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Articles by Adam J. Engler in JoVE

 JoVE Bioengineering

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 2Biomedical Sciences Program, University of California, San Diego, 3Department of Bioengineering, University of California, San Diego


JoVE 50018

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Other articles by Adam J. Engler on PubMed

Patterning, Prestress, and Peeling Dynamics of Myocytes

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, V(peel), was measured as a continuously increasing function of the imposed tension, T(peel), which ranges from approximately 0 to 50 nN/ micro m. For each cell, peeling proved highly heterogeneous, with V(peel) fluctuating between 0 micro m/s ( approximately 80% of time) and approximately 10 micro m/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.

Targeted Worm Micelles

Giant and stable worm micelles formed from poly(ethylene glycol) (PEG)-based diblock copolymer amphiphiles have the potential advantage compared to smaller assemblies for delivery of a large quantity of hydrophobic drugs or dyes per carrier. Here we show that worm micelles can be targeted to cells with internalization and delivery of nontoxic dyes as well as cytotoxic drugs. Constituent copolymers are end-biotinylated to mediate high affinity binding of worm micelles to both avidin-bearing surfaces and biotin-specific receptors on smooth muscle cells. Pristine worm micelles, that lack biotin, show much less frequent and nonspecific point attachments to the same surfaces. Biotinylated worm micelles prove stable in aqueous solution for at least a month and also prove capable of loading, retaining, and delivering hydrophobic dyes and drugs. The results thus demonstrate the feasibility of targeted delivery by polymeric worm micelles.

Elasticity of Native and Cross-linked Polyelectrolyte Multilayer Films

Mechanical properties of polyelectrolyte multilayer films were studied by nanoindentation using the atomic force microscope (AFM). Force-distance measurements using colloidal probe tips were systematically obtained for supported films of poly(L-lysine) and hyaluronan that are suited to bio-application. Both native and covalently cross-linked films were studied as a function of increasing layer number, which increases film thickness. The effective Young's modulus perpendicular to the film, Eperpendicular, was determined to be a function of film thickness, cross-linking, and sample age. Thick PEM films exhibited a lower Eperpendicular than thinner PEM, whereas the Young's modulus of cross-linked films was more than 10-fold larger than native films. Moduli range from approximately 20 kPa for native films up to approximately 800 kPa for cross-linked ones. Young's moduli increased slightly with sample age, plateauing after approximately 4 weeks. Spreading of smooth muscle cells on these substrates with pre-attached collagen proved to be highly dependent on film rigidity with stiffer films giving greater cell spreading.

Myotubes Differentiate Optimally on Substrates with Tissue-like Stiffness: Pathological Implications for Soft or Stiff Microenvironments

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E approximately 12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.

Power-law Rheology of Isolated Nuclei with Deformation Mapping of Nuclear Substructures

Force-induced changes in genome expression as well as remodeling of nuclear architecture in development and disease motivate a deeper understanding of nuclear mechanics. Chromatin and green fluorescent protein-lamin B dynamics were visualized in a micropipette aspiration of isolated nuclei, and both were shown to contribute to viscoelastic properties of the somatic cell nucleus. Reversible swelling by almost 200% in volume, with changes in salt, demonstrates the resilience and large dilational capacity of the nuclear envelope, nucleoli, and chromatin. Swelling also proves an effective way to separate the mechanical contributions of nuclear elements. In unswollen nuclei, chromatin is a primary force-bearing element, whereas swollen nuclei are an order of magnitude softer, with the lamina sustaining much of the load. In both cases, nuclear deformability increases with time, scaling as a power law-thus lacking any characteristic timescale-when nuclei are either aspirated or indented by atomic force microscopy. The nucleus is stiff and resists distortion at short times, but it softens and deforms more readily at longer times. Such results indicate an essentially infinite spectrum of timescales for structural reorganization, with implications for regulating genome expression kinetics.

Mesenchymal Stem Cell Injection After Myocardial Infarction Improves Myocardial Compliance

Cellular therapy for myocardial injury has improved ventricular function in both animal and clinical studies, though the mechanism of benefit is unclear. This study was undertaken to examine the effects of cellular injection after infarction on myocardial elasticity. Coronary artery ligation of Lewis rats was followed by direct injection of human mesenchymal stem cells (MSCs) into the acutely ischemic myocardium. Two weeks postinfarct, myocardial elasticity was mapped by atomic force microscopy. MSC-injected hearts near the infarct region were twofold stiffer than myocardium from noninfarcted animals but softer than myocardium from vehicle-treated infarcted animals. After 8 wk, the following variables were evaluated: MSC engraftment and left ventricular geometry by histological methods, cardiac function with a pressure-volume conductance catheter, myocardial fibrosis by Masson Trichrome staining, vascularity by immunohistochemistry, and apoptosis by TdT-mediated dUTP nick-end labeling assay. The human cells engrafted and expressed a cardiomyocyte protein but stopped short of full differentiation and did not stimulate significant angiogenesis. MSC-injected hearts showed significantly less fibrosis than controls, as well as less left ventricular dilation, reduced apoptosis, increased myocardial thickness, and preservation of systolic and diastolic cardiac function. In summary, MSC injection after myocardial infarction did not regenerate contracting cardiomyocytes but reduced the stiffness of the subsequent scar and attenuated postinfarction remodeling, preserving some cardiac function. Improving scarred heart muscle compliance could be a functional benefit of cellular cardiomyoplasty.

A Hemoglobin Fragment Found in Cervicovaginal Fluid from Women in Labor Potentiates the Action of Agents That Promote Contraction of Smooth Muscle Cells

We employed a proteomic approach to search for peptides that have a physiological role in labor. Cervicovaginal secretions were collected at term from women in labor and women at term not in labor. Samples were spotted onto weak cation exchange chips (WCX-2) and analyzed using Surface-Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). Spectra were obtained for each sample and Biomarker Wizard analysis revealed 25 peaks that had significantly different peak intensity between the labor and non-laboring women. The sequences of five peaks that were significantly elevated in the labor cohort were determined using Protein Chip Interface Quadruple Time-of-Flight Mass Spectrometry (PCI-QTOF-MS). All of these peaks were identified as fragments of alpha or beta-hemoglobin (Hb). A 2.022 kDa fragment of alpha-Hb (amino acids 110-128, NH2-AAHLPAEFTPAVHASLDKF-COOH) was found to potentiate smooth muscle cell contraction in response to bradykinin, oxytocin and prostaglandin-F2alpha. This peptide may promote vasoconstriction and augment normal labor through enhancing the action of uterotonins.

Matrix Elasticity Directs Stem Cell Lineage Specification

Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.

Microtissue Elasticity: Measurements by Atomic Force Microscopy and Its Influence on Cell Differentiation

It is increasingly appreciated that the mechanical properties of the microenvironment around cells exerts a significant influence on cell behavior, but careful consideration of what is the physiologically relevant elasticity for specific cell types is required to produce results that meaningfully recapitulate in vivo development. Here we outline methodologies for excising and characterizing the effective microelasticity of tissues; but first we describe and validate an atomic force microscopy (AFM) method as applied to two comparatively simple hydrogel systems. With tissues and gels sufficiently understood, the latter can be appropriately tuned to mimic the desired tissue microenvironment for a given cell type. The approach is briefly illustrated with lineage commitment of stem cells due to matrix elasticity.

Cell Responses to the Mechanochemical Microenvironment--implications for Regenerative Medicine and Drug Delivery

Soft-tissue cells are surprisingly sensitive to the elasticity of their microenvironment, suggesting that traditional culture plastic and glass are less relevant to tissue regeneration and chemotherapeutics than might be achieved. Cells grown on gels that mimic the elasticity of tissue reveal a significant influence of matrix elasticity on adhesion, cytoskeletal organization, and even the differentiation of human adult derived stem cells. Cellular forces and feedback are keys to how cells feel their mechanical microenvironment, but detailed molecular mechanisms are still being elucidated. This review summarizes our initial findings for multipotent stem cells and also the elasticity-coupled effects of drugs on cancer cells and smooth muscle cells. The drugs include the contractility inhibitor blebbistatin, the proliferation inhibitor mitomycin C, an apoptotis-inducing antibody against CD47, and the translation inhibitor cycloheximide. The differential effects not only lend insight into mechano-sensing of the substrate by cells, but also have important implications for regeneration and molecular therapies.

Fibronectin Expression Modulates Mammary Epithelial Cell Proliferation During Acinar Differentiation

The mammary gland consists of a polarized epithelium surrounded by a basement membrane matrix that forms a series of branching ducts ending in hollow, sphere-like acini. Essential roles for the epithelial basement membrane during acinar differentiation, in particular laminin and its integrin receptors, have been identified using mammary epithelial cells cultured on a reconstituted basement membrane. Contributions from fibronectin, which is abundant in the mammary gland during development and tumorigenesis, have not been fully examined. Here, we show that fibronectin expression by mammary epithelial cells is dynamically regulated during the morphogenic process. Experiments with synthetic polyacrylamide gel substrates implicate both specific extracellular matrix components, including fibronectin itself, and matrix rigidity in this regulation. Alterations in fibronectin levels perturbed acinar organization. During acinar development, increased fibronectin levels resulted in overproliferation of mammary epithelial cells and increased acinar size. Addition of fibronectin to differentiated acini stimulated proliferation and reversed growth arrest of mammary epithelial cells negatively affecting maintenance of proper acinar morphology. These results show that expression of fibronectin creates a permissive environment for cell growth that antagonizes the differentiation signals from the basement membrane. These effects suggest a link between fibronectin expression and epithelial cell growth during development and oncogenesis in the mammary gland.

Embryonic Cardiomyocytes Beat Best on a Matrix with Heart-like Elasticity: Scar-like Rigidity Inhibits Beating

Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A 'cysteine shotgun' method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development.

Stem Cell Fate Dictated Solely by Altered Nanotube Dimension

Two important goals in stem cell research are to control the cell proliferation without differentiation and to direct the differentiation into a specific cell lineage when desired. Here, we demonstrate such paths by controlling only the nanotopography of culture substrates. Altering the dimensions of nanotubular-shaped titanium oxide surface structures independently allowed either augmented human mesenchymal stem cell (hMSC) adhesion or a specific differentiation of hMSCs into osteoblasts by using only the geometric cues, absent of osteogenic inducing media. hMSC behavior in response to defined nanotube sizes revealed a very dramatic change in hMSC behavior in a relatively narrow range of nanotube dimensions. Small (approximately 30-nm diameter) nanotubes promoted adhesion without noticeable differentiation, whereas larger (approximately 70- to 100-nm diameter) nanotubes elicited a dramatic stem cell elongation (approximately 10-fold increased), which induced cytoskeletal stress and selective differentiation into osteoblast-like cells, offering a promising nanotechnology-based route for unique orthopedics-related hMSC treatments.

Multiscale Modeling of Form and Function

Topobiology posits that morphogenesis is driven by differential adhesive interactions among heterogeneous cell populations. This paradigm has been revised to include force-dependent molecular switches, cell and tissue tension, and reciprocal interactions with the microenvironment. It is now appreciated that tissue development is executed through conserved decision-making modules that operate on multiple length scales from the molecular and subcellular level through to the cell and tissue level and that these regulatory mechanisms specify cell and tissue fate by modifying the context of cellular signaling and gene expression. Here, we discuss the origin of these decision-making modules and illustrate how emergent properties of adhesion-directed multicellular structures sculpt the tissue, promote its functionality, and maintain its homeostasis through spatial segregation and organization of anchored proteins and secreted factors and through emergent properties of tissues, including tension fields and energy optimization.

A Novel Mode of Cell Detachment from Fibrillar Fibronectin Matrix Under Shear

Cells within tissues are surrounded by fibrillar extracellular matrix (ECM) that supports cell adhesion via integrin receptors. The strength of cell interactions with fibrillar matrix and the effects of force on these interactions have not been quantified. To this end, we used a spinning disc device to apply radially increasing shear to human HT1080 fibrosarcoma cells attached to a cell-derived fibrillar fibronectin (FN) matrix. The shear required to detach 50% of HT1080 cells was eight times greater on a FN-coated, rigid glass substrate than on fibrillar FN matrix. Covalent crosslinking of the FN matrix increased its stiffness tenfold and produced a modest increase in shear detachment force for these cells. On FN-coated surfaces, cells detach by releasing interactions between alpha5beta1 integrin and FN. By contrast, cell detachment from fibrillar matrix occurred through a novel mechanism of fibril breakage, which left holes in the matrix visible by fluorescence microscopy. These results show that cells require less force to detach from fibrillar matrix than from FN adsorbed on glass and that detachment occurs through breaking fibrils instead of by release of integrin-matrix bonds. Thus, ECM fibril breakage is another molecular feature to consider when understanding cell and tissue homeostasis.

Matrix Strains Induced by Cells: Computing How Far Cells Can Feel

Many tissue cells exert contractile forces that mechanically couples them to elastic matrices and that influence cell adhesion, cytoskeletal organization, and even cell differentiation. However, strains within the depths of matrices are often unclear and are likely relevant not only to the fact that some matrices such as so-called basement membranes are thin relative to cell dimensions but also to defining how far cells can 'feel'. Here we briefly present experimental results for cell spreading on thin, ligand-coated gels and for prestress in stem cells in relation to gel stiffness. We then introduce a finite element computation in which a cell is placed on an elastic matrix, while matrix elasticity and thickness are varied in order to compute and compare elastostatic deformations within the matrix. Average interfacial strains between cell and matrix show large deviations only when soft matrices are a fraction of the height and width of a cell, proving consistent with experiments. Three-dimensional (3D) cell morphologies that model stem cell-derived neurons, myoblasts, and osteoblasts show that a cylinder-shaped myoblast induces the highest strains, consistent with the prominent contractility of muscle. Groups of such cells show a weak crosstalk in matrix strains, but the cells must be much closer than a cell-width. Cells thus feel on length scales closer to that of adhesions than on cellular scales or higher.

Intrinsic Extracellular Matrix Properties Regulate Stem Cell Differentiation

One of the recent paradigm shifts in stem cell biology has been the discovery that stem cells can begin to differentiate into mature tissue cells when exposed to intrinsic properties of the extracellular matrix (ECM), such as matrix structure, elasticity, and composition. These parameters are known to modulate the forces a cell can exert upon its matrix. Mechano-sensitive pathways subsequently convert these biophysical cues into biochemical signals that commit the cell to a specific lineage. Just as with well-studied growth factors, ECM parameters are extremely dynamic and are spatially- and temporally-controlled during development, suggesting that they play a morphogenetic role in guiding differentiation and arrangement of cells. Our ability to dynamically regulate the stem cell niche as the body does is likely a critical requirement for developing differentiated cells from stem cells for therapeutic applications. Here, we present the emergence of stem cell mechanobiology and its future challenges with new biomimetic, three-dimensional scaffolds that are being used therapeutically to treat disease.

Innovations in Cell Mechanobiology

Cell Rheology: Stressed-out Stem Cells

Preparation of Hydrogel Substrates with Tunable Mechanical Properties

The modulus of elasticity of the extracellular matrix (ECM), often referred to in a biological context as "stiffness," naturally varies within the body, e.g., hard bones and soft tissue. Moreover, it has been found to have a profound effect on the behavior of anchorage-dependent cells. The fabrication of matrix substrates with a defined modulus of elasticity can be a useful technique to study the interactions of cells with their biophysical microenvironment. Matrix substrates composed of polyacrylamide hydrogels have an easily quantifiable elasticity that can be changed by adjusting the relative concentrations of its monomer, acrylamide, and cross-linker, bis-acrylamide. In this unit, we detail a protocol for the fabrication of statically compliant and radial-gradient polyacrylamide hydrogels, as well as the functionalization of these hydrogels with ECM proteins for cell culture. Included as well are suggestions to optimize this protocol to the choice of cell type or stiffness with a table of relative bis-acrylamide and acrylamide concentrations and expected elasticity after polymerization.

Nanoscopic Mechanical Anisotropy in Hydrogel Surfaces

The bulk mechanical properties of soft materials have been studied widely, but it is unclear to what extent macroscopic behavior is reflected in nanomechanics. Using an atomic force microscopy (AFM) imaging method called force spectroscopy mapping (FSM), it is possible to map the nanoscopic spatial distribution of Young's modulus, i.e. "stiffness," and determine if soft or stiff polymer domains exist to correlate nano- and macro-mechanics. Two model hydrogel systems typically used in cell culture and polymerized by a free radical polymerization process, i.e. poly (vinyl pyrrolidone) (PVP) and poly(acrylamide) (PAam) hydrogels, were found to have significantly different nanomechanical behavior despite relatively similar bulk stiffness and roughness. PVP gels contained a large number of soft and stiff nanodomains, and their size was inversely related to crosslinking density and changes in crosslinking efficiency within the hydrogel. In contrast, PAam gels displayed small nanodomains occuring at low frequency, indicating relatively uniform polymerization. Given the responsiveness of cells to changes in gel stiffness, inhomogeneities found in the PVP network indicate that careful nanomechanical characterization of polymer substrates is necessary to appreciate complex cell behavior.

Detecting Cell-adhesive Sites in Extracellular Matrix Using Force Spectroscopy Mapping

The cell microenvironment is composed of extracellular matrix (ECM), which contains specific binding sites that allow the cell to adhere to its surroundings. Cells employ focal adhesion proteins, which must be able to resist a variety of forces to bind to ECM. Current techniques for detecting the spatial arrangement of these adhesions, however, have limited resolution and those that detect adhesive forces lack sufficient spatial characterization or resolution. Using a unique application of force spectroscopy, we demonstrate here the ability to determine local changes in the adhesive property of a fibronectin substrate down to the resolution of the fibronectin antibody-functionalized tip diameter, ~20 nm. To verify the detection capabilities of force spectroscopy mapping (FSM), changes in loading rate and temperature were used to alter the bond dynamics and change the adhesion force. Microcontact printing was also used to pattern fluorescein isothiocyanate-conjugated fibronectin in order to mimic the discontinuous adhesion domains of native ECM. Fluorescent detection was used to identify the pattern while FSM was used to map cell adhesion sites in registry with the initial fluorescent image. The results show that FSM can be used to detect the adhesion domains at high resolution and may subsequently be applied to native ECM with randomly distributed cell adhesion sites.

Hydrogels with Time-dependent Material Properties Enhance Cardiomyocyte Differentiation in Vitro

Tissue-specific elastic modulus (E), or 'stiffness,' arises from developmental changes in the extracellular matrix (ECM) and suggests that progenitor cell differentiation may be optimal when physical conditions mimic tissue progression. For cardiomyocytes, maturing from mesoderm to adult myocardium results in a 9-fold stiffening originating in part from a change in collagen expression and localization. To mimic this temporal stiffness change in vitro, thiolated-hyaluronic acid (HA) hydrogels were crosslinked with poly(ethylene glycol) diacrylate, and their dynamics were modulated by changing crosslinker molecular weight. With the hydrogel appropriately tuned to stiffen as heart muscle does during development, pre-cardiac cells grown on collagen-coated HA hydrogels exhibit a 3-fold increase in mature cardiac specific markers and form up to 60% more maturing muscle fibers than they do when grown on compliant but static polyacrylamide hydrogels over 2 weeks. Though ester hydrolysis does not substantially alter hydrogel stiffening over 2 weeks in vitro, model predictions indicate that ester hydrolysis will eventually degrade the material with additional time, implying that this hydrogel may be appropriate for in vivo applications where temporally changing material properties enhance cell maturation prior to its replacement with host tissue.

Stiffness Gradients Mimicking in Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate

Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ∼8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ∼0.6±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior.

Controlling Polymersome Surface Topology at the Nanoscale by Membrane Confined Polymer/polymer Phase Separation

Nature has the exquisite ability to design specific surface patterns and topologies on both the macro- and nanolength scales that relate to precise functions. Following a biomimetic approach, we have engineered fully synthetic nanoparticles that are able to self-organize their surface into controlled domains. We focused on polymeric vesicles or "polymersomes"; enclosed membranes formed via self-assembly of amphiphilic block copolymers in water. Exploiting the intrinsic thermodynamic tendency of dissimilar polymers to undergo phase separation, we mixed different vesicle-forming block copolymers in various proportions in order to obtain a wide range of polymersomes with differing surface domains. Using a combination of confocal laser scanning microscopy studies of micrometer-sized polymersomes, and electron microscopy, atomic force microscopy, and fluorescence spectroscopy on nanometer-sized polymersomes, we find that the domains exhibit similar shapes on both the micro- and nanolength scales, with dimensions that are linearly proportional to the vesicle diameter. Finally, we demonstrate that such control over the surface "patchiness" of these polymersomes determines their cell internalization kinetics for live cells.

More Than a Feeling: Discovering, Understanding, and Influencing Mechanosensing Pathways

The ability of cells to extract biophysical information from their extracellular environment and convert it to biochemical signals is known as mechanotransduction. Here we detail three passive, 'inside-out' mechanotransduction mechanisms with an emphasis on the mechanosensing pathways involved in creating these signal: Rho/ROCK, stretch-activated channels, and 'Molecular Strain Gauges.' We also examine how molecular tools have been used to perturb these pathways to better understand their interconnectivity. However, perturbing pathways may have unintended confounding effects, which must also be addressed. By discovering and understanding mechanosensitive pathways, the ability to influence them for clinical applications increases.

Wet Nanoscale Imaging and Testing of Polymersomes

In Situ Mechanical Analysis of Myofibrillar Perturbation and Aging on Soft, Bilayered Drosophila Myocardium

Drosophila melanogaster is a genetically malleable organism with a short life span, making it a tractable system in which to study mechanical effects of genetic perturbation and aging on tissues, e.g., impaired heart function. However, Drosophila heart-tube studies can be hampered by its bilayered structure: a ventral muscle layer covers the contractile cardiomyocytes. Here we propose an atomic force microscopy-based analysis that uses a linearized-Hertz method to measure individual mechanical components of soft composite materials. The technique was verified using bilayered polydimethylsiloxane. We further demonstrated its biological utility via its ability to resolve stiffness changes due to RNA interference to reduce myofibrillar content or due to aging in Drosophila myocardial layers. This protocol provides a platform to assess the mechanics of soft biological composite systems and, to our knowledge, for the first time, permits direct measurement of how genetic perturbations, aging, and disease can impact cardiac function in situ.

Mechanical Derivation of Functional Myotubes from Adipose-derived Stem Cells

Though reduced serum or myoblast co-culture alone can differentiate adipose-derived stem cells (ASCs) into mesenchymal lineages, efficiency is usually not sufficient to restore function in vivo. Often when injected into fibrotic muscle, their differentiation may be misdirected by the now stiffened tissue. Here ASCs are shown to not just simply reflect the qualitative stiffness sensitivity of bone marrow-derived stem cells (BMSCs) but to exceed BMSC myogenic capacity, expressing the appropriate temporal sequence of muscle transcriptional regulators on muscle-mimicking extracellular matrix in a tension and focal adhesion-dependent manner. ASCs formed multi-nucleated myotubes with a continuous cytoskeleton that was not due to misdirected cell division; microtubule depolymerization severed myotubes, but after washout, ASCs refused at a rate similar to pre-treated values. BMSCs never underwent stiffness-mediated fusion. ASC-derived myotubes, when replated onto non-permissive stiff matrix, maintained their fused state. Together these data imply enhanced mechanosensitivity for ASCs, making them a better therapeutic cell source for fibrotic muscle.

Measuring Passive Myocardial Stiffness in Drosophila Melanogaster to Investigate Diastolic Dysfunction

Aging is marked by a decline in left ventricular diastolic function, which encompasses abnormalities in diastolic relaxation, chamber filling and/or passive myocardial stiffness. Genetic tractability and short life span make Drosophila melanogaster an ideal organism to study the effects of aging on heart function, including senescent-associated changes in gene expression and in passive myocardial stiffness. However, use of the Drosophila heart tube to probe deterioration of diastolic performance is subject to at least two challenges: the extent of genetic homology to mammals and the ability to resolve mechanical properties of the bilayered fly heart, which consists of a ventral muscle layer that covers the contractile cardiomyocytes. Here we argue for wide-spread use of Drosophila as a novel myocardial aging model by 1) describing diastolic dysfunction in flies, 2) discussing how critical pathways involved in dysfunction are conserved across species, and 3) demonstrating the advantage of an atomic force microscopy-based analysis method to measure stiffness of the multilayered Drosophila heart tube versus isolated myocytes from other model systems. By using powerful Drosophila genetic tools we aim to efficiently alter changes observed in factors that contribute to diastolic dysfunction to understand how one might improve diastolic performance at advanced ages in humans. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

The Alignment and Fusion Assembly of Adipose-derived Stem Cells on Mechanically Patterned Matrices

Cell patterning is typically accomplished by selectively depositing proteins for cell adhesion only on patterned regions; however in tissues, cells are also influenced by mechanical stimuli, which can also result in patterned arrangements of cells. We developed a mechanically-patterned hydrogel to observe and compare it to extracellular matrix (ECM) ligand patterns to determine how to best regulate and improve cell type-specific behaviors. Ligand-based patterning on hydrogels was not robust over prolonged culture, but cells on mechanically-patterned hydrogels differentially sorted based on stiffness preference: myocytes and adipose-derived stem cells (ASCs) underwent stiffness-mediated migration, i.e. durotaxis, and remained on myogenic hydrogel regions. Myocytes developed aligned striations and fused on myogenic stripes of the mechanically-patterned hydrogel. ASCs aligned and underwent myogenesis, but their fusion rate increased, as did the number of cells fusing into a myotube as a result of their alignment. Conversely, neuronal cells did not exhibit durotaxis and could be seen on soft regions of the hydrogel for prolonged culture time. These results suggest that mechanically-patterned hydrogels could provide a platform to create tissue engineered, innervated micro-muscles of neural and muscle phenotypes juxtaposed next to each other in order better recreate a muscle niche.

Control of Stem Cell Fate and Function by Engineering Physical Microenvironments

The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell–cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niche environments and regulate stem cell fate and function.

Cell Instructive Microporous Scaffolds Through Interface Engineering

The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil-water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner.

Dynamic and Reversible Surface Topography Influences Cell Morphology

Microscale and nanoscale surface topography changes can influence cell functions, including morphology. Although in vitro responses to static topography are novel, cells in vivo constantly remodel topography. To better understand how cells respond to changes in topography over time, we developed a soft polyacrylamide hydrogel with magnetic nickel microwires randomly oriented in the surface of the material. Varying the magnetic field around the microwires reversibly induced their alignment with the direction of the field, causing the smooth hydrogel surface to develop small wrinkles; changes in surface roughness, ΔR(RMS) , ranged from 0.05 to 0.70 μm and could be oscillated without hydrogel creep. Vascular smooth muscle cell morphology was assessed when exposed to acute and dynamic topography changes. Area and shape changes occurred when an acute topographical change was imposed for substrates exceeding roughness of 0.2 μm, but longer-term oscillating topography did not produce significant changes in morphology irrespective of wire stiffness. These data imply that cells may be able to use topography changes to transmit signals as they respond immediately to changes in roughness. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Mesenchymal Stem Cell Durotaxis Depends on Substrate Stiffness Gradient Strength

Mesenchymal stem cells (MSCs) respond to niche elasticity, which varies between and within tissues. Stiffness gradients result from pathological conditions but also occur through normal variation, e.g. muscle. MSCs undergo directed migration even in response to shallow stiffness gradients before differentiating. More refined gradients of both stiffness range and strength are needed to better understand mechanical regulation of migration in normal and disease pathologies. We describe polyacrylamide stiffness gradient fabrication using three distinct systems that generate stiffness gradients of physiological (1 Pa/μm), pathological (10 Pa/μm), and step (≥ 100 Pa/μm) strength spanning physiologically relevant stiffness for most soft tissue, i.e. 1-12 kPa. MSCs migrated to the stiffest region for each gradient. Time-lapse microscopy revealed that migration velocity scaled directly with gradient strength. Directed migration was reduced in the presence of the contractile agonist lysophosphatidic acid (LPA) and cytoskeletal-perturbing drugs nocodazole and cytochalasin; LPA- and nocodazole-treated cells remained spread and protrusive, while cytochalasin-treated cells did not. Untreated and nocodazole-treated cells spread in a similar manner, but nocodazole-treated cells had greatly diminished traction forces. These data suggest that actin is required for migration whereas microtubules are required for directed migration. The data also imply that in vivo, MSCs may have a more significant contribution to repairs in stiffer regions where they may preferentially accumulate.

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