specifically delivering an antitumor payload.

Measuring Traction Forces of Motile Dendritic Cells on Micropost Arrays

Biophysical Journal. Dec, 2011  |  Pubmed ID: 22261049

Dendritic cells (DCs) migrate from sites of inflammation to secondary lymphoid organs where they initiate the adaptive immune response. Although motility is essential to DC function, the mechanisms by which they migrate are not fully understood. We incorporated micropost array detectors into a microfluidic gradient generator to develop what we consider to be a novel method for probing low magnitude traction forces during directional migration. We found migration of primary murine DCs is driven by short-lived traction stresses at the leading edge or filopodia. The traction forces generated by DCs are smaller in magnitude than found in neutrophils, and of similar magnitude during chemotaxis and chemokinesis, at 18 ± 1.4 and 16 ± 1.3 nN/cell, respectively. The characteristic duration of local DC traction forces was 3 min. The maximum principal stress in the cell occurred in the plane perpendicular to the axis of motion, forward of the centroid. We illustrate that the spatiotemporal pattern of traction stresses can be used to predict the direction of future DC motion. Overall, DCs show a mode of migration distinct from both mesenchymal cells and neutrophils, characterized by rapid turnover of traction forces in leading filopodia.

MiR-125b is an Adhesion-Regulated MicroRNA That Protects Mesenchymal Stem Cells from Anoikis

Stem Cells (Dayton, Ohio). Feb, 2012  |  Pubmed ID: 22331826

Mesenchymal stem cells (MSCs) have the capacity for multilineage differentiation and are being explored as a source for stem cell-based therapies. Previous studies have shown that adhesion to extracellular matrix plays a critical role in guiding MSC differentiation to distinct lineages. Here we conducted a focused screen of microRNAs to reveal one microRNA, miR-125b, whose expression changes as a function of cell adhesion. miR-125b expression was upregulated by limiting cell-matrix adhesion using micropatterned substrates, knocking down beta5 integrin, or placing cells in suspension culture. Interestingly, we noted that suspending hMSCs did not induce substantial apoptosis (anoikis) as is typically observed in adherent cells. Although miR-125b appeared to have some effects of on hMSC differentiation, we demonstrated a striking role for miR-125b in protecting hMSCs from anoikis. Knockdown of miR-125b increased anoikis while expressing a mimic protected cells. Mechanistic studies demonstrated that miR-125b protected against anoikis by increasing ERK phosphorylation and by suppressing p53. Lastly, we found that miR-125b expression is quite limited in endothelial cells and MEFs. The rapid anoikis normally observed in these cells is antagonized by expressing a miR-125b mimic, and induced pluripotent stem (iPS) cells generated from the MEFs led to upregulated miR-125b expression. Together, these observations demonstrate a novel link between cell-matrix adhesion, miR-125b expression, and a stem-cell specific survival program triggered in adhesion-limited contexts such as might occur in early development and wound healing.

A Microfabricated Platform to Measure and Manipulate the Mechanics of Engineered Cardiac Microtissues

Tissue Engineering. Part A. Jan, 2012  |  Pubmed ID: 22092279

Engineered myocardial tissues can be used to elucidate fundamental features of myocardial biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged heart tissue in vivo. However, a key limitation is an inability to test the wide range of parameters (cell source, mechanical, soluble and electrical stimuli) that might impact the engineered tissue in a high-throughput manner and in an environment that mimics native heart tissue. Here we used microelectromechanical systems technology to generate arrays of cardiac microtissues (CMTs) embedded within three-dimensional micropatterned matrices. Microcantilevers simultaneously constrain CMT contraction and report forces generated by the CMTs in real time. We demonstrate the ability to routinely produce ∼200 CMTs per million cardiac cells (<1 neonatal rat heart) whose spontaneous contraction frequency, duration, and forces can be tracked. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that both the dynamic force of cardiac contraction as well as the basal static tension within the CMT increased with boundary or matrix rigidity. Cell alignment is, however, reduced within a stiff collagen matrix; therefore, despite producing higher force, CMTs constructed from higher density collagen have a lower cross-sectional stress than those constructed from lower density collagen. We also study the effect of electrical stimulation on cell alignment and force generation within CMTs and we show that the combination of electrical stimulation and auxotonic load strongly improves both the structure and the function of the CMTs. Finally, we demonstrate the suitability of our technique for high-throughput monitoring of drug-induced changes in spontaneous frequency or contractility in CMTs as well as high-speed imaging of calcium dynamics using fluorescent dyes. Together, these results highlight the potential for this approach to quantitatively demonstrate the impact of physical parameters on the maturation, structure, and function of cardiac tissue and open the possibility to use high-throughput, low volume screening for studies on engineered myocardium.

Matrix Rigidity Regulates a Switch Between TGF-β1-induced Apoptosis and Epithelial-mesenchymal Transition

Molecular Biology of the Cell. Jan, 2012  |  Pubmed ID: 22238361

The transforming growth factor-β (TGF-β) signaling pathway is often misregulated during cancer progression. In early stages of tumorigenesis, TGF-β acts as a tumor suppressor by inhibiting proliferation and inducing apoptosis. However, as the disease progresses, TGF-β switches to promote tumorigenic cell functions, such as epithelial-mesenchymal transition (EMT) and increased cell motility. Dramatic changes in the cellular microenvironment are also correlated with tumor progression, including an increase in tissue stiffness. However, it is unknown if these changes in tissue stiffness can regulate the effects of TGF-β. To this end, we examined NMuMG and MDCK epithelial cells cultured on polyacrylamide gels with varying rigidity and treated with TGF-β1. Interestingly, changing matrix rigidity switched the functional response to TGF-β1. Decreasing rigidity increased TGF-β1-induced apoptosis, while increasing rigidity resulted in EMT. Matrix rigidity did not change Smad signaling, but instead regulated the PI3K/Akt signaling pathway. Direct genetic and pharmacologic manipulations further demonstrated a role for PI3K/Akt signaling in the apoptotic and EMT responses. These findings demonstrate that matrix rigidity regulates a previously undescribed switch in TGF-β-induced cell functions and provide insight into how changes in tissue mechanics during disease may contribute to the cellular response to TGF-β.

Probing Cellular Traction Forces with Magnetic Nanowires and Microfabricated Force Sensor Arrays

Nanotechnology. Feb, 2012  |  Pubmed ID: 22260885

In this paper, the use of magnetic nanowires for the study of cellular response to force is demonstrated. High-aspect ratio Ni rods with diameter 300 nm and lengths up to 20 μm were bound to or internalized by pulmonary artery smooth muscle cells (SMCs) cultured on arrays of flexible micropost force sensors. Forces and torques were applied to the cells by driving the nanowires with AC magnetic fields in the frequency range 0.1-10 Hz, and the changes in cellular contractile forces were recorded with the microposts. These local stimulations yield global force reinforcement of the cells' traction forces, but this contractile reinforcement can be effectively suppressed upon addition of a calcium channel blocker, ruthenium red, suggesting the role of calcium channels in the mechanical response. The responsiveness of the SMCs to actuation depends on the frequency of the applied stimulation. These results show that the combination of magnetic nanoparticles and micropatterned, flexible substrates can provide new approaches to the study of cellular mechanotransduction.