Epithelial Sodium Channel in a Human Trophoblast Cell Line (BeWo)

Atomic force microscopy was used to image Bacillus thuringiensis (Bt) toxins interacting with their natural targets, Manduca sexta midgut brush border membranes (BBMs), as well as with dipalmitoylphosphatidylcholine-dioleoylphosphatidylcholine (DPPC-DOPC) solid-supported lipid bilayers. In lipid bilayers, Cry1Aa formed structures 30-60 nm wide and 3-7 nm high, mostly at the interface of domains formed by the two different lipids or at the edge of DOPC-enriched domains. BBM vesicles, in the absence of toxin, formed flat membrane fragments of up to 25 microm(2) and 4.2 nm high, with irregular embedded structures. After incubation with Cry1Aa, Cry1Ac and Cry1C, which are active against M. sexta, new structures, 35 nm wide and 5.1-6.7 nm high, were observed in some membrane fragments, sometimes only in particular regions. Their density, which reached a plateau within 4 h, was toxin- and concentration-dependent. The structures formed by Cry1Ac were often grouped into dense, two-dimensional arrangements. No such specific interactions were observed with Cry1Ba, which is inactive against M. sexta. This study provides the first visual demonstration of specific interactions of Bt toxins with insect midgut BBMs at the nanometric scale. The observed structures likely represent the protein complexes forming functional Bt pores in target membranes.     

Role of Epithelial Sodium Channels and Their Regulators in Hypertension

The kidney has a central role in the regulation of blood pressure, in large part through its role in the regulated reabsorption of filtered Na⁺. Epithelial Na⁺ channels (ENaCs) are expressed in the most distal segments of the nephron and are a target of volume regulatory hormones. A variety of factors regulate ENaC activity, including several aldosterone-induced proteins that are present within an ENaC regulatory complex. Proteases also regulate ENaC by cleaving the channel and releasing intrinsic inhibitory tracts. Polymorphisms or mutations within channel subunits or regulatory pathways that enhance channel activity may contribute to an increase in blood pressure in individuals with essential hypertension.     

The Migratory Capacity of Human Trophoblastic BeWo Cells: Effects of Aldosterone and the Epithelial Sodium Channel

Aldosterone is a key regulator of the epithelial sodium channel (ENaC) and stimulates protein methylation on the β-subunit of the ENaC. We found that aldosterone (100 nM) promotes cellular migration in a wound-healing model in trophoblastic BeWo cells. Here, we tested if the positive influence of aldosterone on wound healing is related to methylation reactions. Cell migration and proliferation were measured in BeWo cells at 6 h, when mitosis is still scarce. Cell migration covered 12.4, 25.3, 19.6 and 45.1 % of the wound when cultivated under control, aldosterone (12 h), 8Br-cAMP and aldosterone plus 8Br-cAMP, respectively. Amiloride blocked the effects of aldosterone alone or in the presence of 8Br-cAMP on wound healing. Wound healing decreased in aldosterone (plus 8Br-cAMP) coexposed with the methylation inhibitor 3-deaza-adenosine (3-DZA, 12.9 % reinvasion of the wound). There was an increase in wound healing in aldosterone-, 8Br-cAMP- and 3-DZA-treated cells in the presence of AdoMet, a methyl donor, compared to cells in the absence of AdoMet (27.3 and 12.9 % reinvasion of the wound, respectively). Cell proliferation assessed with the reagent MTT was not changed in any of these treatments, suggesting that cellular migration is the main factor for reinvasion of wound healing. Electrophysiological studies showed an increase in ENaC current in the presence of aldosterone. This effect was higher with 8Br-cAMP, and there was a decrease when 3-DZA was present. AdoMet treatment partially reversed this phenomenon. We suggest that aldosterone positively influences wound healing in BeWo cells, at least in part through methylation of the ENaC.     

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, αENaC, and γENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high P_K⁺/P_Na⁺ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K⁺ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, αENaC, or γENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.Gliomas are the most common primary tumors of the central nervous system. These tumors arise either from astrocytes or their progenitor cells (1). Gliomas are divided into four grades based on the degree of malignancy. Glioblastoma multiforme (GBM),² Grade IV, is the most frequently occurring, most invasive, and has the worst prognostic outcome with a median survival of approximately one year from diagnosis (2).We have previously reported the presence of an amiloride-sensitive current in glioblastoma cells that is not seen in normal astrocytes or low grade gliomas (3). Amiloride is a potassium sparing diuretic that inhibits sodium channels composed of subunits from the epithelial sodium channel (ENaC)/Degenerin (Deg) family. Amiloride-sensitive Na⁺ channels are essential for the regulation of Na⁺ transport into cells and tissues throughout the body. These channels are found in all body tissues; from epithelia, endothelia, osteoblasts, keratinocytes, taste cells, lymphocytes, and brain (4). Apart from the ENaCs, the ENaC/Deg family also includes acid-sensing ion channels (ASICs) which have been found predominantly in neurons (4–6). Primary malfunctions of ENaC/Deg family members underlie or are involved in the pathophysiology of several human diseases such as salt-sensitive hypertension (7, 8), pseudohypoaldosteronism type I (7), cystic fibrosis (9), chronic airway diseases (10, 11), and flu (12).The ENaC/Deg family subunits share the same structural topology. They all have short intracellular N and C termini, two transmembrane spanning domains, and a large extracellular cysteine-rich loop (4, 5). There are five ENaC subunits termed α, β, γ, δ, and ϵ. Functional ion channels arise from a multimeric assembly of these subunits. The prototypical ENaC channel of the collecting duct principal cell is thought to be αβγENaC (13, 14). The α-ENaC subunit appears to be the core conducting element, whereas the β- and γ-ENaC subunits are associated with trafficking and insertion of the channel in the cell membrane (13, 15, 16). ASICs are homologous to ENaCs and are most prevalently expressed in the brain and nervous system (17–19), although they are also found in the retina (20–22), testes (23), pituitary gland (24), lung epithelia (22), and bone and cartilage (25). Four ASIC genes have been identified so far, ASIC1–4. Of these, ASIC1–3 has multiple splice variants (19, 22). The crystal structure of chicken ASIC1 has revealed it to be a homotrimer (26). ASICs differ from their ENaC counterparts in that they are transiently activated by extracellular acid (19) and are much less sensitive to inhibition by amiloride (27, 28). Also ASIC1 is inhibited with high affinity by psalmotoxin 1 (PcTX-1), a 40-amino acid peptide found in the venom of the West Indies tarantula, Psalmopoeus Cambridgei (29). ASICs, because they are activated by acidic pH, have been suggested to play a role in chemical pain associated with increased tissue acidification as occurs in ischemia (30, 31). They have also been implicated in touch sensation (32), taste (33), fear-conditioning (6), and learning and memory (34).Our laboratory has proposed that ENaC/Deg channels underlie the basally activated cation current measured in high grade glioma cells (3). We hypothesize that the channels forming this current pathway are composed of a mixture of ASIC and ENaC subunits. RNA profiling of a large number of GBM-derived cell lines and freshly resected tumors have revealed the presence of a myriad of ASIC/ENaC components (3). The basally active current seen in GBM cells can be significantly reduced by amiloride or benzamil (a higher affinity amiloride analog), both of which are inhibitors of the ENaC/Deg family of ion channels (3). PcTX1, a selective ASIC1 blocker, also effectively abolishes the basally active GBM current (35). We have previously shown that ENaC and ASIC subunits can form cross-clade interactions in a heterologous expression system (36). This study aims to probe the composition of the novel ENaC/Deg heteromer in a glioma cell line, D54-MG. Our study postulates that a change in GBM cell electrophysiological properties after subunit knockdown would be indicative of that subunit being a part of the GBM channel. We have sequentially knocked down different ENaC/Deg subunits from the D54-MG glioma cells and measured amiloride-sensitive whole cell current using patch clamp. We found that knocking down various ENaC/Deg subunits significantly reduced the whole cell patch clamp current in glioma cells and changed the resting Na⁺/K⁺ permeability of the these cells. After subunit knockdown, glioma cells showed a reduced cell migration as compared with control cells, consistent with our hypothesis that ENaC/Deg subunits play an important role in glioma cell pathophysiology.     

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.     

New Model for Studying the Migration of Immune Cells into Intestinal Epithelial Cell Monolayers

A novel cell culture system was constructed to analyze the direct interaction between intestinal epithelial cells and immune cells. Human intestinal epithelial Caco-2 cells were monolayer-cultured on the under side of a permeable membrane (12 μm pore size) in a Millicell insert. Integrated monolayers of Caco-2 cells had formed after 12 days of culture. Human monocyte/macrophage-like THP-1 cells were then added to the upper chamber of the insert, and their migration into the Caco-2 cell monolayers was observed by confocal laser scanning microscopy, after staining the cells with specific antibodies. When MCP-1, a β-chemokine, was added to the apical side of the monolayer, a greater number of THP-1 cells migrated into the Caco-2 cell monolayers. This cell culture system will be useful for studying the behavior of macrophages in the intestinal epithelial cell monolayers at the initial stage of an intestinal immune reaction.     

CPT-cGMP Is A New Ligand of Epithelial Sodium Channels

Epithelial sodium channels (ENaC) are localized at the apical membrane of the epithelium, and are responsible for salt and fluid reabsorption. Renal ENaC takes up salt, thereby controlling salt content in serum. Loss-of-function ENaC mutations lead to low blood pressure due to salt-wasting, while gain-of-function mutations cause impaired sodium excretion and subsequent hypertension as well as hypokalemia. ENaC activity is regulated by intracellular and extracellular signals, including hormones, neurotransmitters, protein kinases, and small compounds. Cyclic nucleotides are broadly involved in stimulating protein kinase A and protein kinase G signaling pathways, and, surprisingly, also appear to have a role in regulating ENaC. Increasing evidence suggests that the cGMP analog, CPT-cGMP, activates αβγ-ENaC activity reversibly through an extracellular pathway in a dose-dependent manner. Furthermore, the parachlorophenylthio moiety and ribose 2''-hydroxy group of CPT-cGMP are essential for facilitating the opening of ENaC channels by this compound. Serving as an extracellular ligand, CPT-cGMP eliminates sodium self-inhibition, which is a novel mechanism for stimulating salt reabsorption in parallel to the traditional NO/cGMP/PKG signal pathway. In conclusion, ENaC may be a druggable target for CPT-cGMP, leading to treatments for kidney malfunctions in salt reabsorption.     

Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

Gastrulation, the process that puts the three major germlayers, the ectoderm, mesoderm and endoderm in their correct topological position in the developing embryo, is characterised by extensive highly organised collective cell migration of epithelial and mesenchymal cells. We discuss current knowledge and insights in the mechanisms controlling these cell behaviours during gastrulation in the chick embryo. We discuss several ideas that have been proposed to explain the observed large scale vortex movements of epithelial cells in the epiblast during formation of the primitive streak. We review current insights in the control and execution of the epithelial to mesenchymal transition (EMT) underlying the formation of the hypoblast and the ingression of the mesendoderm cells through the streak. We discuss the mechanisms by which the mesendoderm cells move, the nature and dynamics of the signals that guide these movements, as well as the interplay between signalling and movement that result in tissue patterning and morphogenesis. We argue that instructive cell-cell signaling and directed chemotactic movement responses to these signals are instrumental in the execution of all phases of gastrulation.     

Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo.     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999     

Identification of Anion-selective Channels in the Basolateral Membrane of Mitochondria-rich Epithelial Cells

Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl^- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl^- channels. The two major populations constituted Ohmic Cl^- channels with limiting conductance (γ_125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl^- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (±0.1) × 10^-14, 5.7 (±0.7) × 10^-14, and 32 (±2) × 10^-14 cm³/sec, for the 10, 30 and 150 pS Cl^- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl^- channel was studied in patches generating `noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near E _Cl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl^- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl^- channels are organized in clusters. Received: 10 October 1996/Revised: 8 January 1997     

Tubular Bridges for Bronchial Epithelial Cell Migration and Communication

Background

Biological processes from embryogenesis to tumorigenesis rely on the coordinated coalescence of cells and synchronized cell-to-cell communication. Intercellular signaling enables cell masses to communicate through endocrine pathways at a distance or by direct contact over shorter dimensions. Cellular bridges, the longest direct connections between cells, facilitate transfer of cellular signals and components over hundreds of microns in vitro and in vivo.

Methodology/Principal Findings

Using various cellular imaging techniques on human tissue cultures, we identified two types of tubular, bronchial epithelial (EP) connections, up to a millimeter in length, designated EP bridges. Structurally distinct from other cellular connections, the first type of EP bridge may mediate transport of cellular material between cells, while the second type of EP bridge is functionally distinct from all other cellular connections by mediating migration of epithelial cells between EP masses. Morphological and biochemical interactions with other cell types differentially regulated the nuclear factor-κB and cyclooxygenase inflammatory pathways, resulting in increased levels of inflammatory molecules that impeded EP bridge formation. Pharmacologic inhibition of these inflammatory pathways caused increased morphological and mobility changes stimulating the biogenesis of EP bridges, in part through the upregulation of reactive oxygen species pathways.

Conclusions/Significance

EP bridge formation appears to be a normal response of EP physiology in vitro, which is differentially inhibited by inflammatory cellular pathways depending upon the morphological and biochemical interactions between EP cells and other cell types. These tubular EP conduits may represent an ultra long-range form of direct intercellular communication and a completely new mechanism of tissue-mediated cell migration.     

Self-Inhibition in Amiloride-sensitive Sodium Channels in Taste Receptor Cells

Electrophysiological recording techniques were used to study the Na⁺ dependence of currents through amiloride-sensitive sodium channels (ASSCs) in rat taste cells from the fungiform and vallate papillae. Perforated patch voltage clamp recordings were made from isolated fungiform and vallate taste receptor cells (TRCs) and Na⁺ transport was measured across lingual epithelia containing fungiform or vallate taste buds in a modified Ussing chamber. In isolated fungiform TRCs that contain Na⁺ currents sensitive to the diuretic amiloride, Na⁺ ions inhibit their own influx through ASSCs, a process known as sodium self-inhibition. Due to the interaction between self-inhibition and the driving force for Na⁺ entry, self-inhibition is most evident in whole-cell recordings at Na⁺ concentrations from 50 to 75 mM. In amiloride-sensitive cells, the Na permeability is significantly higher in extracellular solutions containing 35 mM Na⁺ than in 70 or 140 mM Na⁺. Compared with the block by amiloride, the development of self-inhibition is slow, taking up to 15 s to become maximally inhibited. Approximately one third of fungiform TRCs and all vallate TRCs lack functional ASSCs. These amiloride-insensitive TRCs show no signs of self-inhibition, tying this phenomenon to the presence of ASSCs. The sulfhydryl reagent, p-hydroxymercuribenzoate (p-HMB; 200 μM), reversibly removed self-inhibition from amiloride-sensitive Na⁺ currents, apparently by modifying cysteine residues in the ASSC. Na⁺ currents in amiloride-insensitive TRCs were unaffected by p-HMB. In sodium transport studies in fungiform taste bud–containing lingual epithelia, ∼40% of the change in short-circuit current (I_sc) after addition of 500 mM NaCl to the mucosal chamber is amiloride sensitive (0.5 mM). p-HMB significantly enhanced mucosal NaCl-induced changes in these epithelia at mucosal Na⁺ concentrations of 50 mM and above. In contrast, the vallate-containing epithelia, which are insensitive to amiloride, showed no enhancement of I_sc during p-HMB treatment. These findings suggest that sodium self-inhibition is present in ASSCs in taste receptor cells where it may play a crucial role in performance of salt-sensitive pathways in taste tissue during sodium stimulation. This phenomenon may be important in the process of TRC adaptation, in the conservation of cellular resources during chronic sodium exposure, or in the gustatory response to water.     

Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca²⁺] _i ) associated with periodic Ca²⁺ oscillations during prolonged ACh applications. The early transient rise in [Ca²⁺] _i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca²⁺ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca²⁺] _i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca²⁺-sensitive K⁺ currents. These K⁺ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca²⁺ from intracellular stores which in turn activate Ca²⁺-dependent (BK-type) K⁺ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000     

Silk Film Topography Directs Collective Epithelial Cell Migration

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.