A cell culture substrate with biologically relevant size-scale topography and compliance of the basement membrane.

A growing body of literature broadly documents that a wide array of fundamental cell behaviors are modulated by the physical attributes of the cellular microenvironment, yet in vitro assays are typically carried out using tissue culture plastic or glass substrates that lack the 3-dimensional topography present in vivo and have stiffness values that far exceed that of cellular and stromal microenvironments. This work presents a method for the fabrication of thin hydrogel films that can replicate arbitrary topographies with a resolution of 400 nm that possess an elastic modulus of approximately 250 kPa. Material characterization including swelling behavior and mechanics were performed and reported. Cells cultured on these surfaces patterned with anisotropic ridges and grooves react to the biophysical cues present and show an alignment response.

Nuclear and cellular alignment of primary corneal epithelial cells on topography.

The basement membrane of the corneal epithelium presents biophysical cues in the form of topography and compliance that can modulate cytoskeletal dynamics, which, in turn, can result in altering cellular and nuclear morphology and alignment. In this study, the effect of topographic patterns of alternating ridges and grooves on nuclear and cellular shape and alignment was determined. Primary corneal epithelial cells were cultured on either planar or topographically patterned (400-4000 nm pitch) substrates. Alignment of individual cell body was correlated with respective nucleus for the analysis of orientation and elongation. A biphasic response in alignment was observed. Cell bodies preferentially aligned perpendicular to the 800 nm pitch; and with increasing pitch, cells increasingly aligned parallel to the substratum. Nuclear orientation largely followed this trend with the exception of those on 400 nm. On this biomimetic size scale, some nuclei oriented perpendicular to the topography while their cytoskeleton elements aligned parallel. Both nuclei and cell bodies were elongated on topography compared to those on flat surfaces. Our data demonstrate that nuclear orientation and shape are differentially altered by topographic features that are not mandated by alignment of the cell body. This novel finding suggests that nuanced differences in alignment of the nucleus versus the cell body exist and that these differences could have consequences on gene and protein regulation that ultimately regulate cell behaviors. A full understanding of these mechanisms could disclose novel pathways that would better inform evolving strategies in cell, stem cell, and tissue engineering as well as the design and fabrication of improved prosthetic devices.

The influence of a biologically relevant substratum topography on human aortic and umbilical vein endothelial cells.

A topographically patterned substrate with stochastic surface order that closely mimics the topographic features of native basement membranes has been fabricated to investigate the influence of topographic biophysical cueing on human aortic and umbilical vein endothelial cells. The stochastic substrate was fabricated by first generating a highly porous polyelectrolyte multilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) followed by replicate production of this biomimetic topography via soft lithography. These substrates, which are easy to prepare and replicate, possess a number of prominent features associated with in vivo vascular basement membrane (interwoven ridges and grooves, bumps, and pores), which have typically been studied as singular features that frequently possess anisotropic surface order (e.g., alternating ridges and grooves). When compared to a flat surface of identical chemistry, these biomimetic topographies influenced a number of important cellular behaviors associated with the homeostasis and degradation of vascular tissues. These include modulating cell migration rate and directional persistence, proliferation rate, and gene expression associated with regulation and remodeling of vascular tissues as well as inflammation.

Interaction forces between colloidal particles in a solution of like-charged, adsorbing nanoparticles.

We have measured the force between a weakly charged micron-sized colloidal particle and flat substrate in the presence of highly charged nanoparticles of the same sign under solution conditions such that the nanoparticles physically adsorb to the colloidal particle and substrate. The objective was to investigate the net effect on the force profile between the microparticle and flat substrate arising from both nanoparticle adsorption and nanoparticles in solution. The experiments used colloidal probe atomic force microscopy (CP-AFM) to measure the force profile between a relatively large (5 µm) colloidal probe glass particle and a planar glass substrate in aqueous solutions at varying concentrations of spherical nanoparticles. At very low nanoparticle concentrations, the primary effect was an increase in the electrostatic repulsion between the surfaces due to adsorption of the more highly charged nanoparticles. As the nanoparticle concentration is increased, a depletion attraction formed, followed by longer-range structural forces at the highest nanoparticle concentrations studied. These results suggest that, depending on their concentration, such nanoparticles can either stabilize a dispersion of weakly-charged colloidal particles or induce flocculation. This behavior is qualitatively different from that in nonadsorbing systems, where the initial effect is the development of an attractive depletion force.

Topographic modulation of the orientation and shape of cell nuclei and their influence on the measured elastic modulus of epithelial cells.

The influence of nucleus shape and orientation on the elastic modulus of epithelial cells was investigated with atomic force microscopy. The shape and orientation were controlled by presenting the epithelial cells with anisotropic parallel ridges and grooves of varying pitch at the cell substratum. As the cells oriented to the underlying topography, the volume of the nucleus increased as the pitch of the topography increased from 400 nm to 2000 nm. The increase in nucleus volume was reflected by an increase in the measured elastic modulus of the topographically aligned cells. Significant alterations in the shape of the nucleus, by intimate contact with the topographic ridge and grooves of the underlying cell, were also observed via confocal microscopy, indicating that the nucleus may also act as a direct mechanosensor of substratum topography.

Substratum compliance regulates human trabecular meshwork cell behaviors and response to latrunculin B.

PURPOSE: To determine the impact of substratum compliance and latrunculin-B (Lat-B), both alone and together, on fundamental human trabecular meshwork (HTM) cell behavior. Lat-B is a reversible actin cytoskeleton disruptor that decreases resistance to aqueous humor outflow and decreases intraocular pressure. METHODS: HTM cells were cultured on polyacrylamide hydrogels possessing values for compliance that mimic those reported for normal and glaucomatous HTM, or tissue culture plastic (TCP). Cells were treated with 0.2 µM or 2.0 µM Lat-B in dimethyl sulfoxide (DMSO) or DMSO alone. The impact of substratum compliance and/or Lat-B treatment on cell attachment, proliferation, surface area, aspect ratio, and migration were investigated. RESULTS: HTM cells had profoundly decreased attachment and proliferation rates when cultured on hydrogels possessing compliance values that mimic those found for healthy HTM. The effect of Lat-B treatment on HTM cell surface area was less for cells cultured on more compliant hydrogels compared with TCP. HTM cell migration was increased on stiffer hydrogels that mimic the compliance of glaucomatous HTM and on TCP in comparison with more compliant hydrogels. Lat-B treatment decreased cellular migration on all surfaces for at least 7 hours after treatment. CONCLUSIONS: Substratum compliance profoundly influenced HTM cell behaviors and modulated the response of HTM cells to Lat-B. The inclusion of substratum compliance that reflects healthy or glaucomatous HTM results in cell behaviors and responses to therapeutic agents in vitro that may more accurately reflect in vivo conditions.

The role of substratum compliance of hydrogels on vascular endothelial cell behavior.

Cardiovascular disease (CVD) remains a leading cause of death both within the United States (US) as well as globally. In 2006 alone, over one-third of all deaths in the US were attributable to CVD. The high prevalence, mortality, morbidity, and socioeconomic impact of CVD has motivated a significant research effort; however, there remain significant knowledge gaps regarding disease onset and progression as well as pressing needs for improved therapeutic approaches. One critical area of research that has received limited attention is the role of biophysical cues on the modulation of endothelial cell behaviors; specifically, the impact of local compliance, or the stiffness, of the surrounding vascular endothelial extracellular matrix. In this study, the impact of substratum compliance on the modulation of cell behaviors of several human primary endothelial cell types, representing different anatomic sites and differentiation states in vivo, were investigated. Substrates used within our studies span the range of compliance that has been reported for the vascular endothelial basement membrane. Differences in substratum compliance had a profound impact on cell attachment, spreading, elongation, proliferation, and migration. In addition, each cell population responded differentially to changes in substratum compliance, documenting endothelial heterogeneity in the response to biophysical cues. These results demonstrate the importance of incorporating substratum compliance in the design of in vitro experiments as well as future prosthetic design. Alterations in vascular substratum compliance directly influence endothelial cell behavior and may participate in the onset and/or progression of CVDs.

Indentation versus tensile measurements of Young's modulus for soft biological tissues.

In this review, we compare the reported values of Young's modulus (YM) obtained from indentation and tensile deformations of soft biological tissues. When the method of deformation is ignored, YM values for any given tissue typically span several orders of magnitude. If the method of deformation is considered, then a consistent and less ambiguous result emerges. On average, YM values for soft tissues are consistently lower when obtained by indentation deformations. We discuss the implications and potential impact of this finding.

The effect of biophysical attributes of the ocular trabecular meshwork associated with glaucoma on the cell response to therapeutic agents.

Glaucoma is a devastating neurodegenerative disease, which can lead to vision loss and is associated with irreversible damage to retinal ganglion cells. Although the mechanism of disease onset remains unknown, we have recently demonstrated that the stiffness of the ocular trabecular meshwork (HTM) increases dramatically in human donor eyes with a history of glaucoma. Here we report that polyacrylamide hydrogels, which mimic the compliant conditions of normal and glaucomatous HTM, profoundly modulate cytoskeletal dynamics and the elastic modulus of the overlying HTM cells. Substratum compliance also modulates HTM cell response to Latrunculin-B, a cytoskeletal disrupting agent currently in human clinical trials for the treatment of glaucoma. Additionally, we observed a compliance-dependent rebound effect of Latrunculin-B with an unexpected increase in HTM cell elastic modulus being observed upon withdrawal of the drug. The results predict that cytoskeletal disrupting drugs may be more potent in advanced stages of glaucoma.

Refractive index of thin, aqueous films between hydrophobic surfaces studied using evanescent wave atomic force microscopy.

We have studied the refractive index of a thin aqueous film between microscopic hydrophobic surfaces using evanescent wave atomic force microscopy (EW-AFM). An evanescent wave, generated at a solid-liquid interface, is scattered by AFM tips or glass particles attached to AFM cantilevers. The scattering of this wave is used to determine the refractive index as a function of separation between these surfaces. Measurements were performed on surfaces that were rendered hydrophobic with octadecyltrichlorosilane, which produces solid-water contact angles in excess of 90 degrees. For AFM tips, the average refractive index in the thin film was always equal to that of water when the film was thicker than approximately 100 nm. At smaller separations, the refractive index was always greater than or equal to that of water. This is inconsistent with the formation of air or vapor films and consistent with a small amount of organic material between the surfaces. For colloidal spheres (R approximately 10 microm), we were not able to detect changes in the refractive index of the thin film between the sphere and plate.

Relationship between scattered intensity and separation for particles in an evanescent field.

We describe measurements of the scattering of visible light from an evanescent field by both spherical particles (R = 1-10 mum) that are glued to atomic force microscopy (AFM) cantilevers, and by sharp tips (R < 60 nm) that were incorporated onto the cantilevers during manufacture. The evanescent wave was generated at the interface between a flat plate and an aqueous solution, and an atomic force microscope was used to accurately control the separation, h, between the particle and the flat plate. We find that, for sharp tips, the intensity of scattered light decays exponentially with separation between the tip and the plate all the way down to h approximately 0. The measured decay length of scattered intensity, delta, is the same as the theoretical decay length of the evanescent intensity in the absence of the sharp tip. For borosilicate particles, (R = 1-10 mum), the scattering also decays exponentially with separation at large separations. However, when the separation is less than roughly 3delta, the measured scattering intensity is smaller in magnitude than that which would be predicted by extrapolating the exponential decay observed at large separations. For these particles, the scattering approximately fits the sum of two exponentials. The magnitude of the deviation from exponential at contact was roughly 10-15% for R = 1 mum particles and about 30% for larger particles and is larger for s-polarized light. Preliminary experiments on polystyrene particles shows that the scattering is also smaller than exponential at small separations but that the deviation from exponential is larger for p-polarized light. In evanescent wave AFM (EW-AFM) the scattering-separation can be calibrated for situations where the scattering is not exponential. We discuss possible errors that could be introduced by assuming that exponential decay of scattering continues down to h = 0.