Christopher J. Murphy

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.

Surfaces modified with nanometer-thick silver-impregnated polymeric films that kill bacteria but support growth of mammalian cells.

Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (approximately 100 microg/cm(2)) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with approximately 0.4 microg/cm(2) to approximately 23.6 microg/cm(2) of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as approximately 0.4 microg/cm(2) of silver in the polymeric films caused a reduction of 6log(10)CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing approximately 0.4 microg/cm(2) of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.

Synergistic effects of substance P with insulin-like growth factor-1 on epithelial migration of the cornea

We find that substance P (SP) and insulin‐like growth factor‐1 (IGF‐1) demonstrate a synergistic effect on the stimulation of rabbit corneal epithelial migration in an organ culture. The addition of either SP or IGF‐1 alone did not affect epithelial migration, while the combination of SP and IGF‐1 stimulated epithelial migration in a dose‐dependent fashion. The synergistic effects of SP and IGF‐1 on corneal epithelial migration were nulled by the addition of a SP antagonist or enkephalinase. Among neurotransmitters (vasoactive intestinal peptide, calcitonin gene‐related peptide, acethylcholine chloride, norepinephrine, serotonin) or tachykinins (neurokinin A, neurokinin B, kassinin, eledoisin, physalaemin), only SP demonstrated a synergistic effect with IGF‐1 on cellular migration. In contrast, the combination of SP and IGF‐1 did not affect the incorporation of 3H‐thymidine into corneal epithelial cells. The attachment of the corneal epithelial cells to fibronectin, collagen type IV, and laminin matrices increased after treatment of the cells with SP and IGF‐1, but SP or IGF‐1 by themselves did not affect the attachment of the cells to these extracellular matrix proteins. An identical synergistic effect on corneal epithelial migration was observed when an NK‐1 receptor agonist was used in place of SP, suggesting the synergistic effect of SP and IGF‐1 might be mediated through the NK‐1 receptor system. These results suggest that the maintenance of the normal integrity of the corneal epithelium might be regulated by both humoral and neural factors.

Determining the mechanical properties of human corneal basement membranes with Atomic Force Microscopy

Biophysical cues such as substrate modulus have been shown to influence a variety of cell behaviors. We have determined the elastic modulus of the anterior basement membrane and Descemets membrane of the human cornea with atomic force microscopy (AFM). A spherical probe was used with a radius approximating that of a typical cell focal adhesion. Values obtained for the elastic modulus of the anterior basement membrane range from 2 to 15 kPa, with a mean of 7.5+/-4.2 kPa. The elastic modulus of Descemets membrane was found to be slightly higher than those observed for the anterior basement membrane, with a mean of 50+/-17.8 kPa and a range of 20-80 kPa. The topography of Descemets membrane has been shown to be similar to that of the anterior basement, but with smaller pore sizes resulting in a more tightly packed structure. This structural difference may account for the observed modulus differences. The determination of these values will allow for the design of a better model of the cellular environment as well as aid in the design and fabrication of artificial corneas.

The elastic modulus of Matrigel as determined by atomic force microscopy.

Recent studies indicate that the biophysical properties of the cellular microenvironment strongly influence a variety of fundamental cell behaviors. The extracellular matrixs (ECM) response to mechanical force, described mathematically as the elastic modulus, is believed to play a particularly critical role in regulatory and pathological cell behaviors. The basement membrane (BM) is a specialization of the ECM that serves as the immediate interface for many cell types (e.g. all epithelial cells) and through which cells are connected to the underlying stroma. Matrigel is a commercially available BM-like complex and serves as an easily accessible experimental simulant of native BMs. However, the local elastic modulus of Matrigel has not been defined under physiological conditions. Here we present the procedures and results of indentation tests performed on Matrigel with atomic force microscopy (AFM) in an aqueous, temperature controlled environment. The average modulus value was found to be approximately 450 Pa. However, this result is considerably higher than macroscopic shear storage moduli reported in the scientific literature. The reason for this discrepancy is believed to result from differences in test methods and the tendency of Matrigel to soften at temperatures below 37 degrees C.

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 ...

Photopigment basis for dichromatic color vision in the horse

Horses, like other ungulates, are active in the day, at dusk, dawn, and night; and, they have eyes designed to have both high sensitivity for vision in dim light and good visual acuity under higher light levels (Walls, 1942). Typically, daytime activity is associated with the presence of multiple cone classes and color-vision capacity (Jacobs, 1993). Previous studies in other ungulates, such as pigs, goats, cows, sheep and deer, have shown that they have two spectrally different cone types, and hence, at least the photopigment basis for dichromatic color vision (Neitz & Jacobs, 1989; Jacobs, Deegan II, Neitz, Murphy, Miller, & Marchinton, 1994; Jacobs, Deegan II, & Neitz, 1998). Here, electroretinogram flicker photometry was used to measure the spectral sensitivities of the cones in the domestic horse (Equus caballus). Two distinct spectral mechanisms were identified and are consistent with the presence of a short-wavelength-sensitive (S) and a middle-to-long-wavelength-sensitive (M/L) cone. The spectral sensitivity of the S cone was estimated to have a peak of 428 nm, while the M/L cone had a peak of 539 nm. These two cone types would provide the basis for dichromatic color vision consistent with recent results from behavioral testing of horses (Macuda & Timney, 1999; Macuda & Timney, 2000; Timney & Macuda, 2001). The spectral peak of the M/L cone photopigment measured here, in vivo, is similar to that obtained when the gene was sequenced, cloned, and expressed in vitro (Yokoyama & Radlwimmer, 1999). Of the ungulates that have been studied to date, all have the photopigment basis for dichromatic color vision; however, they differ considerably from one another in the spectral tuning of their cone pigments. These differences may represent adaptations to the different visual requirements of different species.

Response of Human Trabecular Meshwork Cells to Topographic Cues on the Nanoscale Level

UNLABELLED purpose To determine how primary human trabecular meshwork (HTM) cells are influenced by their interaction with nanopatterned substrates. METHODS HTM cells from several individuals were grown on planar or anisotropically ordered nanopatterned surfaces. Microscopy was used to measure cellular elongation and alignment. Cells were also incubated with 10(-7) M dexamethasone for comparison to control cells. Quantitative PCR for myocilin and versican isoforms was performed in addition to Western blots of myocilin and alphaB-crystallin. RESULTS Cells on anisotropically ordered nanopatterned substrates aligned with the surface nanopatterns and displayed actin filaments that were parallel to the patterned ridges and grooves. The cells became more elongated on the nanogrooved surfaces compared with the planar control cells. Myocilin mRNA and protein levels increased when HTM cells were plated onto 400-nm pitch surfaces. With some HTM cells, myocilin increased to a greater extent when untreated cells were plated on nanosurfaces compared with the cells grown on planar surfaces with dexamethasone. The V0 and V1 isoforms of versican had increased expression on patterned surfaces. CONCLUSIONS Nanopatterned surfaces containing biomimetic length scale features clearly influenced cellular behavior of HTM cells. Increased mRNA and protein levels of myocilin were observed when cells were grown on 400-nm pitch surfaces, suggesting that the reduction of myocilin mRNA when cells are plated onto flat tissue culture plastic is an artifact of a nonphysiologic culture environment that lacks appropriate topographic cues.