Effect of upstream priming on transient downstream platelet-substrate interactions.

Upstream exposure of platelets to activating proteins 'primes' platelets for increased downstream adhesion, though the mechanics of platelet translocation before permanently arresting are not well understood. To investigate platelet translocation on platelet-binding proteins, primed platelets' transient contacts with immobilized proteins were recorded and analyzed. Using a microfluidic channel, representative of a vascular graft, platelet-activating proteins were covalently attached to the upstream priming, center, and downstream capture positions. Image sequences of platelet interactions with the center protein were captured as platelet-rich plasma (PRP) was perfused through the channel. There was an increase in both platelet pause events and net platelet adhesion on von Willebrand factor, collagen, or fibrinogen following upstream exposure to the same protein. Upstream priming also caused a decrease in average platelet velocity. The duration of transient platelet arrests on the protein-coated surface and the distance that platelets travel between pause events depended on the protein with which they were interacting. The most significant increase in platelet pause events frequency and decrease in average velocity occurred on immobilized von Willebrand factor, compared to the control with no upstream priming. These results demonstrate that platelet priming increases downstream platelet-protein interactions prior to permanent adhesion.

Microfluidic assay of antiplatelet agents for inhibition of shear-induced platelet adhesion and activation.

We have developed a microfluidic system to perfuse whole blood through a flow channel with an upstream stenotic region and a downstream protein capture region. This flow-based system was used to assay how effectively antiplatelet agents suppress shear-induced platelet adhesion and activation downstream of the stenotic region. Microcontact printing was used to covalently attach one of three platelet binding proteins [fibrinogen, collagen, or von Willebrand factor (vWf)] to the surface of the downstream capture region. Whole blood with an antiplatelet agent was transiently exposed to an upstream high wall shear rate (either 4860 s-1 or 11 560 s-1), and subsequently flowed over the downstream capture region where the platelet adhesion was measured. Several antiplatelet agents (acetylsalicylic acid, tirofiban, eptifibatide, anti-vWf, and anti-GPIbα) were evaluated for their efficacy in attenuating downstream adhesion. Following antibody blocking of vWf or GPIbα, downstream platelet activation was also assessed in perfused blood by flow cytometry using two activation markers (active GPIIb/IIIa and P-selectin). Acetylsalicylic acid demonstrated its inability to diminish shear-induced platelet adhesion to all three binding proteins. GPIIb/IIIa inhibitors (tirofiban and eptifibatide) significantly reduced platelet adhesion to fibrinogen. Antibody blocking of vWf or GPIbα effectively diminished platelet adhesion to all three capture proteins as well as platelet activation in perfused blood, indicating an essential role of vWf-GPIbα interaction in mediating shear-induced platelet aggregation.

Effects of elapsed time on downstream platelet adhesion following transient exposure to elevated upstream shear forces.

Transient exposure to elevated shear forces is known to prime platelets for enhanced downstream adhesion, but how far downstream these priming effects persist is not known. In the present study, the platelet capture regions, prepared by immobilizing fibrinogen, collagen, or von Willebrand factor, were placed at three different distances from the upstream stenotic region to vary the elapsed time of circulating platelets downstream. Platelet adhesion increased with the increase of upstream wall shear rates from 1620 s-1 to 11,560 s-1 for all three downstream proteins, but only the adhesion to fibrinogen increased significantly with the distance between the upstream stenotic region and the downstream capture region. In contrast, platelet adhesion to downstream collagen remained essentially independent on the distance and the adhesion to von Willebrand factor marginally increased with the distance after transient platelet exposure to upstream wall shear rates of 2145 s-1 and 11,560 s-1. The results implied that the activation of fibrinogen receptor GPIIb/IIIa by transient exposure to high upstream wall shear rates progresses in a time-dependent manner during the downstream flow of platelets. The highly elevated upstream wall shear rate of 11,560 s-1 altered the morphology of many platelets adhered to downstream fibrinogen from their native ellipsoidal to spread circular form. The platelet shape analysis showed that longer periods of post-stenotic flow increased the surface coverage fraction of ellipsoidal platelet population and decreased the surface coverage fraction of fully spread platelets on fibrinogen for both transiently elevated upstream wall shear rates.

Downstream platelet adhesion and activation under highly elevated upstream shear forces.

Elevated shear force caused by an anastomotic stenosis is a common complication at the blood vessel-vascular implant interface. Although elevated shear forces were found to cause platelet aggregation around a stenotic region, transient platelet exposure to elevated shear forces and subsequent downstream events occurring under lower shear force were not extensively studied. We hypothesize that effects of elevated shear forces on pre-activation of platelets for downstream adhesion and activation are relevant in understanding the increased thrombotic risk associated with blood-contacting devices. We designed a microfluidic flow system to mimic the hemodynamic environment of vasculature with an upstream anastomotic stenosis with five wall shear strain rates ranging from 1620 s-1 to 11560 s-1. Under shear flow conditions, transient exposure of whole blood to elevated shear forces resulted in higher downstream platelet adhesion onto three different immobilized platelet agonists: fibrinogen, collagen, or von Willebrand factor. Platelet expression of four activation markers (P-selectin, GPIIb/IIIa, lysosomal glycoprotein, and phosphatidylserine) significantly increased after transient exposure to higher upstream wall shear strain rates of 2975-11560 s-1. A significant lysis was observed when platelets were primed by upstream wall shear strain rate of 11560 s-1. These experimental results could be helpful to understand how altered hemodynamics around an anastomotic stenosis promotes thrombus formation downstream. STATEMENT OF SIGNIFICANCE: Studying the downstream response of platelets following transient exposure to an upstream agonist is important because of significant clinical implications to the implantation of vascular devices. Due to intimal fibrous hyperplasia, vascular biomaterials such as synthetic small-diameter vascular grafts sometimes become stenotic (narrow), leading to transient platelet exposure to elevated shear forces. In this study, a microfluidic flow system was developed to mimic a stenosed vascular graft and to investigate how highly elevated, transient upstream shear forces, typically found in severe stenosis, results in the pre-activation of platelets for downstream adhesion and activation. The findings of the present study have implications for optimizing the design of blood-contacting biomaterials in order to minimize thrombotic risk associated with transiently elevated shear forces. The findings also provide additional insights into the mechanisms of thrombus formation at the post-stenotic regions of vascular implants.

Effects of upstream shear forces on priming of platelets for downstream adhesion and activation.

Platelets in flowing blood are sometimes exposed to elevated shear forces caused by anastomotic stenosis at the blood vessel-vascular implant interface. The objective of this study was to determine how effective upstream shear forces are in priming platelets for downstream adhesion and activation. Flow chambers with upstream stenotic regions (shear rates of 400-1000 s-1) were manufactured by relief molding of polydimethylsiloxane. Downstream from the stenotic regions, microcontact printing was used to covalently immobilize three different proteins (fibrinogen, collagen, or von Willebrand factor) to serve as platelet capture agents. Anticoagulated whole blood was perfused through the flow chambers and platelet adhesion to the downstream capture region was quantified. It was found that transient exposure of platelets to increased shear forces resulted in higher platelet adhesion on all three proteins. The duration of the platelet exposure to elevated shear forces was varied by changing the length of the stenotic regions. The results indicated that, in addition to the magnitude of shear forces, the duration of exposure to these forces was also an important factor in priming platelets. The effect of upstream shear forces on platelet activation was assessed by quantifying P-selectin, integrin αIIbß3, lysosomal glycoprotein, and phosphatidylserine exposure using flow cytometry. The results suggested that increased shear forces were capable of increasing the priming of platelets for downstream activation. This study implicates the anastomotic region(s) of vascular implants as a locus of platelet pre-activation that may lead to thrombus formation downstream. STATEMENT OF SIGNIFICANCE: A synthetic small-diameter vascular graft can often become stenotic due to intimal fibrous hyperplasia, either generally along the inside of the graft or at the anastomotic regions, leading to an increased shear force on flowing platelets. Our lab is studying how the upstream platelet preactivation (aka "priming") in flowing blood affects their downstream adhesion and activation. This manuscript describes a study in which priming of platelets is achieved by upstream stenotic narrowing in a microfluidic flow chamber. Such experimental design was intended to mimic a vascular implant with stenotic upstream anastomosis and downstream exposed platelet protein agonists. Understanding how the pre-activated platelets respond to imperfect vascular implant surfaces downstream is an important factor in designing better vascular implants.

Silica Nanoparticle-Endothelial Interaction: Uptake and Effect on Platelet Adhesion under Flow Conditions.

Silica nanoparticles are extensively used in biomedical applications and consumer products. Little is known about the interaction of these NPs with the endothelium and effect on platelet adhesion under flow conditions in circulation. In this study, we investigated the effect of silica nanoparticles on the endothelium and its inflammation, and subsequent adhesion of flowing platelets in vitro. Platelet counts adhered onto the surface of endothelial cells in the presence of nanoparticles increased at both low and high concentrations of nanoparticles. Preincubation of endothelial cells with nanoparticles also increased platelet adhesion. Interestingly, platelet adhesion onto TNF-α-treated endothelial cells decreased in the presence of nanoparticles at different concentrations as compared with the absence of nanoparticles. We monitored the expression of different endothelial proteins, known to initiate platelet adhesion, in the presence and absence of silica nanoparticles. We found that silica nanoparticles caused changes in the endothelium such as overexpression of PECAM that promoted platelet adhesion to the endothelial cell.

Astrocyte spreading and migration on aggrecan-laminin dot gradients.

The surface concentration gradient of two extracellular matrix (ECM) macromolecules was developed to study the migratory and morphological responses of astrocytes to molecular cues typically found in the central nervous system injury environment. The gradient, prepared using microcontact printing, was composed of randomly positioned micrometer-sized dots of aggrecan (AGG) printed on a substrate uniformly coated with laminin (LN). AGG dots were printed in an increasing number along the 1000 µm long and 50 µm wide gradient area which had on each end either a full surface coverage of AGG or LN. Each dot gradient was surrounded by a 100 µm-wide uniform field of AGG printed over laminin. Seeded astrocytes were found to predominantly attach to LN regions on the gradient. Cellular extensions of these cells were longer than the similar processes for cells seeded on uniform substrates of AGG or LN serving as controls. Astrocyte extensions were the largest and spanned a distance of 150 µm when the cells were attached to the mixed AGG+LN patches on the gradient. As evidenced by their increased area and perimeter, the cells extended processes in a stellate fashion upon initial attachment and maintained extensions when seeded in AGG+LN regions but not on uniform laminin controls. The cells migrated short distances, ∼20-35 µm, over 24 h and in doing so preferentially shifted from AGG areas to higher LN surface coverage regions. The results indicated that presenting mixed ECM cues caused astrocytes to sample larger areas of the substrate and made the cells to preferentially relocate to a more permissive ECM region.

Binary agonist surface patterns prime platelets for downstream adhesion in flowing whole blood.

As platelets encounter damaged vessels or biomaterials, they interact with a complex milieu of surface-bound agonists, from exposed subendothelium to adsorbed plasma proteins. It has been shown that an upstream, surface-immobilized agonist is capable of priming platelets for enhanced adhesion downstream. In this study, binary agonists were integrated into the upstream position of flow cells and the platelet priming response was measured by downstream adhesion in flowing whole blood. A nonadditive response was observed in which platelets transiently exposed to two agonists exhibited greater activation and downstream adhesion than that from the sum of either agonist alone. Antibody blocking of one of the two upstream agonists eliminated nonadditive activation and downstream adhesion. Crosstalk between platelet activation pathways likely led to a synergistic effect which created an enhanced activation response in the platelet population. The existence of synergy between platelet priming pathways is a concept that has broad implications for the field of biomaterials hemocompatibility and platelet activity testing.

The Biomechanical Effects of Resuscitation Colloids on the Compromised Lung Endothelial Glycocalyx.

BACKGROUND: The endothelial glycocalyx is an important component of the vascular permeability barrier, forming a scaffold that allows serum proteins to create a gel-like layer on the endothelial surface and transmitting mechanosensing and mechanotransduction information that influences permeability. During acute inflammation, the glycocalyx is degraded, changing how it interacts with serum proteins and colloids used during resuscitation and altering its barrier properties and biomechanical characteristics. We quantified changes in the biomechanical properties of lung endothelial glycocalyx during control conditions and after degradation by hyaluronidase using biophysical techniques that can probe mechanics at (1) the aqueous/glycocalyx interface and (2) inside the glycocalyx. Our goal was to discern the location-specific effects of albumin and hydroxyethyl starch (HES) on glycocalyx function. METHODS: The effects of albumin and HES on the mechanical properties of bovine lung endothelial glycocalyx were studied using a combination of atomic force microscopy and reflectance interference contrast microscopy. Logistic regression was used to determine the odds ratios for comparing the effects of varying concentrations of albumin and HES on the glycocalyx with and without hyaluronidase. RESULTS: Atomic force microscopy measurements demonstrated that both 0.1% and 4% albumin increased the thickness and reduced the stiffness of glycocalyx when compared with 1% albumin. The effect of HES on glycocalyx thickness was similar to albumin, with thickness increasing significantly between 0.1% and 1% HES and a trend toward a softer glycocalyx at 4% HES. Reflectance interference contrast microscopy revealed a concentration-dependent softening of the glycocalyx in the presence of albumin, but a concentration-dependent increase in stiffness with HES. After glycocalyx degradation with hyaluronidase, stiffness was increased only at 4% albumin and 1% HES. CONCLUSIONS: Albumin and HES induced markedly different effects on glycocalyx mechanics and had notably different effects after glycocalyx degradation by hyaluronidase. We conclude that HES is not comparable with albumin for studies of vascular permeability and glycocalyx-dependent signaling. Characterizing the molecular and biomechanical effects of resuscitation colloids on the glycocalyx should clarify their indicated uses and permit a better understanding of how HES and albumin affect vascular function.

Functional assay of antiplatelet drugs based on margination of platelets in flowing blood.

A novel functional assay of antiplatelet drug efficacy was designed by utilizing the phenomena of platelet margination in flowing blood and transient platelet contacts with surface-immobilized platelet agonists. Flow margination enhances transient contacts of platelets with the walls of flow chambers covered with surface-immobilized proteins. Depending on the type and the surface density of the immobilized agonists, such transient interactions could "prime" the marginated platelet subpopulation for enhanced activation and adhesion downstream. By creating an upstream surface patch with an immobilized platelet agonist, platelet flow margination was used to test how effective antiplatelet drugs are in suppressing downstream platelet activation and adhesion. The platelet adhesion downstream was measured by a so-called "capture" patch region close to the distal end of the flow chamber. Platelet adhesion downstream was found to be dose-dependent on the upstream surface coverage of the "priming" patch, with immobilized fibrinogen acting as a platelet agonist. Several antiplatelet agents (acetylsalicylic acid, eptifibatide, and tirofiban) were evaluated for their efficacy in attenuating downstream adhesion after upstream platelet priming. The activation of the platelet population was found to be dependent on both the extent of the upstream agonist stimulus and the antiplatelet drug concentration. Such a relationship provides an opportunity to measure the efficacy of specific antiplatelet agents against the type and concentration of upstream platelet agonists.

Two surface gradients of polyethylene glycol for a reduction in protein adsorption.

Polyethylene glycol (PEG) coatings have been commonly used in reducing protein adsorption with the intent of improving a biomaterial's biocompatibility. To elucidate the role of PEG surface density in reducing protein adsorption, two types of grafted PEG surface density gradients were evaluated for the adsorption and desorption of albumin and fibrinogen, two blood proteins. PEG density gradients were characterized using contact angle measurements and X-ray photoelectron spectroscopy. Total internal reflection fluorescence was used to measure protein adsorption kinetics and adsorption profiles on the two types of PEG gradients. The PEG gradient generated by the flow method decreased adsorption of both proteins in proportion to the PEG surface density; however, their desorption by buffer solution from the grafted PEG layer was not complete. In contrast, desorption of two proteins from the grafted PEG layer generated by a UV oxidation method resulted in near-zero adsorbed amount. The difference between the two types of gradients might have originated from counter-diffusion of PEG and water molecules occurring during the flow method procedure.

Cell substrate patterning with glycosaminoglycans to study their biological roles in the central nervous system.

Microcontact printing (µCP) based techniques have been developed for creating cell culture substrates with discrete placement of CNS-expressed molecules. These substrates can be used to study various components of the complex molecular environment in the central nervous system (CNS) and related cellular responses. Macromolecules such as glycosaminoglycans (GAGs), proteoglycans (PGs), or proteins are amenable to printing. Detailed protocols for both adsorption based as well as covalent reaction printing of cell culture substrates are provided. By utilizing a modified light microscope, precise placement of two or more types of macromolecules by sequential µCP can be used to create desired spatial arrangements containing multicomponent PG, GAG, and protein surface patterns for studying CNS cell behavior. Examples of GAG stripe assays for neuronal pathfinding and directed outgrowth, and dot gradients of PG + laminin for astrocyte migration studies are provided.

Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels.

Exploiting differential surface display of chondroitin sulfate variants for directing neuronal outgrowth.

Chondroitin sulfate (CS) proteoglycans (CSPGs) are known to be primary inhibitors of neuronal regeneration at scar sites. However, a variety of CSPGs are also involved in neuronal growth and guidance during other physiological stages. Sulfation patterns of CS chains influence their interactions with various growth factors in the central nervous system (CNS), thus influencing neuronal growth, inhibition, and pathfinding. This report demonstrates the use of differentially sulfated CS chains for neuronal navigation. Surface-immobilized patterns of CS glycosaminoglycan chains were used to determine neuronal preference toward specific sulfations of five CS variants: CS-A, CS-B (dermatan sulfate), CS-C, CS-D, and CS-E. Neurons preferred CS-A, CS-B, and CS-E and avoided CS-C containing lanes. In addition, significant alignment of neurites was observed using underlying lanes containing CS-A, CS-B, and CS-E chains. To utilize differential preference of neurons toward the CS variants, a binary combinations of CS chains were created by backfilling a neuro-preferred CS variant between the microcontact printed lanes of CS-C stripes, which are avoided by neurons. The neuronal outgrowth results demonstrate for the first time that a combination of sulfation variants of CS chains without any protein component of CSPG is sufficient for directing neuronal outgrowth. Biomaterials with surface immobilized GAG chains could find numerous applications as bridging devices for tackling CNS injuries where directional growth of neurons is critical for recovery.

Astrocytes specifically remove surface-adsorbed fibrinogen and locally express chondroitin sulfate proteoglycans.

Surface-adsorbed fibrinogen (FBG) was recognized by adhering astrocytes, and was removed from the substrates in vitro by a two-phase removal process. The cells removed adsorbed FBG from binary proteins' surface patterns (FBG+laminin, or FBG+albumin) while leaving the other protein behind. Astrocytes preferentially expressed chondroitin sulfate proteoglycan (CSPG) at the loci of fibrinogen stimuli; however, no differences in overall CSPG production as a function of FBG surface coverage were identified. Removal of FBG by astrocytes was also found to be independent of transforming growth factor type ß (TGF-ß) receptor based signaling as cells maintained CSPG production in the presence of TGF-ß receptor kinase inhibitor, SB 431542. The inhibitor decreased CSPG expression, but did not abolish it entirely. Because blood contact and subsequent FBG adsorption are unavoidable in neural implantations, the results indicate that implant-adsorbed FBG may contribute to reactive astrogliosis around the implant as astrocytes specifically recognize adsorbed FBG.

Sugar glues for broken neurons.

Proteoglycans (PGs) regulate diverse functions in the central nervous system (CNS) by interacting with a number of growth factors, matrix proteins, and cell surface molecules. Heparan sulfate (HS) and chondroitin sulfate (CS) are two major glycosaminoglycans present in the PGs of the CNS. The functionality of these PGs is to a large extent dictated by the fine sulfation patterns present on their glycosaminoglycan (GAG) chains. In the past 15 years, there has been a significant expansion in our knowledge on the role of HS and CS chains in various neurological processes, such as neuronal growth, regeneration, plasticity, and pathfinding. However, defining the relation between distinct sulfation patterns of the GAGs and their functionality has thus far been difficult. With the emergence of novel tools for the synthesis of defined GAG structures, and techniques for their characterization, we are now in a better position to explore the structure-function relation of GAGs in the context of their sulfation patterns. In this review, we discuss the importance of GAGs on CNS development, injury, and disorders with an emphasis on their sulfation patterns. Finally, we outline several GAG-based therapeutic strategies to exploit GAG chains for ameliorating various CNS disorders.

Use of reflectance interference contrast microscopy to characterize the endothelial glycocalyx stiffness.

Reflectance interference contrast microscopy (RICM) was used to study the mechanics of the endothelial glycocalyx. This technique tracks the vertical position of a glass microsphere probe that applies very light fluctuating loads to the outermost layer of the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx. Fluctuations in probe vertical position are used to estimate the effective stiffness of the underlying layer. Stiffness was measured before and after removal of specific glycocalyx components. The mean stiffness of BLMVEC glycocalyx was found to be ~7.5 kT/nm(2) (or ~31 pN/nm). Enzymatic digestion of the glycocalyx with pronase or hyaluronan with hyaluronidase increased the mean effective stiffness of the glycocalyx; however, the increase of the mean stiffness on digestion of heparan sulfate with heparinase III was not significant. The results imply that hyaluronan chains act as a cushioning layer to distribute applied forces to the glycocalyx structure. Effective stiffness was also measured for the glycocalyx exposed to 0.1%, 1.0%, and 4.0% BSA; glycocalyx compliance increased at two extreme BSA concentrations. The RICM images indicated that glycocalyx thickness increases with BSA concentrations. Results demonstrate that RICM is sensitive to detect the subtle changes of glycocalyx compliance at the fluid-fiber interface.

The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans.

Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteoglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief ß1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding.

Inducing alignment in astrocyte tissue constructs by surface ligands patterned on biomaterials.

Planar substrates with patterned ligands were used to induce astrocyte alignment whereas substrates with uniform fields of ligand were used to produce random cell orientation. DRG neurons plated on top of oriented astrocyte monolayers exhibited directional outgrowth along aligned astrocytes, demonstrating that purely biological cues provided by the oriented astrocytes were sufficient to provide guidance cues. Antibody blocking studies demonstrated that astrocyte associated FN played a major mechanistic role in directing engineered neurite extension. Our results show that nanometer level surface cues are sufficient to direct nerve outgrowth through an intervening organized astrocyte cell layer. In other studies, we showed that patterned ligands were able to transmit organization cues through multiple cell layers to control the overall alignment of an astrocyte tissue construct, demonstrating how natural scar tissue may develop in situ into potent barriers. In such constructs the spatial organization of astrocyte derived FN maintained its organizational anisotropy throughout the thickness of multilayered astrocyte constructs. These in vitro studies suggest possible roles for such constructs as bridging substrates for neuroregenerative applications.

Multiprotein microcontact printing with micrometer resolution.

Depositing multiple proteins on the same substrate in positions similar to the natural cellular environment is essential to tissue engineering and regenerative medicine. In this study, the development and verification of a multiprotein microcontact printing (µCP) technique is described. It is shown that patterns of multiple proteins can be created by the sequential printing of proteins with micrometer precision in registration using an inverted microscope. Soft polymeric stamps were fabricated and mounted on a microscope stage while the substrate to be stamped was placed on a microscope objective and kept at its focal distance. This geometry allowed for visualization of patterns during the multiple stamping events and facilitated the alignment of multiple stamped patterns. Astrocytes were cultured over stamped lane patterns and were seen to interact and align with the underlying protein patterns.