Dynamic blood cell contact with biomaterials: validation of a flow chamber system according to international standards

The increasing number of patients requiring prosthetic substitution of segments of the vascular system strongly supports the need to optimize a relevant, standardized testing panel for new materials designed for synthetic vascular prostheses. The ISO gives the standard requirements for testing biomaterials provided for implantation. Our primary interest was the establishment of a reliable in vitro panel as a useful and relevant screening system for vascular implant devices to evaluate blood/device interactions under flow conditions. The aim of the present study was to evaluate influences of different flow conditions on blood cell–biomaterial interactions with special emphasis on the interactions of human granulocytes (PMN) and polymeric surfaces. PMN were isolated and vital cells were quantified by flow cytometrical analysis directly before, as well as immediately after the experiments. The viscosity of the final cellular suspension was analysed by using a computerized cone-plate rheometer. As reference materials we used FEP-teflon, PVC-DEHD, PU, PP and PE. Dacron and ePTFE synthetic vascular protheses were tested in a comparative way to those references. The adhesion processes were observed over a period of 40 minutes under arterial (shear stress 0.74 Pa) and venous (shear stress 0.16 Pa) flow conditions in a parallel plate flow chamber system under highly standardized conditions and laminar flow. The cells were observed with the help of inverse light microscopy. Cell behaviour was recorded and analysed in both analogue (video) and digital (imaging system) modes. Samples of the cell suspensions were obtained at regular time intervals and analysed by enzyme linked immuno sorbent assay (ELISA) to quantify LTB4 release. Irrespective of the material, approximately 3 to 4 times more PMN adhered to the biomaterial surfaces under venous flow conditions compared to the arterial. Shear intensity did not influence the running order of biomaterials with respect to cell numbers. This response in descending order at the end of the experiments was as follows: PU, PVC-DEHD, PP, PE and ePTFE. The biochemical analyses indicate that in the system used only a weak effect on LTB4 release induced by the different materials could be determined. A significant effect caused by flow conditions was not observed. Further experiments, both static as well as dynamic, must be performed for multiple, relevant parameters of haemocompatibility, for potential biomaterials as well as those currently in use in vascular prostheses.     

A novel strategy to graft RGD peptide on biomaterials surfaces for endothelization of small-diamater vascular grafts and tissue engineering blood vessel

To improve the performance of small-diamater vascular grafts, endothelization of biomaterials surfaces and tissue engineering are more promising strategies to fabricate small-diamater vascular grafts. In this study, a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide was grafted on the surfaces of poly(carbonate urethane)s (PCUs), with photoactive 4-benzoylbenzoic acid (BBA) by UV irradiation. The photoactive peptides (BBM-GRGDSP) were synthesized with classical active ester of peptide synthesis. The modified surfaces of PCU with the photoactive RGD peptides were characterized by water contact angle measurement and X-ray Photoelectron Spectroscopy (XPS), which results suggested that the peptides were successfully grafted on the PCU surfaces. The effect of these modified surfaces on endothelial cells (ECs) adhesion and proliferation was examined over 72 h. PCU surfaces coupled with the synthetic photoactive RGD peptides, as characterized with phase contrast microscope and the metabolic activity (MTT) assay enhanced ECs proliferation and spreading with increasing concentration of RGD peptides grafted on their surfaces. Increased retention of ECs was also observed on the polymers surfaces under flow shear stress conditions. The results demonstrated that GRGDSP peptides grafted on the surfaces of polymers with photoactive 4-benzoylbenzoic acids could be an efficient method of fabrication for artificial small-diamater blood vessels. The modified polymer is expected to be used for small-diamater vascular grafts and functional tissue engineered blood vessels to improve ECs adhesion and retention on the polymer surfaces under flow shear stress conditions.     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Adhesion status of platelets under flow conditions by digital image processing

Abstract

The kinetics and distribution of platelet deposition on protein‐coated surfaces were studied in an in vitro flow system. When fluorescence‐labeled platelets in whole blood flow through a flow chamber that is composed of a glass plate, platelets will deposit onto the protein‐coated surface in a time‐and location‐dependent manner. A combination of fluorescence video microscopy and a digital image processing system allowed us to systematically study thrombosis kinetics under various flow conditions, with different biomaterials and forms of blood.

In this paper, the image processing and pattern recognition techniques have been developed to quantify adhered platelets from numerous video frames. The dynamic adhesion status (newly attached, staying, detached) has been calculated automatically by software developed on an IBM/PC/386. These morphometric parameters as a function of flow rate and type of biomaterials can be determined experimentally.     

Effect of hyaluronic acid incorporation method on the stability and biological properties of polyurethane–hyaluronic acid biomaterials

The high failure rate of small diameter vascular grafts continues to drive the development of new materials and modification strategies that address this clinical problem, with biomolecule incorporation typically achieved via surface-based modification of various biomaterials. In this work, we examined whether the method of biomolecule incorporation (i.e., bulk versus surface modification) into a polyurethane (PU) polymer impacted biomaterial performance in the context of vascular applications. Specifically, hyaluronic acid (HA) was incorporated into a poly(ether urethane) via bulk copolymerization or covalent surface tethering, and the resulting PU–HA materials characterized with respect to both physical and biological properties. Modification of PU with HA by either surface or bulk methods yielded materials that, when tested under static conditions, possessed no significant differences in their ability to resist protein adsorption, platelet adhesion, and bacterial adhesion, while supporting endothelial cell culture. However, only bulk-modified PU–HA materials were able to fully retain these characteristics following material exposure to flow, demonstrating a superior ability to retain the incorporated HA and minimize enzymatic degradation, protein adsorption, platelet adhesion, and bacterial adhesion. Thus, despite bulk methods rarely being implemented in the context of biomolecule attachment, these results demonstrate improved performance of PU–HA upon bulk, rather than surface, incorporation of HA. Although explored only in the context of PU–HA, the findings revealed by these experiments have broader implications for the design and evaluation of vascular graft modification strategies.     

Influence of nanoporesize on platelet adhesion and activation

In this study we have evaluated the influence of biomaterial nano-topography on platelet adhesion and activation. Nano-porous alumina membranes with pore diameters of 20 and 200 nm were incubated with whole blood and platelet rich plasma. Platelet number, adhesion and activation were determined by using a coulter hematology analyzer, scanning electron microscopy, immunocytochemical staining in combination with light microscopy and by enzyme immunoassay. Special attention was paid to cell morphology, microparticle generation, P-selectin expression and beta-TG production. Very few platelets were found on the 200 nm alumina as compared to the 20 nm membrane. The platelets found on the 20 nm membrane showed signs of activation such as spread morphology and protruding filipodia as well as P-selectin expression. However no microparticles were detected on this surface. Despite the fact that very few platelets were found on the 200 nm alumina in contrast to the 20 nm membrane many microparticles were detected on this surface. Interestingly, all microparticles were found inside circular shaped areas of approximately 3 mum in diameter. Since this is the approximate size of a platelet we speculate that this is evidence of transient, non-adherent platelet contact with the surface, which has triggered platelet microparticle generation. To the authors knowledge, this is the first study that demonstrates how nanotexture can influence platelet microparticle generation. The study highlights the importance of understanding molecular and cellular events on nano-level when designing new biomaterials.     

Modification of human polymorphonuclear neutrophilic cell (PMN)-adhesion on biomaterial surfaces by protein preadsorption under static and flow conditions

Biomaterials induce a specific reaction after implantation in the human body. This reaction depends on the chemical and physico-chemical properties of the material as well as on the site and type of implantation. We have used a dynamic model, the parallel-plate flow-chamber, to examine the interactions of different biomaterials with polymorphonuclear neutrophilic cell (PMN) and how these interactions are influenced by protein preadsorption. Our results clearly show that for hydrophobic materials, glass and PE, which induce a prominent adhesion of PMN, the mixture of albumin and fibrinogen induces the best inhibitory effect. On hydrophilic biomaterial surfaces, untreated TCPS and PC-coated TCPS, reveal only a minor influence of adsorbed proteins on PMN adhesion because of a primary low adhesive surface for PMN and proteins as well. Human citrated plasma leads only to a slight inhibition of PMN adhesion. On the hydrophobic materials, glass and PE, bovine serum albumin (BSA) had the best anti-adhesive potential with respect to PMN. The coating using phosphorylcholine is an excellent surface modification to prevent PMN-adhesion and protein adsorption. The results of our experiments suggest that investigations under static and flow conditions are also needed to determine the influence of protein adsorption on other relevant blood cell populations, for example, platelets and monocytes.     

Improved thrombogenicity on oxygen etched Ti6Al4V surfaces

Thrombus formation on blood contacting biomaterials continues to be a key factor in initiating a critical mode of failure in implantable devices, requiring immediate attention. In the interest of evaluating a solution for one of the most widely used biomaterials, titanium and its alloys, this study focuses on the use of a novel surface oxidation treatment to improve the blood compatibility. This study examines the possibility of using oblique angle ion etching to produce a high quality oxide layer that enhances blood compatibility on medical grade titanium alloy Ti6Al4V. An X-ray photoelectron spectroscopy (XPS) analysis of these oxygen-rich surfaces confirmed the presence of TiO₂ peaks and also indicated increased surface oxidation as well as a reduction in surface defects. After 2 h of contact with whole human plasma, the oxygen etched substrates demonstrated a reduction in both platelet adhesion and activation as compared to bare titanium substrates. The whole blood clotting behavior was evaluated for up to 45 min, showing a significant decrease in clot formation on oxygen etched substrates. Finally, a bicinchoninic acid (BCA) total protein assay and XPS were used to evaluate the degree of key blood serum protein (fibrinogen, albumin, immunoglobulin G) adsorption on the substrates. The results showed similar protein levels for both the oxygen etched and control substrates. These results indicate that oblique angle oxygen etching may be a promising method to increase the thrombogenicity of Ti6Al4V.     

Reduced platelet adhesion and blood coagulation on cross-linked albumin films

Surface-induced thrombosis is a major complication associated with blood-contacting biomaterials. Cross-linked albumin films possessing native albumin characteristics such as resistance to cell adhesion and drug binding ability are available for improving the blood compatibility of biomaterial surfaces. In the present study, we aimed to evaluate the blood compatibility of cross-linked albumin films. Platelet adhesion analysis showed that albumin film coated substrates exhibited very low platelet adhesion, and platelet adhesion was further suppressed by loading the antiplatelet drug, cilostazol, into the film. Monitoring the coagulation process of whole blood using a coaxial-cylinder rotational viscometer showed that the initial time of coagulation in albumin film coated cylinders was delayed compared with that in uncoated cylinders, suggesting that activation of the intrinsic coagulation cascade was reduced on the albumin film coated surface. Thus, surface coating with cross-linked albumin films is a promising approach to conferring biomaterials with antithrombogenic surfaces due to the resistance to platelet adhesion and the antiplatelet drug-releasing capability afforded by the films.     

Evaluation of water sorption property and in vitro blood compatibility of poly(2-hydroxyethyl methacrylate) (PHEMA) based semi interpenetrating polymer networks (IPNs)

pH responsive smart biomaterials of gelatin and poly(2-hydroxyethyl methacrylate-co-acrylic acid) were synthesized by redox polymerization and characterized by FTIR, Environmental Scanning Electron Microscopy (ESEM). The prepared environmental responsive biomaterials containing polyelectrolyte segments were assessed for their water sorption potential under varying experimental conditions. The diffusion mechanism of transport of water molecules arising due to solvent-polymer interaction was also analysed to predict the behaviour of continuously relaxing macromolecular chains. The in vitro blood compatibility of the prepared polymeric hydrophilic materials was evaluated by methods such as blood clot formation, platelet adhesion, percent haemolysis and protein-adsorption study on the surface of the prepared biomaterials.     

Acoustic radiation force enhances targeted delivery of ultrasound contrast microbubbles: in vitro verification

Recent research has shown that targeted ultrasound contrast microbubbles achieve specific adhesion to regions of intravascular pathology, but not in areas of high flow. It has been suggested that acoustic radiation can be used to force free-stream microbubbles toward the target, but this has not been verified for actual targeted contrast agents. We present evidence that acoustic radiation indeed increases the specific targeted accumulation of microbubbles. Lipid microbubbles bearing an antibody as a targeting ligand were infused through a microcapillary flow chamber coated with P-selectin as the target protein. A 2.0 MHz ultrasonic pulse was applied perpendicular to the flow direction. Microbubble accumulation was observed on the flow chamber surface opposite the transducer. An acoustic pressure of 122 kPa enhanced microbubble adhesion up to 60-fold in a microbubble concentration range of 0.25 x 10(6) to 75 x 106) ml(-1). Acoustic pressure mediated the greatest adhesion enhancement at concentrations within the clinical dosing range. Acoustic pressure enhanced targeting nearly 80-fold at a wall shear rate of 1244 s(-1), suggesting that this mechanism is appropriate for achieving targeted microbubble delivery in high-flow vessels. Microbubble adhesion increased with the square of acoustic pressure between 25 and 122 kPa, and decreased substantially at higher pressures.     

Controlling Enamel Remineralization by Amyloid-Like Amelogenin Mimics

In situ regeneration of the enamel-like structure of hydroxyapatite (HAp) crystals under oral conditions is significant for dental caries treatment. However, it is still a challenge for dentists to duplicate the elegant and well-aligned apatite structure bonding to the surface of demineralized enamel. A biocompatible amelogenin-inspired matrix, a phase-transited lysozyme (PTL) film mimicking an N-terminal amelogenin with central domain (N-Ame) combined with synthetic peptide (C-AMG) based on the functional domains of C-terminal telopeptide (C-Ame) is shown here, which is formed by amyloid-like lysozyme aggregation at the enamel interface through a rapid one-step aqueous coating process. In the PTL/C-AMG matrix, C-AMG facilitated the oriented arrangement of amorphous calcium phosphate (ACP) nanoparticles and their transformation to ordered enamel-like HAp crystals, while PTL served as a strong interfacial anchor to immobilize the C-AMG peptide and PTL/C-AMG matrix on versatile substrate surfaces. PTL/C-AMG film-coated enamel induced both of the in vivo and in vitro synthesis of HAp crystals, facilitated epitaxial growth of HAp crystals and recovered the highly oriented structure and mechanical properties to levels nearly identical to those of natural enamel. This work underlines the importance of amyloid-like protein aggregates in the biomineralization of enamel, providing a promising strategy for treating dental caries.     

Synergistic effect of hydrophobic and anionic surface groups triggers blood coagulation in vitro

Biomaterial induced coagulation encompasses plasmatic and cellular processes. The functional loss of biomedical devices possibly resulting from these thrombotic reactions motivates the need for a better understanding of processes occurring at blood–biomaterial interfaces. Well defined model surfaces providing specific chemical–physical properties (self assembled monolayers (SAMs)) displaying hydrophobic or/and acidic terminal groups were used to uncover initial mechanisms of biomaterial induced coagulation. We investigated the influence of electrical charge and wettability on platelet- and contact activation, the two main actors of blood coagulation, which are often considered as separate mechanisms in biomaterials research. Our results show a dependence of contact activation on acidic surface groups and a correlation of platelet adhesion to surface hydrophobicity. Clot formation resulting from the interplay of blood platelets and contact activation was only found on surfaces combining both acidic and hydrophobic surface groups but not on monolayers displaying extreme hydrophobic/acidic properties.     

聚乙二醇及其衍生物改性生物医用材料表面的血液相容性

综述了PEG及其衍生物在生物医用材料表面血液相容性改性中的应用,并介绍了对改性后的表面进行表征的常用方法和手段,如反射红外,水接触角,光电子能谱,表面等离子共振,椭圆偏振仪,蛋白质亲和印迹,蛋白质标记等。     

The development of in vitro biocompatibility tests for the evaluation of intraocular biomaterials

Recent developments in ocular implant technology require the in vitro evaluation of ocular compatibility in early stage development programs. This requires an understanding and appreciation of the biological interactions which occur in the ocular environment and their relevance with respect to the clinical complications associated with surgical implantation of devices. This paper describes the development of a series of clinically reflective in vitro assays for assessing the potential ocular compatibility of novel intraocular lens materials. Staphylococcus epidermidis attachment, fibrinogen adsorption, mouse embryo fibroblast 3T3 adhesion and proliferation, primary rabbit lens cell adhesion, human peripheral blood macrophage adhesion and granulocyte activation tests were employed to evaluate two widely used intraocular biomaterials poly(methyl methacrylate) (PMMA) and silicone, and a novel biomimetic phosphorylcholine-based coating (PC). The performance of these materials in the in vitro assays was compared to their ability to reduce postoperative inflammation in vivo in a rabbit model. The results demonstrated that the in vitro assays described here are predictive of in vivo ocular compatibility. These assays offer a more relevant means of assessing the ocular compatibility of biomaterials than those presently required by the authorities for regulatory approval of medical devices and implants. © 1999 Kluwer Academic Publishers     

An improved peel test method for measurement of adhesion to biomaterials

Adhesion of tissues to biomaterials is desirable to prevent bacterial proliferation and for epithelial/transmucosal sealing of transcutaneous appliances, but can be counter-productive elsewhere, e.g. implants contacting tendons or maxillofacial subcutaneous tissue. It is therefore important to gauge adhesion strength of tissues to biomaterials before clinical use. Peel-testing is widely used for industrial product adhesion monitoring, but has rarely been applied biomedically. Here we describe peel-testing instrumentation designed for testing adherence of soft tissues to biomaterials. It offers the advantage that a 90° angle between peel and substrate is maintained, simplifying determination of applied normal forces separating tissue layers from material surfaces. The device is portable and can be brought directly to the specimen removal site. This minimizes time delays between explantation and testing, maintaining the tissue/biomaterial interface in the freshest possible state closely approximating in vivo conditions, and so avoids measurement artifacts. So far, the instrument has been used to test adhesion of tape to a biomaterial surface (for determining the devices technical performance), assess strength of tissue adhesives, and measure adhesion of subcutaneous tissue to orthopaedic biomaterials. However, its versatility suggests additional applications for the peel-tester where adhesion of soft tissue to biomaterials is of interest.     

Adaptive Liquid Interfacially Assembled Protein Nanosheets for Guiding Mesenchymal Stem Cell Fate

There is a growing interest in the development of dynamic adaptive biomaterials for regulation of cellular functions. However, existing materials are limited to two-state switching of the presentation and removal of cell-adhesive bioactive motifs that cannot emulate the native extracellular matrix (ECM) in vivo with continuously adjustable characteristics. Here, tunable adaptive materials composed of a protein monolayer assembled at a liquid–liquid interface are demonstrated, which adapt dynamically to cell traction forces. An ultrastructure transition from protein monolayer to hierarchical fiber occurs through interfacial jamming. Elongated fibronectin fibers promote formation of elongated focal adhesion structures, increase focal adhesion kinase activation, and enhance neuronal differentiation of stem cells. Cell traction force results in spatial rearrangement of ECM proteins, which feeds back to alter stem cell fate. The reported biomimetic adaptive liquid interface enables dynamic control of stem cell behavior and has potential translational applications.     

Integrating blood cell mechanics,platelet adhesive dynamics and coagulation cascade for modelling thrombus formation in normal and diabetic blood

Normal haemostasis is an important physiological mechanism that prevents excessive bleeding during trauma, whereas the pathological thrombosis especially in diabetics leads to increased incidence of heart attacks and strokes as well as peripheral vascular events. In this work, we propose a new multiscale framework that integrates seamlessly four key components of blood clotting, namely transport of coagulation factors, coagulation kinetics, blood cell mechanics and platelet adhesive dynamics, to model the development of thrombi under physiological and pathological conditions. We implement this framework to simulate platelet adhesion due to the exposure of tissue factor in a three-dimensional microchannel. Our results show that our model can simulate thrombin-mediated platelet activation in the flowing blood, resulting in platelet adhesion to the injury site of the channel wall. Furthermore, we simulate platelet adhesion in diabetic blood, and our results show that both the pathological alterations in the biomechanics of blood cells and changes in the amount of coagulation factors contribute to the excessive platelet adhesion and aggregation in diabetic blood. Taken together, this new framework can be used to probe synergistic mechanisms of thrombus formation under physiological and pathological conditions, and open new directions in modelling complex biological problems that involve several multiscale processes.     

Hypergravity Effects on Dendritic Cells and Vascular Wall Interactions

Dendritic cells (DCs), the most potent antigen-presenting cells inducing specific immune responses, are involved in the pathogenesis of atherosclerosis. In this inflammatory disease, DCs increase in number, being particularly abundant in the shoulder regions of plaques. Since the exposure to altered gravitational conditions results in a significant impairment of the immune function, the aim of this study was to investigate the effects of hypergravity on both the function of DCs and their interactions with the vascular wall cells. Monocytes from peripheral blood mononuclear cells of healthy volunteers were sorted by CD14+ magnetic beads selection, cultured for 6 days in medium supplemented with GM-CSF and IL-4, followed by a further maturation stimulus. DC phenotype, assessed by flow cytometry, showed a high expression of the specific DC markers CD80, CD86, HLA-DR and CD83. The DCs obtained were then exposed to hypergravitational stimuli and their phenotype, cytoskeleton, ability to activate lymphocytes and interaction with vascular wall cells were investigated. The findings showed that the exposure to hypergravity conditions resulted in a significant impairment of DC cytoskeletal organization, without affecting the expression of DC markers. Moreover, an increase in DC adhesion to human vascular smooth muscle cells and in their ability to activate lymphocytes was observed.     

Blood compatibility of zinc–calcium phosphate conversion coating on Mg–1.33Li–0.6Ca alloy

Magnesium alloys as a new class of biomaterials possess biodegradability and biocompatibility in comparison with currently used metal implants. However, their rapid corrosion rates are necessary to be manipulated by appropriate coatings. In this paper, a new attempt was used to develop a zinc-calcium phosphate (Zn-Ca-P) conversion coating on Mg-1.33Li-0.6Ca alloys to increase the biocompatibility and improve the corrosion resistance. In vitro blood biocompatibility of the alloy with and without the Zn-Ca-P coating was investigated to determine its suitability as a degradable medical biomaterial. Blood biocompatibility was assessed from the hemolysis test, the dynamic cruor time test, blood cell count and SEM observation of the platelet adhesion to membrane surface. The results showed that the Zn-Ca-P coating on Mg-1.33Li-0.6Ca alloys had good blood compatibility, which is in accordance with the requirements for medical biomaterials.