Evelyn K.F. Yim

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Youngs moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Effects of nanoimprinted patterns in tissue-culture polystyrene on cell behavior.

Tissue engineering seeks to develop functional tissues in a biomimetic environment in vitro. As the extracellular environment in vivo is composed of numerous nanostructures, fabrication of nanostructured substrates will be valuable for tissue engineering applications. In this article, we report a simple nanoimprint lithography (NIL) process to pattern nanostructures directly on tissue-culture polystyrene plates. By repeating this NIL process, three-dimensional scaffolds consisting of multiple-layer nanostructures were also fabricated. Bovine pulmonary artery smooth muscle cells were cultured on imprinted gratings ranging from 350 nm to 10 μm. The smooth muscle cells attached and proliferated well on these imprinted substrates without additional surface treatment. Cell elongation and alignment were observed on the micro- and nanopatterns, with the effect significantly more pronounced on the nanostructures.

In Vitro Topographical Model of Fuchs Dystrophy for Evaluation of Corneal Endothelial Cell Monolayer Formation

A common indication for corneal transplantation, which is the most transplanted tissue, is a dysfunctional corneal endothelium due to Fuchs endothelial dystrophy (FED). FED is diagnosed by the presence of in vivo pathological microtopography on the Descemet membrane, which is called corneal guttata. Minimally invasive corneal endothelial cell regenerative procedures such as endothelial cell injection therapy and Rho kinase inhibitor pharmacotherapy have been proposed as alternatives to conventional corneal transplantation for FED patients. However, the effect of guttata on monolayer reformation following such therapies is unknown and there is no equivalent in vitro or animal model to study monolayer reformation. Using a synthetic guttata FED disease model, the formation of the monolayer is investigated to evaluate the efficacy of both therapies. Results obtained suggest that guttata dimensions, density, and spacing greatly affect the fate of corneal endothelial cells in terms of migratory behavior and monolayer reformation. Densely packed synthetic guttata mimicking late-stage FED hinders monolayer reformation, while synthetic guttata of lower height and density show improved monolayer formation. These results suggest that severity of the FED, as determined by height and density of existing guttata, can potentially attenuate corneal endothelial monolayer formation of corneal cell injection therapy and pharmacotherapy.

Cell–Substrate Interactions

Abstract In most cases, tissue engineering inxa0vitro or inxa0vivo requires a scaffold to provide the optimal microenvironment for seeded cells. There is a growing trend toward using synthetic substrates to mimic natural, physiological systems for tissue engineering or regenerative medicine. Cell–substrate interactions are fundamentally important to studies geared toward designing biomimetic substrates that may replace damaged vital organs or tissues or assist in the natural healing processes of the body. This chapter reviews cell interactions with the extracellular matrix. This information is followed by sections detailing the modification of cell behavior by different aspects of a biomimetic substrate, such as its physical, chemical, and biological properties. The role of surface topography in modulating cell interactions is also discussed. Many studies have underscored the necessity of a three-dimensional environment to yield physiologically relevant data; thus, a section is dedicated to the effect of dimensionality on cell behavior. The chapter concludes with a discussion on the importance of mechanical stress in tissue development at both the cellular and tissue levels.

Contribution of actin filaments and microtubules to cell elongation and alignment depends on the grating depth of microgratings.

BackgroundIt has been reported that both chemical and physical surface patterns influence cellular behaviors, such as cell alignment and elongation. However, it still remains unclear how actin filament and microtubules (MTs) differentially respond to these patterns.ResultsWe examined the effects of chemical and physical patterns on cell elongation and alignment by observing actin filament and MTs of retinal pigment epithelium-1(RPE-1) cells, which were cultured on either fibronectin (FN)-line pattern (line width and spacing: 1xa0μm) or FN-coated 1xa0μm gratings with two different depths (0.35 or 1xa0μm). On the surface with either FN-line pattern or micrograting structure, the cell aspect ratios were at least two times higher than those on the surface with no pattern. Cell elongation on the gratings depended on the depth of the gratings. Cell elongation and alignment on both FN-line pattern and 1xa0μm gratings with 0.35xa0μm depth were perturbed either by inhibition of actin polymerization or MT depletion, while cell elongation and alignment on 1xa0μm gratings with 1xa0μm depth were perturbed only by MT depletion.ConclusionsOur results suggest that the contribution of actin filaments and MTs to the elongation and alignment of epithelial cells on microgratings depends on the groove depth of these gratings.

Molecular organization of integrin-based adhesion complexes in mouse Embryonic Stem Cells

The mechanical microenvironment serves as an important factor influencing stem cell differentiation. Mechanobiological responses depend strongly on actomyosin contractility and integrin-based cell-extracellular matrix (ECM) interactions mediated by adhesive structures such as focal adhesions (FAs). While the roles of FAs in mechanobiology have been intensively studied in many mesenchymal and migratory cell types, recently it has been recognized that certain pluripotent stem cells (PSCs) exhibited significantly attenuated FA-mediated mechanobiological responses. FAs in such PSCs are sparsely distributed and much less prominent in comparison to “classical” FAs of typical adherent cells. Despite these differences, insights into how FAs in PSCs are structurally organized to perform their functions are still elusive. Using mouse embryonic stem cells (mESCs) to study PSC-ECM interactions, here we surveyed the molecular composition and nanostructural organization of FAs. We found that despite being small in size, mESC FAs appeared to be compositionally mature, containing markers such as vinculin, zyxin, and α-actinin, and dependent on myosin II contractility. Using super-resolution microscopy, we revealed that mESC FAs were organized into a conserved multilayer nanoscale architecture. However, the nanodomain organization was compressed in mESCs, with the force transduction layer spanning ∼ 10 nm, significantly more compact than in FAs of other cell types. Furthermore, we found that the position and orientation of vinculin, a key mechanotransduction protein, were modulated in an ECM-dependent manner. Our analysis also revealed that while most core FA genes were expressed, the expression of LIM domain proteins was comparatively lower in PSCs. Altogether our results suggest that while core structural and mechanosensitive elements are operational in mESC FAs, their structural organization and regulatory aspects may diverge significantly from “classical” FAs, which may account for the attenuated mechanobiological responses of these cell types.

Reactive Ion Plasma Modification of Poly(Vinyl-Alcohol) Increases Primary Endothelial Cell Affinity and Reduces Thrombogenicity

Bulk material properties and luminal surface interaction with blood determine the clinical viability of vascular grafts, and reducing intimal hyperplasia is necessary to improve their long-term patency. Here, the authors report that the surface of a biocompatible hydrogel material, poly(vinyl alcohol) (PVA) can be altered by exposing it to reactive ion plasma (RIP) in order to increase primary endothelial cell attachment. The power and the carrier gas of the RIP treatment are varied and the resultant surface nitrogen, water contact angle, as well as the ability of the RIP-treated surfaces to support primary endothelial colony forming cells is characterized. Additionally, in a clinically relevant shunt model, the amounts of platelet and fibrin attachment to the surface were quantified during exposure to non-anticoagulated blood. Treatments with all carrier gases resulted in an increase in the surface nitrogen. Treating PVA with O2 , N2 , and Ar RIP increased affinity to primary endothelial colony forming cells. The RIP treatments did not increase the thrombogenicity compared to untreated PVA and had significantly less platelet and fibrin attachment compared to the current clinical standard of expanded polytetrafluoroethylene (ePTFE). These findings indicate that RIP-treatment of PVA could lead to increased patency in synthetic vascular grafts.

Anisotropic traction stresses and focal adhesion polarization mediates topography-induced cell elongation

Cell elongation and differentiation has been shown to be modulated by topographical cues provided by grating substratum. However, little is known about the mechanisms and forces involved in the grating-induced cell elongation, due to the difficulty in fabricating soft elastic gels that allow 3-dimensional (3D) cell traction stress measurements. In this paper, we present a method to fabricate soft elastic polyacrylamide grating substrates, using an imprinted polyethylene terephthalate mould, for 3D cell traction stress measurements. Fibroblasts were observed to form protrusions in the grating grooves, and elongate and align parallel to the grating direction on the soft polyacrylamide grating substrates. Focal adhesions were also found to be aligned parallel to the grating direction as compared to cells on flat substrates, suggesting that grating grooves restrict focal adhesion growth perpendicular to the grating direction. The 3D traction stress measurements revealed that highly elongated fibroblasts on grating substrates exert anisotropic traction stresses, in the direction parallel to the grating direction. We propose that focal adhesion alignment along the grating direction may result in increased actin stress fibre formation in the direction parallel to the grating, leading to polarized traction stresses which drive cell elongation.