Response of human trabecular meshwork cells to topographic cues on the nanoscale level

The trabecular meshwork of the human eye is believed to be the tissue that regulates intraocular pressure.1 Human trabecular meshwork (HTM) cells line the trabeculae of the inner areas of the outflow pathway and also are present in the cribriform area. The HTM cells can be cultured in vitro for several passages, but some characteristic properties of the cells, such as myocilin mRNA expression, are downregulated when the cells are cultured on normal tissue culture plastic.2 One possibility for this loss of myocilin could be the absence of appropriate topographic cues normally provided to the cells in vivo. The in vivo basement membrane on the trabeculae and the extracellular matrix at the cribriform region are the surfaces with which HTM cells interact. In contrast to flat plastic surfaces, basement membranes have been shown to have a topographically rich composition at the nanoscale level so that when cells interact with their physical environment, they receive thousands of topographic cues from the surfaces on which they grow.3– 6 Immunomicroscopy7 and scanning electron microscopy8 studies suggest that the trabecular meshwork cells interact with features on the nanometer-scale level. The phenotypic and molecular changes in cells grown on nanosurfaces demonstrate that the topographic features on which the cells are grown profoundly influence them.5,9 –12 These topographic cues are missing on the flat tissue culture plastic normally used to culture HTM cells. It is now possible to fabricate surfaces that have features in the nanometer range to study the interaction of biochemical and physical cues that cells may obtain from their surrounding milieu. These surfaces can finally be produced in large enough quantities for a variety of assays that allow identification of molecular and biochemical properties of cells. Currently, several nanoscale patterns for cell growth can be fabricated.13,14 Synthetic extracellular matrix surfaces are commercially available; however, these are not of uniform topography (Donaldson, Minneapolis, MN). One of the patterns that has been studied is an anisotropic array of ridges and grooves.15–18 The distance between the ridges is termed pitch and is composed of the width of one ridge plus one groove (Fig. 1). The depth of the groove has been shown to be important in directing contact guidance for cells. Loesberg et al.19 reported a threshold of 35 nm of depth to be necessary before a cell is influenced by the substrate pattern, whereas our laboratory found threshold depth to be cell-type dependent and to range from 75 to 150 nm.20 In the study reported herein, all the surfaces had depths of 300 nm. A biomimetic surface can have a variety of pitches as well as differences between the size of the grooves and the ridges. The surfaces used for this study, however, used pitches that each had equal dimensions for the grooves and ridges. Figure 1 Schematic diagram of a patterned surface. Pitch is defined as the sum of the distance of one ridge and one groove. The patterned surfaces used in this report had equivalent dimensions for the grooves and ridges. The depth for all the patterned surfaces ... The purpose of this study was to determine whether HTM cells would be influenced by nanoscale topography compared with flat surfaces. The effect of growth conditions was investigated using several parameters. The alignment and elongation of the HTM cells were parameters that could be compared on a variety of different pitches. At the molecular level, the influence of nanoscale cues on myocilin expression was determined, since data suggest this protein is secreted by HTM cells.21,22 In addition, isoforms of the protein versican appear to be altered when cells are grown in vitro23 and have recently been shown to be influenced by mechanical stretching.24 Changes in versican isoforms, which possess variable numbers of chondroitin sulfate– binding domains, have the potential to influence glaucoma, since it has been reported that chondroitin sulfate increases with disease.25 Therefore, the influence of topography on the versican isoforms was also investigated.