[Show abstract][Hide abstract] ABSTRACT: The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design.
[Show abstract][Hide abstract] ABSTRACT: To determine the effect of chronic corneal epithelial debridement on epithelial and stromal morphology and extracellular matrix components, and to compare those changes to those in spontaneous chronic corneal epithelial defects (SCCED) in dogs.
Axial corneal epithelial wounds, 10 mm in diameter, were created weekly for 8 weeks in five normal adult laboratory beagles. Slit lamp biomicroscopy and corneal pachymetry were performed weekly before wounding. Three days after the last debridement the dogs were killed humanely, and corneas were processed for light and electron microscopy and immunohistochemistry for collagen IV, collagen VII, fibronectin, and laminin.
No significant changes in corneal thickness were found. All samples demonstrated epithelial dysmaturation adjacent to the wound edge, and, in four of five, a narrow zone of nonadherent epithelium formed adjacent to the exposed stroma. All samples had a stromal acellular zone in the area of the defect and continuing for a short distance under the adjacent attached epithelium. Experimentally wounded dogs did not form the superficial hyaline acellular lamina found in 92% of dogs with SCCED. Laminin, collagen IV, and fibronectin were present on the stromal surface in all samples, and collagen VII was present in four of five samples. Transmission electron microscopy (TEM) demonstrated the presence of basement membrane on the surface of the exposed stroma.
Epithelial changes are similar between experimentally wounded dogs and dogs with SCCED. The stromal acellular zone that forms in experimentally wounded dogs is distinct from the hyaline lamina observed in dogs with SCCED. The difference in the acellular stromal layers between chronically wounded dogs and dogs with SCCED may be of relevance to our understanding of the pathophysiology of persistent epithelial defects.
[Show abstract][Hide abstract] ABSTRACT: To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial cells.
Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose-response curve was generated for norepinephrine in concentrations of 100 nM-100 microM. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25 x 10(-3) cells were plated onto FNC-coated 96-well plates, incubated in 10 nM-100 microM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-microm pore size and FNC-coated filters. Cells (250 x 10(3)) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed.
Norepinephrine stimulated corneal epithelial cell proliferation and migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested.
Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.