George A. Abrams

Effects of synthetic micro- and nano-structured surfaces on cell behavior

Topographical cues, independent of biochemistry, generated by the extracellular matrix may have significant effects upon cellular behavior. Studies have documented that substratum topography has direct effects on the ability of cells to orient themselves, migrate, and produce organized cytoskeletal arrangements. Basement membranes are composed of extracellular matrix proteins and found throughout the vertebrate body, serving as substrata for overlying cellular structures. The topography of basement membranes is a complex meshwork of pores, fibers, ridges, and other features of nanometer sized dimensions. Synthetic surfaces with topographical features have been shown to influence cell behavior. These facts lead to the hypothesis that the topography of the basement membrane plays an important role in regulating cellular behavior in a manner distinct from that of the chemistry of the basement membrane. This paper describes the topography of the basement membrane and reviews the fabrication of synthetic micro- and nano-structured surfaces and the effects of such textured surfaces on cell behavior.

Epithelial contact guidance on well-defined micro- and nanostructured substrates

The human corneal basement membrane has a rich felt-like surface topography with feature dimensions between 20 nm and 200 nm. On the basis of these findings, we designed lithographically defined substrates to investigate whether nanotopography is a relevant stimulus for human corneal epithelial cells. We found that cells elongated and aligned along patterns of grooves and ridges with feature dimensions as small as 70 nm, whereas on smooth substrates, cells were mostly round. The percentage of aligned cells was constant on substrate tomographies with lateral dimensions ranging from the nano- to the micronscale, and increased with groove depth. The presence of serum in the culture medium resulted in a larger percentage of cells aligning along the topographic patterns than when no serum was added to the basal medium. When present, actin microfilaments and focal adhesions were aligned along the substrate topographies. The width of the focal adhesions was determined by the width of the ridges in the underlying substrate. This work documents that biologic length-scale topographic features that model features encountered in the native basement membrane can profoundly affect epithelial cell behavior.

Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells

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.

Nanoscale topography of the corneal epithelial basement membrane and Descemet's membrane of the human

PURPOSE Quantitatively define and compare the nanoscale topography of the corneal epithelial basement membrane (anterior basement membrane) and Descemets membrane (posterior basement membrane) of the human. METHODS Human corneas not suitable for transplantation were obtained from the Wisconsin Eye Bank. The corneas were placed in 2.5 mM EDTA for 2.5 h or 30 min. for removal of the epithelium or endothelium, respectively. After removal of the overlying cells, specimens were fixed in 2% glutaraldehyde and either examined in this state by atomic force microscopy only or dehydrated through an ethanol series and prepared for transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). RESULTS The subepithelial and subendothelial basement membrane surfaces have a similar appearance that consists of an interwoven meshwork of fibers and pores. Topographic feature sizes were found to be in the nanometer size range with the epithelial basement membrane features larger and less densely packed than Descemets membrane features. The topographic features are fractile in nature and increase surface area for cell contact. CONCLUSION With the use of the TEM, SEM, and AFM, a detailed description of the surface topography of corneal epithelial basement membrane and Descemets membrane of the human cornea are provided. The significance of differences in corneal basement membrane topography may reflect differences in function of the overlying cells or may be related to differences in cell migration and turnover patterns between the epithelium and endothelium.

Electron Microscopy of the Canine Corneal Basement Membranes

The purpose of this study was to characterize the surface topographical features of the epithelial and endothelial (Descemet’s) basement membranes of the canine cornea. Corneas were obtained from young, healthy dogs (<2 years old) with no history or evidence of previous ocular disease. The epithelium and endothelium was carefully removed preserving the anterior and posterior basement membranes. The specimens were examined by transmission electron microscopy and scanning electron microscopy. The epithelial and endothelial basement membrane surface topography is an intricate meshwork of pores and fibers measuring in the nanometer size range. The features of the endothelial basement membrane overall are smaller in size than the epithelial basement membrane. These surface topographical features may incite changes in epithelial and endothelial cell behavior.

Cell behavior on lithographically defined nanostructured substrates

Lithographically defined substrates offer unique opportunities for the study of cell behaviors, by allowing the presentation of controlled cell stimuli. We have investigated the effects of substrate topography on the behavior of human corneal epithelial cells using substrates patterned with grooves and ridges of well-defined dimensions. On each substrate, we included feature sizes ranging from the micrometer to the nanometer scale. This work was motivated by the fact that the surface that underlies epithelial tissues in vivo, the basement membrane, has a rich topography with features of nanoscale dimensions. We found that cells responded to topographic features as small as 70 nm wide by aligning with the pattern direction. Additionally, substrate topography affected internal organization of the cell, inducing the alignment of cytoskeletal elements (actin filaments) and adhesive structures (focal adhesions). Therefore, synthetic topographies with feature dimensions of the same length scale as the features ...

Aspergillus Blepharitis and Dermatitis in a Peregrine Falcon-Gyrfalcon Hybrid (Falco peregrinus × Falco rusticolus)

Abstract A 3-year-old female peregrine falcon-gyrfalcon hybrid (Falco peregrinus × Falco rusticolus) was presented for evaluation of a lesion below the right eye. Initial examination revealed an abraded, crusty lesion on the right lower eyelid that was believed to be consistent with trauma and a secondary bacterial infection. Despite initial diagnostic procedures and treatment, the condition progressed to severe blepharitis and dermatitis involving the upper and lower eyelids of both eyes and the head. Septated fungal hyphae were identified by histopathologic analysis in specimens obtained from the lesions and an Aspergillus species was cultured. The blepharitis and dermatitis were treated successfully with oral itraconazole (15 mg/kg q12h) and topical miconazole cream, applied twice daily.

Conjunctivitis in birds.

There are numerous causative agents of avian conjunctivitis, many of which are associated with systemic disease. A thorough evaluation of the patients history and habitat is mandatory, and a complete physical examination and ophthalmic examination are required. The owner should be informed at the initial visit that an orderly sequential approach for obtaining diagnostic specimens is required and that a protracted course of treatment may be required to bring about complete, long-lasting resolution of clinical signs.