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From stents and large-diameter vascular grafts, to mechanical heart valves and blood pumps, blood-contacting devices are enjoying significant clinical success owing to the application of systemic antiplatelet and anticoagulation therapies. On the contrary, research into material and device hemocompatibility aimed at alleviating the need for systemic therapies has suffered a decline. This research area is undergoing a renaissance fueled by recent fundamental insights into coagulation and inflammation that are offering new avenues of investigation, the growing recognition of the limitations facing existing therapeutic approaches, and the severity of the cardiovascular disorders epidemic. This Opinion article discusses clinical needs for hemocompatible materials and the emerging research directions for fulfilling those needs. Based on the 2017 BloodSurf conference that brought together clinicians, scientists, and engineers from academia, industry, and regulatory bodies, its purpose is to draw the attention of the wider clinical and scientific community to stimulate further growth. STATEMENT OF SIGNIFICANCE: The article highlights recent fundamental insights into coagulation, inflammation, and blood-biomaterial interactions that are fueling a renaissance in the field of material hemocompatibility. It will be useful for clinicians, scientists, engineers, representatives of industry and regulatory bodies working on the problem of developing hemocompatible materials and devices for treating cardiovascular disorders.

Integrating acoustic wave sensors into lab-on-a-chip (LoC) devices is a well-known challenge. We address this challenge by designing a microfluidic device housing a monolithic array of 24 high-fundamental frequency quartz crystal microbalance with dissipation (HFF-QCMD) sensors. The device features six 6-&micro, L channels of four sensors each for low-volume parallel measurements, a sealing mechanism that provides appropriate pressure control while assuring liquid confinement and maintaining good stability, and provides a mechanical, electrical, and thermal interface with the characterization electronics. We validate the device by measuring the response of the HFF-QCMD sensors to the air-to-liquid transition, for which the robust Kanazawa&ndash, Gordon&ndash, Mason theory exists, and then by studying the adsorption of model bioanalytes (neutravidin and biotinylated albumin). With these experiments, we show how the effects of the protein&ndash, surface interactions propagate within adsorbed protein multilayers, offering essentially new insight into the design of affinity-based bioanalytical sensors.

Quartz crystal microbalance with dissipation monitoring (QCMD) is a simple and versatile sensing technique with applications in a wide variety of academic and industrial fields, most notably electrochemistry, biophysics, quality control, and environmental monitoring. QCMD is limited by a relatively poor time resolution, which is of the order of seconds with conventional instrument designs at the noise level usually required. In this work, we present a design of an ultrafast QCMD with submillisecond time resolution. It is based on a frequency comb approach applied to a high-fundamental-frequency (HFF) resonator through a multifrequency lock-in amplifier. The combination allows us to reach data acquisition rates >10 kHz. We illustrate the method using a toy model of a glass sphere dropped on the resonator surfaces, bare or coated with liposomes, in liquid. We discuss some interesting features of the results obtained with the dropped spheres, such as bending of the HFF resonators due to the impact, sphere bouncing (or the absence of it), and contact aging.

The aim of this work was to investigate the potential of pegylated poly(anhydride) nanoparticles to enhance the oral bioavailability of docetaxel (DTX). Nanoparticles were prepared after the incubation between the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN), poly(ethylene glycol) (PEG2000 or PEG6000) and docetaxel (DTX). The oral administration of a single dose of pegylated nanoparticles to mice provided sustained and prolonged therapeutic plasma levels of docetaxel for up 48–72 h. In addition, the relative oral bioavailability of docetaxel was around 32%. The organ distribution studies revealed that docetaxel underwent a similar distribution when orally administered encapsulated in nanoparticles as when intravenously as Taxotere®. This observation, with the fact that the clearance of docetaxel when loaded into the oral pegylated nanoparticles was found to be similar to that of intravenous formulation, suggests that docetaxel would be released at the epithelium surface and then absorbed to the circulation.

Annexins are a family of soluble proteins that bind to acidic phospholipids such as phosphatidylserine in a calcium-dependent manner. The archetypical member of the annexin family is annexin A5. For many years, its function remained unknown despite the availability of a high-resolution structure. This, combined with the observations of specific ion conductance in annexin-bound membranes, fueled speculations about the possible membrane-spanning forms of annexins that functioned as ion channels. The channel hypothesis remained controversial and did not gather sufficient evidence to become accepted. Yet, it continues to draw attention as a framework for interpreting indirect (e.g., biochemical) data. The goal of the mini-review is to examine the data on annexin-lipid interactions from the last ~30 years from the point of view of the controversy between the two lines of inquiry: the well-characterized peripheral assembly of the annexins at membranes vs. their putative transmembrane insertion. In particular, the potential role of lipid rearrangements induced by annexin binding is highlighted.

Treatment of osseoimplant surfaces with autologous platelet-rich plasma prepared according to the plasma rich in growth factors (PRGF-Endoret) protocol prior to implantation yields promising results in the clinic. Our objective is to understand the organization of complex interfaces between blood plasma preparations of various compositions and model titania surfaces. Here we present the results of the morphological and chemical characterization of TiO(2) surfaces incubated with four types of blood plasma preparations devoid of leukocytes and red blood cells: either enriched in platelets (PRGF-Endoret) or platelet-depleted, and either activated with CaCl(2) to induce clotting, or not. Chemical characterization was done by time-of-flight secondary ion mass spectrometry with principal component analysis (ToF-SIMS/PCA). The interface morphology was studied with scanning electron and atomic force microscopy. Immunofluorescence microscopy was used to identify platelets and infer their activation state. We observe clear differences among the four types of interfaces by ToF-SIMS/PCA. Some of these could be straightforwardly related to the differences in the sample morphology and known effects of platelet activation, but others are more subtle. Strikingly, it was possible to differentiate between these samples by ToF-SIMS/PCA of the protein species alone. This clearly indicates that the composition, orientation, and/or conformation of the proteins in these specimens depend both on the platelets' presence and on their activation. The ToF-SIMS imaging functionality furthermore provides unique insight into the distribution of phospholipid species in these samples.

The worldwide cardiovascular disease (CVD) epidemic is of grave concern. A major role in the etiology of CVDs is played by the platelets (thrombocytes). Platelets are anuclear cell fragments circulating in the blood. Their primary function is to catalyze clot formation, limiting traumatic blood loss in the case of injury. The same process leads to thrombosis in the case of CVDs, which are commonly managed with antiplatelet therapy. Platelets also have other, nonhemostatic functions in wound healing, inflammation, and tissue regeneration. They play a role in the early stages of atherosclerosis and the spread of cancer through metastases. Much remains to be learned about the regulation of these diverse platelet functions under physiological and pathological conditions. Breakthroughs in this regard are expected to come from single platelet studies and systems approaches. The immobilization of platelets at surfaces is advantageous for developing such approaches, but platelets are activated when they come in contact with foreign surfaces. In this work, we develop and validate a protocol for immobilizing platelets on supported lipid bilayers without activation due to immobilization. Our protocol can therefore be used for studying platelets with a wide variety of surface-sensitive techniques.

Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process.

There is a widely recognized need to improve the performance of vascular implants and external medical devices that come into contact with blood by reducing adverse reactions they cause, such as thrombosis and inflammation. These reactions lead to major adverse cardiovascular events such as heart attacks and strokes. Currently, they are managed therapeutically. This need remains unmet by the biomaterials research community. Recognized stagnation of the blood-biomaterial interface research translates into waning interest from clinicians, funding agencies, and practitioners of adjacent fields. The purpose of this contribution is to stir things up. It follows the 2014 BloodSurf meeting (74th International IUVSTA Workshop on Blood-Biomaterial Interactions), offers reflections on the situation in the field, and a three-pronged strategy integrating different perspectives on the biological mechanisms underlying blood-biomaterial interactions. The success of this strategy depends on reengaging clinicians and on the renewed cooperation of the funding agencies to support long-term efforts.

Quartz crystal microbalance (QCM) is widely used for studying soft interfaces in liquid environment. Many of these interfaces are heterogeneous in nature, in the sense that they are composed of discrete, isolated entities adsorbed at a surface. When characterizing such interfaces, one is interested in determining parameters such as surface coverage and size of the surface-adsorbed entities. The current strategy is to obtain this information by fitting QCM data--shifts in resonance frequency, DeltaF, and bandwidth, DeltaGamma--with the model derived for smooth, homogeneous films using the film acoustic thickness and shear elastic moduli as fitting parameters. Investigating adsorption of liposomes and icosahedral virus particles on inorganic surfaces of titania and gold, we demonstrate that the predictions of this model are at variance with the experimental observations. In particular, while the model predicts that the ratio between the bandwidth and frequency shifts, DeltaGamma/DeltaF (the Df ratio), should increase with both surface coverage and particle size, we observe that this ratio increases with increasing particle size but decreases with increasing surface coverage, demonstrating that QCM response in heterogeneous films, such as those composed of adsorbed colloidal particles, does not conform with the predictions of the homogeneous film model. Employing finite element method (FEM) calculations, we show that hydrodynamic effects are the cause of this discrepancy. Finally, we find that the size of the adsorbed colloidal particles can be recovered from a model-independent analysis of the plot of the DeltaGamma/DeltaF ratio versus the frequency shift on many overtones.

Recent years have been ripe with discoveries of non-haemostatic platelet functions. This led to the appreciation of the significant, previously unknown, role played by the platelets in various pathologies and regenerative processes. As a result, exciting opportunities for clinical applications in fields as diverse as regenerative medicine and cancer treatment are emerging. However, their realization depends on the understanding of the regulatory mechanisms governing these diverse platelet functions, so that particular platelet responses could be artificially tailored to specific clinical situations. Current understanding of the signalling pathways controlling haemostatic responses is rooted in the development of quantitative assays for measuring them and sensitive markers for their quantification. However, the existing assays and markers are not sufficiently sensitive for distinguishing between individual signalling pathways and unravelling inter-pathway connections. Moreover, entirely new approaches are needed for studying non-haemostatic platelet functions, since there are currently no assays or markers for quantifying them. We review the on-going efforts in these directions, including our own recent work on using lectins as sensitive probes for profiling platelet activation.

Supported phospholipid bilayers (SPBs) are useful for studying cell adhesion, cell-cell interactions, protein-lipid interactions, protein crystallization, and applications in biosensor and biomaterial areas. We have recently reported that SPBs could be formed on titanium dioxide, an important biomaterial, from vesicles containing anionic phospholipid phosphatidyl serine (PS) in the presence of calcium. Here, we show that the mobility of the fluorescently labeled PS present in these bilayers is severely restricted, whereas that of the zwitterionic phosphatidyl choline is not affected. Removal of calcium alleviated the restriction on the mobility of PS. Both components were found to be mobile in SPBs of identical compositions prepared in the presence of calcium on silica. To explain these results, we propose that, on TiO2, PS is trapped in the proximal leaflet of the bilayers. This proposal is supported by the results of protein adsorption experiments carried out on bilayers containing various amounts of PS prepared on silica and titania.

Titanium is widely used in biomedical applications. Its mechanical properties and biocompatibility, conferred by a layer of oxide present on its surface, make titanium the material of choice for various implants (artificial hip and knee joints, dental prosthetics, vascular stents, heart valves). Furthermore, the high refractive index of titanium oxide is advantageous in biosensor applications based on optical detection methods. In both of the above fields of application, novel surface modification strategies leading to biointeractive interfaces (that trigger specific responses in biological systems) are continuously sought. In this report, we investigate the interactions between TiO2 and phosphatidyl serine-containing liposomes, present a novel approach for preparing supported phospholipid bilayers (SPBs) of various compositions on TiO2, and use the unique ability of liposomes to distinguish between different surfaces to create SPB corrals on SiO2/TiO2 structured substrates. These results represent an important first step toward the design of biointeractive interfaces on titanium oxide surfaces that are based on a cell membrane-like environment.

Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.

Advancement in various fields ranging from biomaterials to cell biology is fostered by the evolution in patterning technology, that delivers smaller and smaller features exhibiting distinct (bio)chemical contrast and addressability. We have recently introduced a simple and versatile patterning technique based on selective adsorption from aqueous media of multifunctional organic molecules onto oxide substrates pre-patterned by lithographic methods. Here we demonstrate the use of this technique, termed selective molecular assembly patterning, for the preparation of cell-adhesive patterns of arbitrary geometry, and their use to investigate the relationship between pattern geometry and the organization of elements of the cell adhesion apparatus, namely focal contacts and stress fibres. Cell-culture compatible substrates with patterns consisting of ligands for cell-adhesion or other receptors represent a promising experimental tool for investigating cell–surface interactions with the goal of elucidating the mechanisms of how cells sense structural cues of the extracellular matrix.

Interactions between the graft copolymer poly(L-lysine)-g-poly(ethylene glycol), PLL-g-PEG, and two kinds of surface-supported lipidic systems (supported phospholipid bilayers and supported vesicular layers) were investigated by a combination of microscopic and spectroscopic techniques. It was found that the application of the copolymer to zwitterionic or negatively charged supported bilayers in a buffer of low ionic strength led to their decomposition, with the resulting formation of free copolymer–lipid complexes. The same copolymer had no destructive effect on a supported vesicular layer made up of vesicles of identical composition. A comparison between poly(L-lysine), which did not induce decomposition of supported bilayers, and PLL-g-PEG copolymers with various amounts of PEG side chains per backbone lysine unit, suggested that steric repulsion between the PEG chains that developed upon adsorption of the polymer to the nearly planar surface of a supported phospholipid bilayer (SPB) was one of the factors responsible for the destruction of the SPBs by the copolymer. Other factors included the ionic strength of the buffer used and the quality of the bilayers, pointing toward the important role defects present in the SPBs play in the decomposition process.

A quartz crystal microbalance with dissipation, or QCM-D, allows the properties of a loaded quartz oscillator—resonance frequency and dissipation [1/(the quality factor)]—to be monitored on several overtones. The frequency and the dissipation factor of the loaded oscillator are functions of the physical properties (such as thickness, density, viscosity, elasticity, roughness) of the media loading the resonator (e.g., a liquid, a thin polymer film, a phospholipid bilayer, etc.) and the frequency at which the measurement was performed. In this contribution, it is shown that the frequencies measured with the QCM-D instrument on the first, third, fifth, and seventh overtones do not agree with those expected of an ideal, infinite crystal. Therefore, the results of QCM-D measurements cannot be directly compared with theoretical models that require the resonance frequency of an unloaded crystal to be used as one of the input parameters. A phenomenological procedure for dealing with this problem is proposed.

Titanium is a widely used biomaterial, and there is, therefore, considerable interest in studying the processes occurring at its surface. A suitable in situ investigation technique is atomic force ...

A novel patterning technique that combines colloidal patterning with selective adsorption of organic molecules has been used to chemically pattern metal oxide surfaces at length scales down to 50 nm. Lithographic nanofabrication using surface-assembled colloids as etch masks (“colloidal lithography”) was used to create nanopillars of TiO2 (50−90 nm in diameter, ∼20 nm in height) on whole oxidized silicon or quartz wafer substrates. These nanopillars were then rendered hydrophobic by the selective self-assembly of an organophosphate, whereas a poly(ethylene glycol)-grafted copolymer was adsorbed onto the surrounding SiO2 rendering it protein resistant. This resulted in a two-component chemical pattern, displaying contrast with respect to protein adsorption (protein-adhesive pillars on nonadsorbing background). This property allows for efficient translation of the lithographic pattern into a surface protein pattern by two simple dip-and-rinse processes in aqueous solutions. The feasibility of the method and...

A novel patterning technique based on selective self-assembly of alkane phosphates on metal oxide surfaces is presented. Standard photolithography was used to create patterns of titanium dioxide within a matrix of silicon dioxide. Alkane phosphates were found to self-assemble on TiO2, but not on SiO2, surfaces. Subsequent adsorption of poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) rendered the exposed SiO2 surface resistant to protein adsorption. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry were employed to monitor the assembly processes. Protein-adsorption studies by means of fluorescence microscopy conclusively established that the resulting surfaces displayed protein-adhesive, alkyl phosphate modified TiO2 features, arranged within a protein-resistant PLL-g-PEG-modified SiO2 matrix. Human foreskin fibroblasts, incubated in a serum-containing medium, were found to selectively attach to the protein-adhesive areas, where they developed focal contacts. No interac...

We review recent progress in the study of the structure and dynamics of phospholipid membranes and associated proteins, using novel label-free analytical tools. We describe these techniques and illustrate them with examples highlighting current capabilities and limitations. Recent advances in applying such techniques to biological and model membranes for biophysical studies and biosensing applications are presented, and future prospects are discussed.

Annexin A5 is a soluble protein which binds to negatively charged phospholipids, such as dioleoylphosphatidylserine (DOPS), in the presence of Ca2+. A solid-solid phase transition between two two-dimensional crystal forms of annexin A5, p6 and p3, previously identified by electron microscopy (EM), was studied in situ on supported phospholipid bilayers (SPBs) by atomic force microscopy (AFM) and ex situ on lipid monolayers by EM. The ability to directly vary the protein surface density on SPBs allowed a delicate and systematic way to investigate the reversibility of the transition with the AFM. The p6 crystal form was found to form first and to cover the entire lipid surface available, at all DOPS concentrations studied (5-95%). An increase in protein surface density, achievable only on SPBs (or monolayers) containing sufficient amounts of DOPS, destabilized the p6 phase. The transition to the more compact (p3) form occurred via local melting of the p6 phase at defects, such as grain boundaries, initially present in p6 crystals. On mono- or bilayers with lower DOPS contents, the density-dependent p6-p3 transition was not observed, but the p3 crystal form could still be reached by disturbing the system mechanically.

Quartz crystal microbalance (QCM) is emerging as a versatile tool for studying lipid phase behavior. The technique is attractive for fundamental biophysical studies as well applications because of its simplicity, flexibility, and ability to work with very small amounts of material crucial for biomedical studies. Further progress hinges on the understanding of the mechanism, by which a surface-acoustic technique such as QCM, senses lipid phase changes. Here, we use a custom-built instrument with improved sensitivity to investigate phase behavior in solid-supported lipid systems of different geometries (adsorbed liposomes and bilayers). We show that we can detect a model anesthetic (ethanol) through its effect on the lipid phase behavior. Further, through the analysis of the overtone dependence of the phase transition parameters, we show that hydrodynamic effects are important in the case of adsorbed liposomes, and viscoelasticity is significant in supported bilayers, while layer thickness changes make up the strongest contribution in both systems.

The behavior of small liposomes on surfaces of inorganic oxides remains enigmatic. Under appropriate conditions it results in the formation of supported lipid bilayers (SLBs). During this process, some lipids leave the surface (desorb). We were able to visualize this by a combination of time-resolved fluorescence microscopy and fluorescence recovery after photobleaching studies. Our observations also allowed us to analyze the kinetics of bilayer patch growth during the late stages of SLB formation. We found that it entails a balance between desorption of excess lipids and further adsorption of liposomes from solution. These studies were performed with liposomes containing zwitterionic phospholipids (dioleoylphosphatidylcholine alone or a mixture of dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and cholesterol) on TiO2 in the presence of Ca(2+) but in the absence of other salts.

Annexin V is a member of a family of structurally homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca2+-dependent manner. The structure of the soluble form of annexin. V has been solved by X-ray crystallography, while electron crystallography of two-dimensional (2D) crystals has been used to reveal the structure of its membrane-bound form. Two 2D crystal forms of annexin V have been reported to date, with either p6 or p3 symmetry. Atomic force microscopy has previously been used to investigate the growth and the topography of the p6 crystal form on. supported phospholipid bilayers (Reviakine et al., 1998). The surface structure of the second crystal form, p3, is presented in this study, along with an improved topographic map of the p6 crystal form. The observed topography is correlated with the structure determined by X-ray crystallography. (C) 2000 Academic Press.

Theories of crystallization, both in 3-D and 2-D, are still very limited, mainly due to the scarcity of experimental approaches providing pertinent data on elementary phenomena. We present here a novel experimental approach for following, in real time and in situ, the process of 2-D crystallization of proteins on solid supports. Using annexin V as a model of a protein binding by affinity to a lipid matrix, we show that 2-D crystals of proteins can be formed on supported planar lipid bilayers (SPBs). Atomic Force Microscopy (AFM) enables the process of 2-D crystal growth to be visualized. The submolecular organization of the crystals was characterized at a resolution of approximately 2 nanometers, and defects, hitherto not observed in protein crystals, were resolved. These results have potential applications in basic and applied sciences. Copyright 1998 Academic Press.

Solid-supported lipid bilayers are used as cell membrane models and form the basis of biomimetic and biosensor platforms. The mechanism of their formation from adsorbed liposomes is not well-understood. Using membrane-permeable solute glycerol, impermeable solutes sucrose and dextran, and a pore forming peptide melittin, we studied experimentally how osmotic effects affect the kinetics of the adsorbed liposome-to-bilayer transition. We find that its rate is enhanced if adsorbed liposomes are made permeable but is not significantly retarded by impermeable solutes. The results are explained in terms of adsorbed liposome deformation and formation of transmembrane pores.

Bacterial cellulose (BC) has been suggested to be a suitable biomaterial for the development of cardiovascular grafts. The combination of BC with polyvinyl alcohol (PVA) results in nanocomposites with improved properties. Surprisingly, there are very few studies on the BC–blood interaction. This is the focus of this paper. We present the first thorough assessment of the hemocompatibility of the BC/PVA nanocomposite. Whole blood clotting time, plasma recalcification, Factor XII activation, platelet adhesion and activation, hemolytic index and complement activation are all determined. The platelet activation profiles on BC and BC/PVA surfaces are comprehensively characterized. BC and BC/PVA outperformed ePTFE – used as a point of comparison – thus evidencing their suitability for cardiovascular applications., Funding for this work was made possible by the FCT fund-ing through the project PTDC/EBB-EBI/112170/2009, FCT PhDgrant (SFRH/BD/66094/2009) and COST-Short Term Mission (COST-STSM-TD1003-8978). Additionally, funding from the Departmentof Industry of the Basque Government program ETORTEK and fromthe Spanish Ministry of Science and Innovation (basic researchgrant CTQ2009-11245 to IR) are also gratefully acknowledged.

A range of new functional copolymers bearing aldehyde and carboxylic acid groups have been prepared by functionalization of poly(4-methylstyrene). These polymers have molecular weights from 2,000 to 16,500 and contain up to 20% aldehyde and up to 90% carboxylic acid groups. The reaction proceeds by selective catalytic oxidations with molecular oxygen or air in acetic acid/organic cosolvent mixtures in presence of cobalt acetate and sodium bromide or hydrogen bromide. The effects of reaction temperature, catalyst, co-solvents and oxygen partial pressure on the reaction are described. © 1995 John Wiley & Sons, Inc.

In this study, the activation of purified human platelets due to their adhesion on glass and TiO2 in the absence of extracellular calcium was investigated. Differences in α-granule secretion between platelets adhering on the two surfaces were detected by examining the expression and secretion of the α-granule markers P-selectin (CD62P) and β-thromboglobulin. Similarly, differences in the expression of phosphatidylserine (PS), and in the activation of the major integrin GPIIb/IIIa, on the surfaces of the adhering platelets, were also observed. While all of these activation markers were expressed in platelets adhering on glass, the surface markers were not expressed in platelets adhering on TiO2, and β-thromboglobulin secretion levels were substantially reduced. Differences in marker expression and secretion correlated with differences in the intracellular calcium dynamics. Calcium ionophore treatment triggered α-granule secretion and PS expression in TiO2-adhering platelets but had no effect on the activation of GPIIb/IIIa. These results demonstrate specificity in the way surfaces of artificial materials activate platelets, link differences in the intracellular calcium dynamics observed in the platelets adhering on the two surfaces to the differences in some of the platelet responses (α-granule secretion and PS expression), but also highlight the involvement of synergistic, calcium-independent pathways in platelet activation. The ability to control activation in surface-adhering platelets makes this an attractive model system for studying platelet signaling pathways and for tissue engineering applications.

Cagigal 19, 01007 Vitoria, SpainReceived 1 June 2012; revised 1 August 2012; accepted 6 August 2012Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34428Abstract: Plasma rich in growth factors (PRGFs) technologyis an autologous platelet-rich plasma approach that providesa pool of growth factors and cytokines that have been shownto increase tissue regeneration and accelerate dental implantosseointegration. In this framework, the spatiotemporalrelease of growth factors and the establishment of a provi-sional fibrin matrix are likely to be key aspects governing thestimulation of the early phases of tissue regeneration aroundimplants. We investigated the kinetics of growth factorrelease at implant surfaces functionalized either with PRGFsor platelet-poor plasma and correlated the results obtainedwith the morphology of the resulting interfaces. Our mainfinding is that activation and clot formation favors longer res-idence times of the growth factors at the interfaces studied,probably due to their retention in the adsorbed fibrin matrix.The concentration of the platelet-derived growth factorsabove the interfaces becomes negligible after 2–4 days and issignificantly higher in the case of activated interfaces than inthe case of nonactivated ones, whereas that of the plasmatichepatocyte growth factor is independent of platelet concen-tration and activation, and remains significant for up to 9days. Platelet-rich plasma preparations should be activated topermit growth factor release and thereby facilitate implantsurface osseointegration.

Deformation of surface-adsorbed liposomes is an important parameter that governs the kinetics of their transformations, but one that is very difficult to measure in the case of nm-size liposomes. We investigate the deformation of dimyristoyl phosphatidyl choline liposomes by quartz crystal microbalance (QCM) as a function of temperature and show that it follows the dependence of this lipids bending modulus on temperature, as expected from theoretical considerations. To corroborate our approach, we model QCM response from adsorbed liposomes by explicitly considering their shape and mechanical properties. © 2012 American Institute of Physics., We would like to acknowledge the funding from the Department of Industry of the Basque Government (Program ETORTEK) and from the Spanish Ministry of Science and Innovation (MICINN, ref. CTQ2009-11245) to I.R. and thank David Gil Carton from the CIC bioGUNE Electron Microscopy facility (Derio, Spain) for assistance with the use of their CryoTEM equipment.

Deformation of liposomes upon adsorption at solid surfaces is an important parameter that is related to the lipid-surface interactions and that governs the kinetics of surface-adsorbed liposome transformations. It is challenging to study in the case of nm-sized liposomes. The extent of adsorbed liposome deformation is expected to depend on the bilayer bending modulus. This parameter can be varied in a predictable manner by changing the temperature. In this study, we study the adsorption of dimyristoyl phosphatidyl choline (DMPC) liposomes adsorbed TiO2 as a function of temperature by quartz crystal microbalance (QCM). Using model-free analysis of QCM data, We show that the size of surface-adsorbed DMPC liposomes follows the dependence of this lipid's bending modulus on temperature.

Over the last 2 decades, the quartz crystal microbalance (QCM or QCM-D) has emerged as a versatile tool for investigating soft and solvated interfaces between solid surfaces and bulk liquids because it can provide a wealth of information about key structural and functional parameters of these interfaces. In this Feature, we offer QCM users a set of guidelines for interpretation and quantitative analysis of QCM data based on a synthesis of well-established concepts rooted in rheological research of the last century and of new results obtained in the last several years.

Interactions between surfaces of inorganic materials and biological systems are important in numerous technological contexts (implant integration, biosensor development). They also present basic challenges. For example, the role of surface ion equilibrium in the biological response to the material is not well understood, although a casual link between the two has been proposed. Here, we investigate the behaviour of phosphatidyl serine (PS)-containing liposomes on TiO2 as a function of liposome PS contents and solution Ca2+ concentration. We determine a “phase diagram”, where a percolation-type transition between adsorbed liposomes and supported bilayers is observed, describe the driving force for this transition, and identify the role of surface heterogeneities in this porcess. Finally, we quantify the distribution of PS in the resulting supported bilayers by neutron reflectometry.

The quartz crystal microbalance (QCM) has become a popular method to study the formation of surface-confined films that consist of discrete biomolecular objects--such as proteins, phospholipid vesicles, virus particles--in liquids. The quantitative interpretation of QCM data--frequency and bandwidth (or, equivalently, dissipation) shifts--obtained with such films is limited by the lack of understanding of the energy dissipation mechanisms that operate in these films as they are sheared at megahertz frequencies during the QCM experiment. Here, we investigate dissipation mechanisms in such films experimentally and by finite-element method (FEM) calculations. Experimentally, we study the adsorption of globular proteins and virus particles to surfaces with various attachment geometries: direct adsorption to the surface, attachment via multiple anchors, or attachment via a single anchor. We find that the extent of dissipation caused by the film and the evolution of dissipation as a function of surface coverage is not dependent on the internal properties of these particles but rather on the geometry of their attachment to the surface. FEM calculations reproduce the experimentally observed behavior of the dissipation. In particular, a transient maximum in dissipation that is observed experimentally is reproduced by the FEM calculations, provided that the contact zone between the sphere and the surface is narrow and sufficiently soft. Both a small-angle rotation of the sphere in the flow field of the background fluid (rocking) and a small-amplitude slippage (sliding) contribute to the dissipation. At high coverage, lateral hydrodynamic interactions between neighboring spheres counteract these modes of dissipation, which results in a maximum in dissipation at intermediate adsorption times. These results highlight that, in many scenarios of biomolecular adsorption, the dissipation is not primarily determined by the adsorbate itself, but rather by the link by which it is bound to the substrate.

Quartz crystal microbalance (QCM or QCM-D) has become instrumental in life sciences and biosensor research. It is routinely and successfully used for monitoring interfacial processes, such as protein adsorption and conformational changes in the protein-adsorbed films, liposome-surface interactions and supported bilayer formation, and in the development of biosensor platforms. However, quantitative interpretation of QCM data from biological interfaces studied in liquid remains challenging. In vacuum, the so-called Sauerbrey relationship is routinely used to relate QCM frequency shifts due to the adsorbed layer to the mass of the adsorbed layer. Deviations from Sauerbrey relationship are typically observed when studying soft interfaces in liquids; these are interpreted in terms of layer viscoelastic properties. In this study, we develop and use a combined atomic force microscopy (AFM)-QCM setup to investigate the adsorption of protein ferritin on the surface of gold. First, we find that deviations from the Sauerbrey relationship in this system originate almost entirely from the heterogeneity of the protein films caused by the presence of impurities. Second, relying on the ability of AFM to visualize single ferritin molecules adsorbed on the surface, we find that the frequency shifts determined by QCM are not linearly related to the protein surface coverage.

A quartz crystal microbalance was integrated into an AFM in order to monitor the adsorption of biomolecules to the resonator surface with both atomic force microscopy and microgravimetry. The comparison between the two techniques allows the fractional coverage of the surface, theta, to be correlated with the frequency shift of the resonator, deltaf. The adsorbed material was ferritin, which is a spherical protein with a diameter of approximately 12 nm. Even ata coverage below 50%, the protein layer exhibits Sauerbrey-like behavior, meaning that the magnitude in the frequency shift [deltaf] much exceeds the shift in bandwidth and that the normalized frequency shift, deltaf/n (n the overtone order), is similar on the different overtones. The relation between coverage and frequency shift was found to be nonlinear. In order to model this situation, we performed finite element method calculations based on the incompressible Navier-Stokes equation. The comparison between the model and the experiment suggests that the deformation of the protein upon adsorption is small. For low coverage, the volume of the trapped solvent exceeds the volume of the adsorbate itself. The ratio of the two decreases with increasing coverage. This is the cause of the experimentally observed nonlinear relationship between the surface coverage and frequency shift. Comparing frequency shifts at different overtones, one finds that deltaf/n slightly decreases with overtone order. Such a behavior is typically attributed to softness. The model shows that, for the adsorbed spheres, this apparent softness arises through a rocking motion of the spheres at the surface instead of the shear deformation. Also, there is a hydrodynamic contribution (related to roughness) to the overtone dependence of deltaf/n.

A quartz crystal microbalance (QCM) is described, which is based on a torsional resonator, rather than a conventional thickness-shear resonator. Typical applications are measurements of film thickness in the coating industry and monitoring of biofouling. The torsional QCM is about a factor of 100 less sensitive than the conventional QCM. On the other hand, it can probe film thicknesses in the range of hundreds of microns, which is impossible with the conventional QCM due to viscoelastic artifacts. Data acquisition and data analysis proceed in analogy to the conventional QCM. An indicator of the material’s softness can be extracted from the bandwidth of the resonance. Within the small-load approximation, the frequency shift is independent of whether the sample is applied to the face or to the side of the cylinder. Details of the geometry matter if the viscoelastic properties of the sample are of interest.

The work describes the use of readily available and economical quartz crystal torsional resonators for microweighing. The resonance behavior was probed by impedance analysis. Torsional resonators are straightforwardly adapted to the detection electronics usually employed for driving a quartz crystal microbalance. The analysis proceeds as with quartz crystal resonators. The measurement of the viscoelastic parameters of a silicone oil gave results consistent with the corresponding low-frequency data. Thickness determinations on a film formed from a polymer dispersion have confirmed that torsional resonators indeed function as microbalances when the sample is thin enough. Biofilm formation is easily monitored, as well.

During layerwise growth of crystals, capillarity governs the generation of new crystal layers. Theory predicts that the line tension of the layer edge determines, via the characteristic two-dimensional capillary length L(c), the rates of generation and initial growth of the new layers. To test the correlation between L(c) and the rate of layer generation, we used in situ Tapping Mode Atomic Force Microscopy (TM-AFM) to study the generation and spreading of layers during crystallization of rhombohedral, R3, porcine insulin. We show that crystallization of this insulin form is uniquely suitable for such an investigation due to the linear kinetics of step growth it exhibits. This linear kinetics reflects the abundance of the incorporation sites along the rough steps, the lack of long-range step-step interactions, and the transport control of the growth kinetics. The kinetic coefficients are 7 x 10(-)(3) and 4 x 10(-)(2) cm s(-)(1), respectively, in the absence and presence of the cosolvent acetone-somewhat high for proteins and comparable to values for inorganic systems. We show that (i). the relevant capillary length, the size of a critical quadrangular 2D nucleus L(c), is the main scaling factor for the density of growth steps, while (ii). all steps longer than L(c) grow with a rate determined only by the supersaturation and independent of their length. We explain the divergence of (ii). from theoretical predictions with the high supersaturations typical of the growth of this protein system.

Bunching of growth steps plagues layerwise crystallization of materials in laboratory, industrial, and geological environments, and theory predicts that equidistant step trains are unstable under a variety of conditions. Searching for an example of stable equidistant step trains, we monitored the generation and spatiotemporal evolution of step trains on length scales from 100 nm to 1 mm during the crystallization of insulin, using atomic force microscopy and phase-shifting interferometry. We show that near-equidistant step trains are generated by single and cooperating screw dislocation. The lack of step-step interaction and the overall transport-controlled growth regime further regularize the step train and ensure the stability of the obtained equidistant arrangement.

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the alpha-helical state that is formed upon binding to a hydrophobic solid, and the beta-sheet state, which is formed at the air-water interface. Here, we show that the formation of the beta-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the alpha-helical state of SC3. The second intermediate is rich in beta-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.