Synthesis,characterization, and preliminary biological study of poly(3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone)

Poly(3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone) has been synthesized and characterized by gel permeation chromatography, Fourier transform infrared spectroscopy, NMR spectroscopy, and thermal analysis. The polymer is a chemically amplified photoresist. Arrays of lines with 25 microm width and 25 microm spacing were successfully patterned with this polymer by photolithography. Rat fibroblast cells were seeded on these patterned surfaces as well as the smooth glass surface. Phase contrast microscopy showed that cells on the patterned surfaces were strongly aligned and elongated along the grooves as compared to randomly spreading on the smooth surface. Since controlling cell orientation is critical for the development of advanced forms of tissue repair and cell engineering therapies, for example, peripheral nerve repair, production of tendon and ligament substitutes in vitro, and control of microvascular repair, the described polymer may be useful for applications in tissue reconstruction.     

Effect of adsorbed fibronectin on the differential adhesion of osteoblast-like cells and Staphylococcus aureus with and without fibronectin-binding proteins

The influence of fibronectin (Fn) coated surfaces patterned with poly(ethylene glycol) microgels having inter-gel spacings between 0.5 and 3.0 μm on the adhesion of Staphylococcus aureus strains with and without Fn-binding proteins and cellular adhesion/spreading was investigated. Quantitative force measurements between a S. aureus cell and a patterned surface showed that the adhesion force between the bacterium and the patterned surface increased substantially after Fn adsorption, regardless of the strain used, but decreased with decreasing inter-gel spacing. In flow-chamber experiments, the Fn-binding strain adhered at a higher rate after Fn adsorption than the strain lacking Fn-binding proteins. In both cases, the adhesion rates decreased with decreasing inter-gel spacing. Osteoblast-like cells could bind to patterned surfaces despite the microgels, and adsorbed Fn substantially amplified this effect. Even under highly non-adhesive conditions associated with closely spaced microgels, adsorbed Fn preserves a window of inter-gel spacing around 1 μm where the adhesion of staphylococcal cells is hindered while cells can still adhere and spread.     

Two-step cell patterning on planar and complex curved surfaces by precision spraying of polymers

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.     

Oriented Schwann cell growth on microgrooved surfaces

Silicon wafers bearing microgrooved surfaces with various groove width, spacing, and depth were fabricated using microlithography. The orientation of rat Schwann cells along the direction of the grooves was measured at 24 h after seeding the cells. When the width/spacing of the grooves was fixed at 10/10 microm, the mean percentage of aligned cells was 12% for grooves of 0.5 microm depth, 15% for those of 1 microm depth, and 26% for those of 1.5 microm depth (P < 0.05). When the depth of grooves was fixed at 1.5 microm, the mean percentage of aligned cells increased from 26% for width/spacing 10/10 microm, to 33% for 10/20 microm or 20/10 microm, and up to 41% for 20/20 microm (P < 0.05). On the surface with grooves of width/spacing/depth = 20/20/1.5 microm and modified by laminin, the alignment at 24 h approached 60%, versus 51% for collagen-coated surface and 41% for uncoated surface (P < 0.05). At 48 h after seeding, about 66% of the cells were aligned on the above laminin-modified surface. The groove depth influenced orientation of Schwann cells significantly. The cell alignment on 20/20/3 microm microgrooved poly(D,L-lactide-co-glycolide) 90:10 (PLGA) surfaces transferred from silicon reached 72% at 48 h and 92% at 72 h (P < 0.05). Coating this surface with laminin enhanced cell alignment only in short term (67% vs. 62% at 24 h, P < 0.05). The cell alignment guided by surface microgrooves was time dependent.     

Substrate Topography Induces a Crossover from 2D to 3D Behavior in Fibroblast Migration

In a three-dimensional environment, cells migrate through complex topographical features. Using microstructured substrates, we investigate the role of substrate topography in cell adhesion and migration. To do so, fibroblasts are plated on chemically identical substrates composed of microfabricated pillars. When the dimensions of the pillars (i.e., the diameter, length, and spacing) are varied, migrating cells encounter alternating flat and rough surfaces that depend on the spacing between the pillars. Consequently, we show that substrate topography affects cell shape and migration by modifying cell-to-substrate interactions. Cells on micropillar substrates exhibit more elongated and branched shapes with fewer actin stress fibers compared with cells on flat surfaces. By analyzing the migration paths in various environments, we observe different mechanisms of cell migration, including a persistent type of migration, that depend on the organization of the topographical features. These responses can be attributed to a spatial reorganization of the actin cytoskeleton due to physical constraints and a preferential formation of focal adhesions on the micropillars, with an increased lifetime compared to that observed on flat surfaces. By changing myosin II activity, we show that actomyosin contractility is essential in the cellular response to micron-scale topographic signals. Finally, the analysis of cell movements at the frontier between flat and micropillar substrates shows that cell transmigration through the micropillar substrates depends on the spacing between the pillars.     

The modulation of canine mesenchymal stem cells by nano-topographic cues

Mesenchymal stem cells (MSCs) represent a promising cellular therapeutic for the treatment of a variety of disorders. On transplantation, MSCs interact with diverse extracellular matrices (ECMs) that vary dramatically in topographic feature type, size and surface order. In order to investigate the impact of these topographic cues, surfaces were fabricated with either isotropically ordered holes or anisotropically ordered ridges and grooves. To simulate the biologically relevant nano through micron size scale, a series of topographically patterned substrates possessing features of differing pitch (pitch=feature width+groove width) were created. Results document that the surface order and size of substratum topographic features dramatically modulate fundamental MSC behaviors. Topographically patterned (ridge+groove) surfaces were found to significantly impact MSC alignment, elongation, and aspect ratio. Novel findings also demonstrate that submicron surfaces patterned with holes resulted in increased MSC alignment to adjacent cells as well as increased migration rates. Overall, this study demonstrates that the presentation of substratum topographic cues dramatically influence MSC behaviors in a size and shape dependent manner. The response of MSCs to substratum topographic cues was similar to other cell types that have been studied previously with regards to cell shape on ridge and groove surfaces but differed with respect to proliferation and migration. This is the first study to compare the impact of anisotropically ordered ridge and groove topographic cues to isotropically order holed topographic cues on fundamental MSC behaviors across a range of biologically relevant size scales.     

Cells responding to chemoattractant on a structured substrate

Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and detachment from, the substrate. To study the effect of cell-to-substrate adhesion on the chemotactic response of Dictyostelium discoideum cells, we exposed the cells to patterned substrate surfaces consisting of adhesive and inert areas, and forced them by a gradient of chemoattractant to enter the border between the two areas. Wild-type as well as myosin II-deficient cells stop at the border of an adhesive area. They do not detach with their rear part, while on the nonadhesive area they protrude pseudopods at their front toward the source of chemoattractant. Avoidance of the nonadhesive area may cause a cell to move in tangential direction relative to the attractant gradient, keeping its tail at the border of the adhesive surface.     

3-D surface charges modulate protrusive and contractile contacts of chondrosarcoma cells

Up to now, most of the studies addressing the critical roles played by protrusive and contractile cell-matrix contacts in cell adhesion, guidance, migration, matrix assembly, and activation of signaling molecules have been performed on two-dimensional surfaces. Here, we analysed the organization of chondrosarcoma cell contacts in a new three-dimensional environment made of titanium beads. Surface charges were modified by deposition of polyelectrolyte multilayer films built up by alternated polycations poly-(L-lysine) or poly(allylamine hydrochloride) and polyanions poly-(L-glutamic acid) or poly(sodium 4-styrenesulfonate). Negatively charged 3-D titanium surfaces amplified the occurrence and length of cell protrusions. These protrusions had pseudopod characteristics extended to 200 microm in length, growing off the substratum to distant beads. Pseudopod formation is inhibited by the exocytosis inhibitor concanamycin A and is triggered by a secreted factor. Chondrosarcoma cells adhering on uncoated or on negatively charged surfaces contained discrete focal spots of vinculin and actin cables. In cells plated onto these surfaces, phosphorylation of p44/42 MAPK/ERK was twofold increased. In contrast, no cytoskeletal vinculin and actin organization was observed when the surface was positively charged. These data suggest that chondrosarcoma cells adapt a more stable adhesion on uncoated or negatively charged surfaces. This point may be critical in tissue engineering strategies designed for cartilage repair.     

Neutrophil motility in extracellular matrix gels: mesh size and adhesion affect speed of migration.

Polymorphonuclear leukocyte (PMN) migration through tissue extracellular space is an essential step in the inflammatory response, but little is known about the factors influencing PMN migration through gels of extracellular matrix (ECM). In this study, PMN migration within reconstituted gels containing collagen type I or collagen type I supplemented with laminin, fibronectin, or heparin was measured by quantitative direct visualization, resulting in a random motility coefficient (mum a quantitative index for rate of cell dispersion) for the migrating cell population. The random motility coefficient in unsupplemented collagen (0.4 mg/ml) gels was approximately 9 x 10(-9) cm2/s. Supplementing gels with heparin or fibronectin produced a significant decrease in mu, even at the lowest concentrations studied (1 microgram/ml fibronectin or 0.4 microgram/ml heparin). At least 100 micrograms/ml of laminin, or 20% of the total gel protein, was required to produce a similar decrease in mu. Scanning electron microscopy revealed two different gel morphologies: laminin or fibronectin appeared to coat the 150-nm collagen fibers whereas heparin appeared to induce fiber bundle formation and, therefore, larger interstitial spaces. The decrease in mu observed in heparin-supplemented gels correlated with the increased mesh size of the fiber network, but the difference observed in mu for fibronectin- and laminin-supplemented gels did not correlate with either mesh size or the mechanical properties of the gel, as determined by rheological measurements. However, PMNs adhered to fibronectin-coated surfaces in greater numbers than to collagen- or laminin-coated surfaces, suggesting that changes in cell adhesion to protein fibers can also produce significant changes in cell motility within an ECM gel.     

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen Matrices

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.     

Biochemical and mechanical extracellular matrix properties dictate mammary epithelial cell motility and assembly

Biochemical and mechanical cues of the extracellular matrix have been shown to play important roles in cell-matrix and cell-cell interactions. We have experimentally tested the combined influence of these cues to better understand cell motility, force generation, cell-cell interaction, and assembly in an in vitro breast cancer model. MCF-10A non-tumorigenic mammary epithelial cells were observed on surfaces with varying fibronectin ligand concentration and polyacrylamide gel rigidity. Our data show that cell velocity is biphasic in both matrix rigidity and adhesiveness. The maximum cell migration velocity occurs only at specific combination of substrate stiffness and ligand density. We found cell-cell interactions reduce migration velocity. However, the traction forces cells exert onto the substrate increase linearly with both cues, with cells in pairs exerting higher maximum tractions observed over single cells. A relationship between force and motility shows a maximum in single cell velocity not observed in cell pairs. Cell-cell adhesion becomes strongly favored on softer gels with elasticity ≤ 1250 Pascals (Pa), implying the existence of a compliance threshold that promotes cell-cell over cell-matrix adhesion. Finally on gels with stiffness similar to pre-malignant breast tissue, 400 Pa, cells undergo multicellular assembly and division into 3D spherical aggregates on a 2D surface.     

Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.     

Competitive protein adsorption on micro patterned polymeric biomaterials, and viscoelastic properties of tailor made extracellular matrices

Cell adhesion on biomaterial surfaces and the vitality of anchorage dependent cells is affected by several parameters of an adsorbate layer which is intentionally or spontaneously formed. Surface pre-treatments and several conditioning steps prior and during to the cell/biomaterial contact affect the composition, orientation, quantity and viscoelasticity of the interfacing layer between cells and biomaterial. This work was performed to elucidate the response of cells on two modified biomaterial surfaces based on protein or carbohydrate adsorbates: (a) Masked UV irradiations opened a simple route to obtain chemically patterned substrates controlling serum protein adsorption and cell adhesion. It is possible to achieve structures of subcellular size and to produce immobilized gradients. In order to examine the protein matrix deposited on these substrates we applied a quartz microbalance technique (QCM-D) capable to extract viscoelastic data in addition to the mass uptake during plasma protein deposition. It was found that the quantity and viscosity of surface bound albumin is lowered when the surface is modified (patterned) by UV exposure. Hence, the UV modification promotes the competitive adsorption of cell adhesion proteins from the media or upon secretion by the cells and yields to the observed cell patterns. (b) Another tissue engineering technique, using immobilized, modified and/or cross linked hyaluronic acid (HA), an important extra cellular matrix component in vivo, is also examined by QCM-D. Our data demonstrate that HA can be modified by an activation with a carbodiimide, followed by the application of an alpha,omega-bisamino polyethyleneglycol. The QCM-D data can be interpreted as a stiffening of the HA layer combined with the release of hydration water. Further, the hydration state and the viscoelastic behaviour of surface bound ultrathin HA hydrogels was examined. Quantification of viscoelastic parameters of thin films of ECM by QCM-D is valuable for the interpretation of durotaxis, describing effects of mechanical substrate parameters on the adhesion and motility of cells.     

Intracellular Ca2+ imaging for micropatterned cardiac myocytes

The patterning of cardiac myocytes on a micron scale ( approximately 5 microm) was achieved by microcontact printing of fibronectin onto a hydrophobically pretreated glass substrate. The patterned cardiac myocytes conjugated with each other by forming a gap junction, as judged from the synchronized Ca(2+) transition over the pattern, and thus simultaneously contracted. The dynamic change of the Ca(2+) concentration within the patterned tissue was analyzed quantitatively during successive contraction and relaxation using a Nipkow-type high-speed confocal microscope.     

Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength

Although a biphasic dependence of cell migration speed on cell- substratum adhesiveness has been predicted theoretically, experimental data directly demonstrating a relationship between these two phenomena have been lacking. To determine whether an optimal strength of cell- substratum adhesive interactions exists for cell migration, we measured quantitatively both the initial attachment strength and migration speed of human smooth muscle cells (HSMCs) on a range of surface concentrations of fibronectin (Fn) and type IV collagen (CnIV). Initial attachment strength was measured in order to characterize short time- scale cell-substratum interactions, which may be representative of dynamic interactions involved in cell migration. The critical fluid shear stress for cell detachment, determined in a radial-flow detachment assay, increased linearly with the surface concentrations of adsorbed Fn and CnIV. The detachment stress required for cells on Fn, 3.6 +/- 0.2 x 10(-3) mu dynes/absorbed molecule, was much greater than that on CnIV, 5.0 +/- 1.4 x 10(-5) mu dynes/absorbed molecule. Time- lapse videomicroscopy of individual cell movement paths showed that the migration behavior of HSMCs on these substrates varied with the absorbed concentration of each matrix protein, exhibiting biphasic dependence. Cell speed reached a maximum at intermediate concentrations of both proteins, with optimal concentrations for migration at 1 x 10(3) molecules/micron2 and 1 x 10(4) molecules/micron2 on Fn and CnIV, respectively. These optimal protein concentrations represent optimal initial attachment strengths corresponding to detachment shear stresses of 3.8 mu dyne/micron2 on Fn and 1.5 mu dyne/micron2 on CnIV. Thus, while the optimal absorbed protein concentrations for migration on Fn and CnIV differed by an order of magnitude, the optimal initial attachment strengths for migration on these two proteins were very similar. Further, the same minimum strength of initial attachment, corresponding to a detachment shear stress of approximately 1 mu dyne/micron2, was required for movement on either protein. These results suggest that initial cell-substratum attachment strength is a central variable governing cell migration speed, able to correlate observations of motility on substrata differing in adhesiveness. They also demonstrate that migration speed depends in biphasic manner on attachment strength, with maximal migration at an intermediate level of cell-substratum adhesiveness.     

A laminated, flex structure for electronic transport and hybridization of DNA

We have developed the first prototypes of a three-dimensional, electrophoretically driven microlaboratory for the analysis of proteins and DNA. By selecting the appropriate spacing and geometrical configuration, oligonucleotides were transported, in a controlled, rapid fashion, by electrophoresis in free-space. Transport efficiencies over 2 mm distances exceeded 70%. Electrodes of similar design were combined with an electronically addressed DNA hybridization chip to form a fully electrophoretic microlaboratory. In this instance, gold-plated copper electrodes were patterned on a 2 mil thick polyimide substrate. This polyimide layer was stiffened with 20 mil of polyimide to provide support for flip-chip bonding of our standard 100-site Nanochip. This composite structure illustrated three-dimensional transport of target oligonucleotides, through a via in the polyimide, along a series of electrodes and onto the diagnostic chip. Upon reaching the diagnostic chip, electronic hybridization was performed for a competitive reverse dot blot assay. The electronic assay showed a specific to nonspecific ratio in excess of 20:1. These results suggested that this type of structure might be of practical consequence with the development of a microlaboratory for biowarfare applications.     

Microtopography and flow modulate the direction of endothelial cell migration

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 microm in width at a constant depth of 1 microm. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm(2)), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm(2)) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.     

Nano rough micron patterned titanium for directing osteoblast morphology and adhesion

Previous studies have demonstrated greater functions ofosteoblasts (bone-forming cells) on nanophase compared with conventional metals. Nanophase metals possess a biologically inspired nanostructured surface that mimics the dimensions of constituent components in bone, including collagen and hydroxyapatite. Not only do these components possess dimensions on the nanoscale, they are aligned in a parallel manner creating a defined orientation in bone. To date, research has yet to evaluate the effect that organized nanosurface features can have on the interaction of osteoblasts with material surfaces. Therefore, to determine if surface orientation of features can mediate osteoblast adhesion and morphology, this study investigated osteoblast function on patterned titanium substrates containing alternating regions of micron rough and nano rough surfaces prepared by novel electron beam evaporation techniques. This study was also interested in determining whether or not the size of the patterned regions had an effect on osteoblast behavior and alignment. Results indicated early controlled osteoblast alignment on these patterned materials as well as greater osteoblast adhesion on the nano rough regions of these patterned substrates. Interestingly, decreasing the width of the nano rough regions (from 80 microm to 22 microm) on these patterned substrates resulted in a decreased number of osteoblasts adhering to these areas. Changes in the width of the nano rough regions also resulted in changes in osteoblast morphology, thus, suggesting there is an optimal pattern dimension that osteoblasts prefer. In summary, results of this study provided evidence that aligned nanophase metal features on the surface of titanium improved early osteoblast functions (morphology and adhesion) promising for their long term functions, criteria necessary to improve orthopedic implant efficacy.     

Creating biological membranes on the micron scale: forming patterned lipid bilayers using a polymer lift-off technique

We present a new method for creating patches of fluid lipid bilayers with conjugated biotin and other compounds down to 1 microm resolution using a photolithographically patterned polymer lift-off technique. The patterns are realized as the polymer is mechanically peeled away in one contiguous piece in solution. The functionality of these surfaces is verified with binding of antibodies and avidin on these uniform micron-scale platforms. The biomaterial patches, measuring 1 micro m-76 microm on edge, provide a synthetic biological substrate for biochemical analysis that is approximately 100x smaller in width than commercial printing technologies. 100 nm unilamellar lipid vesicles spread to form a supported fluid lipid bilayer on oxidized silicon surface as confirmed by fluorescence photobleaching recovery. Fluorescence photobleaching recovery measurements of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC(18)(3))) stained bilayer patches yielded an average diffusion coefficient of 7.54 +/- 1.25 microm(2) s(-1), equal to or slightly faster than typically found in DiI stained cells. This diffusion rate is approximately 3x faster than previous values for bilayers on glass. This method provides a new means to form functionalized fluid lipid bilayers as micron-scale platforms to immobilize biomaterials, capture antibodies and biotinylated reagents from solution, and form antigenic stimuli for cell stimulation.     

CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia

Transepithelial migration of neutrophils (PMN) is a defining characteristic of active inflammatory states of mucosal surfaces. The process of PMN transepithelial migration, while dependent on the neutrophil beta 2 integrin CD11b/CD18, remains poorly understood. In these studies, we define a monoclonal antibody, C5/D5, raised against epithelial membrane preparations, which markedly inhibits PMN migration across polarized monolayers of the human intestinal epithelial cell line T84 in a bidirectional fashion. In T84 cells, the antigen defined by C5/D5 is upregulated by epithelial exposure to IFN-gamma, and represents a membrane glycoprotein of approximately 60 kD that is expressed on the basolateral membrane. While transepithelial migration of PMN was markedly inhibited by either C5/D5 IgG or C5/D5 Fab fragments, the antibody failed to inhibit both adhesion of PMN to T84 monolayers and adhesion of isolated T84 cells to the purified PMN integrin, CD11b/CD18. Thus, epithelial-PMN interactions blocked by C5/D5 appear to be downstream from initial CD11b/CD18-mediated adhesion of PMN to epithelial cells. Purification, microsequence analysis, and cross-blotting experiments indicate that the C5/D5 antigen represents CD47, a previously cloned integral membrane glycoprotein with homology to the immunoglobulin superfamily. Expression of the CD47 epitope was confirmed on PMN and was also localized to the basolateral membrane of normal human colonic epithelial cells. While C5/D5 IgG inhibited PMN migration even in the absence of epithelial, preincubation of T84 monolayers with C5/D5 IgG followed by antibody washout also resulted in inhibition of transmigration. These results suggest the presence of both neutrophil and epithelial components to CD47-mediated transepithelial migration. Thus, CD47 represents a potential new therapeutic target for downregulating active inflammatory disease of mucosal surfaces.