The influence of substrate topography on the migration of corneal epithelial wound borders.

Currently available artificial corneas can develop post-implant complications including epithelial downgrowth, infection, and stromal melting. The likelihood of developing these disastrous complications could be minimized through improved formation and maintenance of a healthy epithelium covering the implant. We hypothesize that this epithelial formation may be enhanced through the incorporation of native corneal basement membrane biomimetic chemical and physical cues onto the surface of the keratoprosthesis. We fabricated hydrogel substrates molded with topographic features containing specific bio-ligands and developed an in vitro wound healing assay. In our experiments, the rate of corneal epithelial wound healing was significantly increased by 50% in hydrogel surfaces containing topographic features, compared to flat surfaces with the same chemical attributes. We determined that this increased healing is not due to enhanced proliferation or increased spreading of the epithelial cells, but to an increased active migration of the epithelial cells. These results show the potential benefit of restructuring and improving the surface of artificial corneas to enhance epithelial coverage and more rapidly restore the formation of a functional epithelium.

Hydrogels with well-defined peptide-hydrogel spacing and concentration: impact on epithelial cell behavior().

The spacing of peptides away from a hydrogel matrix dramatically impacts their availability and subsequent interactions with cells. Peptides were synthesized with monodisperse poly(ethylene glycol) spacers of different lengths that separate the peptide from the monomeric functionality which reacts during hydrogel polymerization. Specifically, bioactive RGD ligands were conjugated to PEG(5), PEG(11) or PEG(27) spacers via solid phase techniques and then functionalized with an acryloyl end group. These acryloyl-PEGx-RGD conjugates were then copolymerized with PEGDA to form an inert hydrogel network decorated with RGD ligands for cell interactions. As the PEG spacer length increases, the RGD concentration required to support cell attachment and spreading decreases. The competitive detachment of hTCEpi cells in the presence of soluble linear RGD also shows non-linear dependence on the PEG spacer length, as more cells remained attached and spread on gels functionalized with longer PEG-RGD conjugates in comparison to the shorter PEG-RGD conjugates. The strategy and synthetic techniques developed here allow for reproducible control over peptide-hydrogel spacing and peptide concentration, and may be extended for incorporation of multiple peptides and to other hydrogel platforms.

Substratum topography modulates corneal fibroblast to myofibroblast transformation.

PURPOSE: The transition of corneal fibroblasts to the myofibroblast phenotype is known to be important in wound healing. The purpose of this study was to determine the effect of topographic cues on TGFß-induced myofibroblast transformation of corneal cells. METHODS: Rabbit corneal fibroblasts were cultured on nanopatterned surfaces having topographic features of varying sizes. Cells were cultured in media containing TGFß at concentrations ranging from 0 to 10 ng/mL. RNA and protein were collected from cells cultured on topographically patterned and planar substrates and analyzed for the myofibroblast marker α-smooth muscle actin (αSMA) and Smad7 expression by quantitative real time PCR. Western blot and immunocytochemistry analysis for αSMA were also performed. RESULTS: Cells grown on patterned surfaces demonstrated significantly reduced levels of αSMA (P < 0.002) compared with planar surfaces when exposed to TGFß; the greatest reduction was seen on the 1400 nm surface. Smad7 mRNA expression was significantly greater on all patterned surfaces exposed to TGFß (P < 0.002), whereas cells grown on planar surfaces showed equal or reduced levels of Smad7. Western blot analysis and αSMA immunocytochemical staining demonstrated reduced transition to the myofibroblast phenotype on the 1400 nm surface when compared with cells on a planar surface. CONCLUSIONS: These data demonstrate that nanoscale topographic features modulate TGFß-induced myofibroblast differentiation and αSMA expression, possibly through upregulation of Smad7. It is therefore proposed that in the wound environment, native nanotopographic cues assist in stabilizing the keratocyte/fibroblast phenotype while pathologic microenvironmental alterations may be permissive for increased myofibroblast differentiation and the development of fibrosis and corneal haze.

Functionalization of reactive polymer multilayers with RGD and an antifouling motif: RGD density provides control over human corneal epithelial cell-substrate interactions.

Our study demonstrates that substrates fabricated using a "reactive" layer-by-layer approach promote well-defined cell-substrate interactions of human corneal epithelial cells. Specifically, crosslinked and amine-reactive polymer multilayers were produced by alternating "reactive" deposition of an azlactone-functionalized polymer [poly(2-vinyl-4,4-dimethylazlactone)] (PVDMA) and a primary amine-containing polymer [branched poly(ethylene imine)] (PEI). Advantages of our system include a 5- to 30-fold decrease in deposition time compared to traditional polyelectrolyte films and direct modification of the films with peptides. Our films react with mixtures of an adhesion-promoting peptide containing Arg-Gly-Asp (RGD) and the small molecule D-glucamine, a chemical motif which is nonfouling. Resulting surfaces prevent protein adsorption and promote cell attachment through specific peptide interactions. The specificity of cell attachment via immobilized RGD sequences was verified using both a scrambled RDG peptide control as well as soluble-RGD competitive assays. Films were functionalized with monotonically increasing surface densities of RGD which resulted in both increased cell attachment and the promotion of a tri-phasic proliferative response of a human corneal epithelial cell line (hTCEpi). The ability to treat PEI/PVDMA films with peptides for controlled cell-substrate interactions enables the use of these films in a wide range of biological applications.

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.

Alterations in gene expression of human vascular endothelial cells associated with nanotopographic cues.

Human cells in vivo are exposed to a topographically rich, 3-dimenisional environment which provides extracellular cues initiating a cascade of biochemical signals resulting in changes in cell behavior. One primary focus of our group is the development of biomimetic substrates with anisotropic nanoscale topography to elucidate the mechanisms by which physical surface cues are translated into biochemical signals. To investigate changes in gene expression as a result of nanotopographic cues, Human Umbilical Vein Endothelial Cells (HUVECs) were cultured on chemically identical flat and 400 nm pitch nanogrooved surfaces. After 12 h, RNA was harvested for an Affymetrix HG U133 Plus 2.0 gene array. Of over 47,000 possible gene probes, 3171 had at least a two-fold difference in expression between the control flat and 400 nm pitch. The gene ontology groups with the most significant increase in expression are involved in protein modification and maintenance, similar to cells upregulating chaperone and protein synthesis genes in response to physical stresses. The most significant decreases in expression were observed with cell cycle proteins, including cyclins and checkpoint proteins. Extracellular matrix proteins, including integrins, collagens, and laminins, are almost uniformly downregulated on the 400 nm pitch surfaces compared to control. The downregulation of one of these genes, integrin beta 1, was confirmed via quantitative PCR. Together, these gene array data, in addition to our studies of cell behavior on nanoscale surfaces, contribute to our understanding of the signaling pathways modulated by topographical surface cues.

Modulation of human vascular endothelial cell behaviors by nanotopographic cues.

Basement membranes possess a complex three-dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimetic length-scale topographic cues on orientation/elongation, proliferation and migration on four human vascular endothelial cell-types from large and small diameter vessels. We found that all cell-types exhibited orientation and alignment with the most pronounced response on anisotropically ordered ridges > or =800 nm. HUVEC cells were the only cell-type examined to demonstrate a decrease in proliferation in response to the smallest topographic features regardless of surface order. On anisotropically ordered surfaces all cell-types migrated preferentially parallel to the long axis of the ridges, with the greatest increase in cell migration being observed on the 1200 nm pitch. In contrast, cells did not exhibit any preference in direction or increase in migration speed on isotropically ordered surfaces. Overall, our data demonstrate that surface topographic features impact vascular endothelial cell behavior and that the impact of features varies with the cell behavior being considered, topographic feature scale, surface order, and the anatomic origin of the cell being investigated.

Visualization of morphological and molecular features associated with chronic ischemia in bioengineered human skin.

We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure. Multiphoton images of the dermal compartment revealed a decrease in collagen structural order. Immunofluorescence analysis showed changes in matrix metalloproteinase (MMP)-2 protein spatial localization in the epidermis with a shift to the basal layer, and loss of Ki67 expression in proliferative basal cells after 192 h of hypoxic exposure. Upon reoxygenation MMP-2 mRNA levels showed a biphasic response, with restoration of MMP-2 levels and localization. These results indicate that chronic oxygen deprivation causes an overall degeneration in tissue architecture, combined with an imbalance in proteolytic expression and a decrease in proliferative capacity. We propose that these tissue changes are representative of the ischemic condition and that our experimental model system is appropriate for addressing mechanisms of susceptibility to chronic wounds.

The ability of corneal epithelial cells to recognize high aspect ratio nanostructures.

The basement membrane of the human corneal epithelium comprises topographic features including fibers, pores, and elevations with feature dimensions on the order of 20-400 nm. Understanding the impact of sub-micron and nanotopography on corneal cell behavior will contribute to our understanding of biomechanical cues and will assist in the design of improved synthetic corneal implants. We utilized well defined ridge and groove wave-like nanostructures (wave ordered structures, WOS) of 60-140 pitches (30-70 nm ridge widths) and 200 nm depths to assess human corneal epithelial cell (HCEC) contact guidance and to establish HCEC contact acuity defined as the lower limit in feature dimensions at which cells respond to biomimetic topographic cues. Results using the WOS substrates demonstrate that HCEC contact acuity is in the range of 60 nm pitch for cells in a serum-free basal medium (EpiLife) and in the range of 90 nm pitch for cells in epithelial medium. To further investigate the influence of HCEC contact acuity in the presence of larger topographic cues, we fabricated 70 nm pitch WOS-overlaid parallel to the top of the ridges of 800-4000 nm pitch. HCEC cultured in epithelial medium demonstrate a significant increase in the percent of cells aligning to 4000 nm pitch topography with WOS-overlay compared to controls (both flat and 70 nm WOS alone) and 4000 nm pitch topography alone. These results highlight the significance of the lower range of basement membrane scale topographic cues on cell response and allow for improved prosthetic design.

Nanoscale topography-induced modulation of fundamental cell behaviors of rabbit corneal keratocytes, fibroblasts, and myofibroblasts.

PURPOSE: Keratocyte-to-myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast, and myofibroblast cell behavior. METHODS: Primary rabbit corneal keratocytes, fibroblasts, and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400-, 800-, 1200-, 1600-, 2000-, and 4000-nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged, and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours, and individual cell trajectories and migration rates were calculated. RESULTS: Keratocytes, fibroblasts, and myofibroblasts aligned and elongated to pitch sizes larger than 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes, with a transition zone around the 800- to 1200-nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges larger than 1000 nm but lacked directional guidance on submicron and nanoscale topographic features and on planar surfaces. Keratocytes remained essentially immobile. CONCLUSIONS: Corneal stromal cells elongated, aligned, and migrated, differentially guided by substratum topographic features. All cell types failed to respond to topographic features approximating the dimensions of individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.

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 matrix's (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.

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 Descemet's 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 Descemet's 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 Descemet's 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.

Characterization of endothelial basement membrane nanotopography in rhesus macaque as a guide for vessel tissue engineering.

Basement membranes have many features that greatly influence vascular endothelial cell function, including a complex three-dimensional topography. As a first step in the design and development of vascular prosthetics, we undertook a thorough characterization of the topographic features of endothelial vascular basement membranes utilizing transmission electron microscopy and scanning electron microscopy. Specifically, we quantitatively analyzed the topographic features present in the aorta, carotid, saphenous, and inferior vena cava vessels in the rhesus macaque. Our results indicate that vascular basement membranes are composed of a complex meshwork consisting of pores and fibers in the submicron (100-1000 nm) and nanoscale (1-100 nm) range, consistent with what has previously been reported in basement membranes of other tissues. We found significant differences (p<0.05) in basement membrane thickness and pore and fiber diameter depending on the location and physical properties of the vessel. These results have relevance to our fundamental understanding of vascular endothelial cell-matrix interactions in health and disease, evolving strategies in cell and tissue engineering and the design of cardiovascular prosthetic devices.

Cell sorting but not serum starvation is effective for SV40 human corneal epithelial cell cycle synchronization.

SV40 human corneal epithelial cell (HCEC) populations are readily used as a substitute for primary corneal epithelial cells that are difficult to maintain in vitro. To initiate cell-cycle experiments with the SV40-HCEC cells, two separate methods of cell synchronization were compared including serum starvation and sterile cell sorting. We hypothesized that SV40 cells are synchronized at higher efficiencies into each cell cycle phase (G1, S, G2M) when cell sorting is performed when compared to alternative methods of synchronization. SV40 cells were synchronized by deprivation of serum over 96 h or labeled with Höechst 33342 dye and sorted based on DNA content. Cells were synchronized using both methods and harvested at time points up to 72 h after release. To define more precisely the nature of sorted fractions, cells were pulsed with BrdU prior to sorting. SV40-HCEC cells exhibit a well-defined cell cycle profile. Serum deprivation up to 96 h was ineffective for cell synchronization of SV40-HCECs. In comparison, we achieved efficient synchronization of the SV40-HCECs with sterile cell sorting. SV40-HCEC cells gated into G1, S and G2M were synchronized up to 85% following the sort and maintained synchronization up to 24 h. Our findings indicate that serum starvation is not effective for synchronization of the SV40-HCEC cell line. We present a more effective approach, the use of cell sorting for cell synchronization of the SV40-HCEC cells.