In vitro studies of platelet adhesion on laser-treated polyethylene terephthalate surface.

In order to improve blood compatibility, the surfaces of polyethylene terephthalate (PET) were treated using CO2 pulsed and KrF excimer lasers. The physico-chemical characterization of the laser-treated PET surfaces was carried out through attenuated total reflectance infrared spectroscopy and contact-angle measurements. The hemocompatibility of the laser-irradiated PET films was examined in vitro to evaluate their capability of inducing platelet adhesion in comparison with unmodified PET. The number of adhered platelets was determined by lactate dehydrogenase (LDH) activity measurement. Platelet adhesion on the untreated PET was relatively high compared to the laser-treated samples. Laser irradiation of PET surface reduced the number of adherent platelets and prevented platelet spreading on the surface. Reduction of platelet adhesion was attributed to the change in morphology, chemical structure, and crystallinity of the PET surface due to laser irradiation with various numbers of pulses. The morphology of adhered platelets on the PET surfaces was investigated by scanning electron microscopy (SEM). The SEM observations were consistent with the results obtained from LDH activity measurement.     

Platelet adhesion and aggregation on polyethylene: effect of exhaustive exercise

The aim of the present study was to evaluate the effect of an exhaustive exercise on platelet adhesion and aggregation on polyethylene (PE) in relation to changes in plasma cortisol concentration in order to ascertain the effect of physical stress response in the blood-contacting properties of polymeric materials. Twelve healthy sedentary subjects, six males and six females, were studied. Each subject performed an exercise test on a bicycle ergometer at intensity corresponding to 70% VO2 max until exhaustion. One month after the exercise session, each subject participated in a control rest session. In both sessions, blood samples were drawn every 5 min for cortisol, lactate, hemoglobin, and hematocrit determinations and every 15 min for evaluation of platelet adhesion and aggregation. Individual comparisons between the rest and exercise cortisol patterns identified three categories of cortisol responders to exercise: positive responders (C +, showing higher concentrations during exercise than during rest), negative responders (C -, showing lower concentrations during exercise than during rest), and nonresponders (NR, showing similar concentrations during exercise and rest). The results revealed that C + had lower platelet adhesion and aggregation scores during exercise than during rest; moreover C - had higher scores than C + and NR during exercise. The results obtained demonstrated no effects of sex or exercise on either cortisol plasma levels or platelet adhesion and aggregation on PE surface. With regard to cardiovascular risk, the results suggest that exercise favorably affects platelet functions when mechanisms of metabolic adaptation to prolonged muscular work, expressed by a cortisol increase, are activated during exercise.     

Platelet interactions with titanium: modulation of platelet activity by surface topography

Endosseous implants initially come into contact with blood. Thus, the nature of the interactions between blood and implanted endosseous implants may influence subsequent bone healing events in the peri-implant healing compartment. We conducted studies to address the following question: Does implant surface microtexture modulate platelet activity? We used commercially pure Ti (cpTi) disks with four different surface finishes: dual acid-etched (DAE), 320 grit (320G) abraded, machined, and p1200 polished cpTi. Surfaces were characterized by scanning electron microscopy (SEM) and optical profilometry. The DAE and 320G surfaces presented more complex microtextures than the machined or polished surfaces. Platelet activities were measured by quantifying platelet adherence, platelet-derived microparticle (MP) formation, and P-selectin expression as function of surface type. Platelet adhesion, measured using a lactate dehydrogenase (LDH) assay. was increased on DAE and 320G surfaces compared to machined and polished surfaces (p < 0.05). M P formation and P-selectin expression, assayed by flow cytometry, also showed increased activation of platelets on DAE and 320G surfaces. Because increased activation of platelets may lead to up-regulation of osteogenic responses during bone healing, these results may explain the enhanced osteoconductivity known to occur with DAE cpTi surfaces in comparison with machined cpTi surfaces.     

Mathematical model of static platelet adhesion on a solid surface

The scheme of platelet/surface interaction and a kinetic model of platelet adhesion on a solid surface are suggested. The elaborated approach takes into account the platelet activation by the surface and accumulation of free activated cells in the bulk of the liquid phase. This effect has an especially important role in static experimental conditions. The suggested model explains three types of adhesion kinetic curves, obtained in experiments in vitro: sigmoid curves with or without saturation and an exponential curve with saturation. According to the model, the curve shape is determined by material surface properties, platelet functionality, and experimental conditions of the platelet/surface interaction. The data of static platelet adhesion from platelet rich plasma on glass, siliconized glass, hexadecyltrichlorosilane monolayers, and low-density polyethylene are described mathematically with the proposed model. Numerical parameters are calculated from approximation of experimental data by the model. These parameters allow quantitative characterization of platelet interaction with the material surface.     

Interactions between human whole blood and modified TiO2-surfaces: influence of surface topography and oxide thickness on leukocyte adhesion and activation.

An in vitro model (Nygren et al., J Lab Clin Med 129 (1997) 35-46) was used to investigate interactions between leukocytes and four modified TiO2-surfaces. Surface topography was measured using scanning electron microscopy and optical profilometry while Auger electron spectroscopy was used to determine surface composition and oxide thickness. The surfaces were either smooth or rough with either thin or thick oxides. All surfaces consisted of TiO2 covered by a carbonaceous layer. The surfaces were incubated with capillary blood for time periods of between 8 min and 32 h. Immunofluorescence techniques together with computer aided image analysis and chemiluminescence technique were used to detect cell adhesion, expression of adhesion receptors and the zymosan-stimulated respiratory burst response. Leukocyte adhesion to the surfaces increased during the first hours of blood-material contact and then decreased. Polymorphonuclear granulocytes were the dominating leukocytes on all surfaces followed by monocytes. Cells adhering to rough surfaces had higher normalized expression of adhesive receptors than cells on smooth surfaces. Maximum respiratory burst response occurred earlier on the smooth than on the rough surfaces. In conclusion, topography had a greater impact than oxide thickness on most cellular reactions investigated, but the latter often had a dampening effect on the responses.     

Increasing cell adhesion on plasma deposited fluorocarbon coatings by changing the surface topography

In designing new biomaterials, it is of outstanding importance to consider how cells respond to specific chemical and topographical features on the material surface. The behavior of most cell types in vivo is strictly related to specific chemical and topographical cues that characterize the extra cellular environment. In particular, during their lives cells react to topographical patterns such as those of the extracellular matrix (ECM), of micro and/or nanometric dimensions. The production of micrometric and/or nanometric features on artificial materials usually involves expensive and time-consuming methods of manufacturing, such as electron beam and colloidal lithography. In this article, different "Teflon-like" structured surfaces were deposited from tetrafluoroethylene (C(2)F(4))-fed plasmas, for the study of cell adhesion and growth. The reaction of different cell lines to different topographical features was evaluated and compared with cell behavior on flat samples with the same chemical composition. Cell adhesion was calculated from area covered by cells at different time of culture. Beside this, cell proliferation was determined with the MTT test. Cell morphology and filopodia interaction with the nanofeatures were also estimated by optical and scanning electron microscopy. A dramatic difference both in adhesion and growth was found between cells seeded on flat and rough surfaces with the density and spreading of adhered cells varying as a function of the roughness of coatings.     

Reduced platelet adhesion on the surface of polyurethane bearing structure of sulfobetaine

Poly(etherurethane)s are widely used as blood-contacting biomaterials due to their good biocompatibility and mechanical properties. Nevertheless, their blood compatibility is still not adequate for the more demanding applications. Surface modification is an effective way to improve the blood compatibility and retain the bulk properties of biomaterials. The purpose of present study was to design and synthesize a novel nonthrombogenic biomaterial by modifying the surface of poly(etherurethane) with zwitterionic monomer. Films of polyurethane were grafted with sulfobetaine by a three-step procedure. In the first step, the film surfaces were treated with hexamethylene diisocyanate (HDI) in toluene at 50 degrees C in the presence of di-n-butyl tin dilaurate (DBTDL) as a catalyst. The extent of the reaction was measured by ATR-IR spectra; a maximum number of free NCO group was obtained after a reaction time of 90 min. In the second step, the hydroxyl group of 4-dimethylamino-1-butanol (DMAB) was allowed to react in toluene with isocyanate groups bound on the surface. In the third step, sulfobetaine was formed on the surface through the ring-opening reaction between tertiary amine of DMAB and 1,3- propane-sultone (PS). It was characterized by ATR-IR, XPS. The data showed that the grafted surfaces were composed of sulfobetaine. The results of the contact angle measurements showed that they were strongly hydrophilic. The state of platelet adhesion and shape variation for the attached platelets was described. The modified surface shows excellent blood compatibility feature by the low platelet adhesion.     

Platelet adhesion on titanium oxide gels: effect of surface oxidation

The correlations between titanium oxide layers on oxidized titanium (Ti) substrates and platelet adhesion were examined. Ti substrates were prepared by three different oxidation methods: the first one was treated with hydrogen peroxide (H2O2) solution, the second one was heated in air at moderate temperatures, and the third one was processed with both H2O2 and heating. The titanium oxide layers formed on the Ti substrates were characterized by wettability, chemical composition, thickness, and crystal phase. The platelet adhesion on these oxide layers was examined and correlated to the characterizations of the surface layers. The number of adhesive platelets seemingly correlated with the contact angle towards distilled water, because the number increased close to 70-80 degrees of the contact angle. The effect of surface oxidation on platelet adhesion was examined in detail and it was found that the composition and thickness of the oxide layer influenced platelet adhesion rather than wettability. Thick titanium oxide layers formed on Ti substrates by heating displayed less platelet adhesion than thin oxide layers on untreated Ti substrates. The largest number of adhesive platelets was found on H2O2-oxidized substrates; the substrates found on amorphous titanium oxide contained the Ti-peroxide radical species. The number of platelets, on the other hand, could hardly be observed on Ti substrates treated with H2O2 and subsequently heated above 300 degrees C. The titanium oxide layer on the substrate was thick and we found it to consist of only a few radical species. That is, the effect of heat treatment accelerates the growth of the oxide layer, and decomposes or decreases the number of radical species. Ti substrates with H2O2 and heat treatment above 300 degrees C held the least number of platelets, and were concluded to be the most inhibitory for platelet adhesion.     

Fibrinogen surface distribution correlates to platelet adhesion pattern on fluorinated surface-modified polyetherurethane

In previous work, it had been shown that platelet adhesion could be reduced by fluorinating surfaces with oligomeric fluoropolymers, referred to as surface-modifying macromolecules (SMMs). In the current study, two in vitro blood-contacting experiments were carried out on a polyetherurethane modified with three different SMMs in order to determine if altered platelet adhesion levels could be related to the pattern of adsorbed protein and more specifically to the manner in which fibrinogen (Fg) distribution occurs at the surface. In the first experiment, the materials were placed in whole human blood and the adherent platelets were viewed with high-resolution scanning electron microscopy (SEM). In a second experiment, the materials were incubated with human plasma with the absence of platelets. The plasma contained 5% fluorescent-Fg. The materials were then viewed with a fluorescence microscope and images were collected to define the distribution of high-density fluorescent-Fg areas. The SEM and fluorescent-Fg images were imported to Image Pro Plus imaging software to measure the area, length and circularity and a bivariate correlation test was conducted between the two sets of data. For area and length morphology parameters, there were high and significant correlations (r > 0.9, p < 0.05) between the platelets and Fg aggregates. The data suggest that the Fg distribution may serve as a predictor of platelet morphology/activation and provides insight into the non-thrombogenic character of biomaterials containing the fluorinated SMMs.     

The effect of in vitro activation and platelet interaction on the CD9 distribution and adhesion properties of human eosinophils

Objective and Design: The redistribution of CD9 in peripheral blood eosinophils was investigated with respect to the interaction with platelets during in vitro activation, and whether this interaction exerts influence on eosinophil adhesion properties.¶Materials and Methods: Flow cytometry was used to investigate the CD9 expression in purified eosinophils or eosinophils from different whole blood preparations, with or without platelets present. To confirm an eosinophil/platelet interaction fluorescence microscopy was used, and to demonstrate release/shedding of CD9 molecules a biosensor technique was performed.¶Results: Our results show that both intracellular and surface expression of CD9 decrease upon in vitro activation in the absence of platelets, a phenomenon probably caused by release/shedding of soluble forms of CD9 and not due to intracellular degradation. Increased expression of CD9 on eosinophils, stimulated in the presence of platelets, is partly a result of interacting platelets, judged by the increase in platelet specific marker CD61. In our adhesion assay a significant increase in eosinophil adhesion properties to fibronectin was obtained when eosinophils were PMA stimulated and interacting with platelets, as compared to activated eosinophils without platelets.¶Conclusions: Our findings, that CD9 expression on eosinophils is dynamically regulated, support our previous suggestion that CD9 may be a useful activity marker and that platelet interaction acts on eosinophil adhesion.     

Surface characterization and platelet adhesion studies on polyurethane surface immobilized with C60.

Due to its distinctive molecular configuration, C60 and its derivatives have been the research focus in exploring its electrical, mechanical, optical, and even biological applications during the past decades. In this investigation, C60 molecules are grafted onto the polyurethane surface, which is pretreated with oxygen plasma activation, through amine-terminated silane coupling agents. ESCA analysis indicates that the C60 molecules spontaneously grafted onto the amine-terminated PU substrate through nucleophilic additions to the fullerene double bonds which fuse two six-membered rings. More amine functional groups are formed on the PU surface if 4-aminobutyldimethylmethoxysilane is used as the coupling agent. In vitro platelet adhesion assay shows the C60 grafted PU are more platelets activating than the nontreated PU control. This might be attributed to the synergistic effect of the grafted C60 molecules and the few residual amine functional groups which are left after the C60 grafting reaction. Further applications using these C60 grafted polyurethane surfaces as the cell adhesion and cell growth substrates are currently under exploration.     

The influence of surface topography of a porous perfluoropolyether polymer on corneal epithelial tissue growth and adhesion

Design principles for corneal implants are challenging and include permeability which inherently involves pore openings on the polymer surface. These topographical cues can be significant to a successful clinical outcome where a stratified epithelium is needed over the device surface, such as with a corneal onlay or corneal repair material. The impact of polymer surface topography on the growth and adhesion of corneal epithelial tissue was assessed using porous perfluoropolyether membranes with a range of surface topography. Surfaces were characterised by AFM and XPS, and the permeability and water content of membranes was measured. Biological testing of membranes involved a 21-day in vitro tissue assay to evaluate migration, stratification and adhesion of corneal epithelium. Similar parameters were monitored in vivo by surgically implanting membranes into feline corneas for up to 5 months. Data showed optimal growth and adhesion of epithelial tissue in vitro when polymer surface features were below a 150 nm RMS value. Normal processes of tissue growth and adhesion were disrupted when RMS values approached 300 nm. Data from the in vivo study confirmed these findings. Together, outcomes demonstrated the importance of surface topography in the design of implantable devices that depend on functional epithelial cover.     

Bone healing and the effect of implant surface topography on osteoconduction in hyperglycemia

Dental implant failures that occur clinically for unknown reasons could be related to undiagnosed hyperglycemia. The exact mechanisms that underlie such failures are not known, but there is a general consensus that bone growth is compromised in hyperglycemia. Nevertheless, contradictory findings exist related to peri-implant bone healing in hyperglycemia. We hypothesized that hyperglycemia delays early bone healing by impeding osteoconduction, and that the compromised implant integration due to hyperglycemia could be abrogated by using nanotopographically complex implants. Thus we undertook two parallel experiments, an osteotomy model and a bone in-growth chamber model. The osteotomy model tracked temporal bone healing in the femora of euglycemic and hyperglycemic rats using micro computed tomography (microCT) analysis and histology. The bone in-growth chamber model used implant surfaces of either micro- or nanotopographical complexity and measured bone–implant contact (BIC) using backscattered electron imaging in both metabolic groups. Quantitative microCT analyses on bone volume, trabeculae number and trabeculae connectivity density provided clear evidence that bone healing, both reparative trabecular bone formation and remodeling, was delayed in hyperglycemia, and the reparative bone volume changed with time between metabolic groups. Furthermore, fluorochrome labeling showed evidently less mineralized bone in hyperglycemic than euglycemic animals. An increased probability of osteoconduction was seen on nano-compared with microtopographically complex surfaces, independent of metabolic group. The nanotopographically complex surfaces in hyperglycemia outperformed microtopographically complex surfaces in euglycemic animals. In conclusion, the compromised implant integration in hyperglycemia is abrogated by the addition of nanotopographical features to an underlying microtopographically complex implant surface.     

Covalently grafted phospholipid monolayer on silicone catheter surface for reduction in platelet adhesion

We report a novel method of surface grafting a polymeric phospholipid system containing an acryloyl end group (1stearoyl-2-[12-(acryloyloxy)-dodecanoyl]-sn-glycero-3-phosphocholine) onto medical grade silicone catheters. The surface of silicone catheters was functionalized in a sequence of steps: plasma polymerization of allyl alcohol on the catheter surface, grafting acryloyl moieties and in situ polymerization of the pre-assembled acryloyl terminated phospholipids on the acryloyl functionalized catheter surface. The surface morphological changes analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), a sharp decrease in water contact angle, and appearance of N1s peak in XPS analysis indicated a successful monolayer grafting of the phospholipid. In platelet adhesion tests performed using platelets isolated from rabbit plasma, the phospholipid grafted surface showed fewer adhered platelets, without emerging pseudopodes or aggregation. However, ungrafted catheter surface showed large number of platelets in extensively spread and aggregated states. Thus, this modified phospholipid system and its simple grafting technique was very effective with regard to suppressing in vitro platelet adhesion on the silicon catheter surface.     

Influence of implant surface topography on early osseointegration: a histological study in human jaws

The purpose of this study was to evaluate the influence of the oxidized surface on bone-to-implant contact (BIC%), the bone density in the threaded area (BA%), as well as the bone density outside the threaded area (BD%) in human jaws after 2 months of unloaded healing. Thirteen subjects (mean age 42.61 +/- 6.15 years) received two microimplants (2.5 mm diameter and 6 mm length) each, during conventional mandible or maxilla implant surgery. The microimplants with commercially pure titanium surfaces (machined) and oxidized surfaces served as the control and test surfaces, respectively. After 2 months, the microimplants and the surrounding tissue were removed and prepared for histomorphometric analysis. All microimplants, except two machined and one oxidized microimplant surfaces, were found to be clinically stable after the healing period. Histometric evaluation indicated that the mean BIC% was (21.71 +/- 13.11)% and (39.04 +/- 15.75)% for machined and oxidized microimplant surfaces, respectively. The BD% was higher for the oxidized surface, although there was no difference for maxilla and mandible. The oxidized surface impacted the BA% for the type-IV bone. Data suggest that the oxidized surface presented a higher bone-to-implant contact rate compared with machined surfaces under unloaded conditions, after a healing period of 2 months.     

The role of adsorbed fibrinogen in platelet adhesion to polyurethane surfaces: a comparison of surface hydrophobicity, protein adsorption, monoclonal antibody binding, and platelet adhesion

Ten specially synthesized polyurethanes (PUs) were used to investigate the effects of surface properties on platelet adhesion. Surface composition and hydrophilicity, fibrinogen (Fg) and von Willebrand's factor (vWf) adsorption, monoclonal anti-Fg binding, and platelet adhesion were measured. PUs preadsorbed with afibrinogenemic plasma or serum exhibited very low platelet adhesion, while adhesion after preadsorption with vWf deficient plasma was not reduced, showing that Fg is the key plasma protein mediating platelet adhesion under static conditions. Platelet adhesion to the ten PUs after plasma preadsorption varied greatly, but was only partially consistent with Fg adsorption. Thus, while very hydrophilic PU copolymers containing PEG that had ultralow Fg adsorption also had very low platelet adhesion, some of the more hydrophobic PUs had relatively high Fg adsorption but still exhibited lower platelet adhesion. To examine why some PUs with high Fg adsorption had lower platelet adhesion, three monoclonal antibodies (mAbs) that bind to sites in Fg thought to mediate platelet adhesion were used. The antibodies were: M1, specific to gamma-chain C-terminal; and R1 and R2, specific to RGD containing regions in the alpha-chain N- and C-terminal, respectively. Platelet adhesion was well correlated with M1 binding, but not with R1 or R2 binding. When these mAbs were incubated with plasma preadsorbed surfaces, they blocked adhesion to variable degrees. The ability of the R1 and R2 mAbs to partially block adhesion to adsorbed Fg suggests that RGD sites in the alpha chain may also be involved in mediating platelet adhesion and act synergistically with the C-terminal of the gamma-chain.     

Laser surface modification of silicone rubber to reduce platelet adhesion in vitro

To improve the blood compatibility, the surface of polydimethylsiloxane (PDMS) films were irradiated using a CO₂-pulsed laser. Acrylamide (AAm) was grafted onto a pre-irradiated surface. The AAm-grafted and laser-treated films were characterized using different techniques. Platelet adhesion and activation onto the AAm-grafted PDMS, laser-treated (ungrafted) and unmodified PDMS film surfaces were compared. Data from in vitro assays indicated that the platelet adhesion was reduced on the AAm-grafted PDMS and laser treated PDMS films in comparison with the unmodified PDMS. The laser-irradiated sample showed the minimum platelet adhesion. It seems that laser irradiation onto a silicone rubber surface is a versatile technique to produce anti-thrombogenic surface for biomaterial applications.     

Amidine surface modification of poly(acrylonitrile-co-vinyl chloride) reduces platelet adhesion

The surface nitrile groups of solvent-cast polyacrylonitrile-co-vinyl chloride (PAN-VC) films were converted into amidine groups through a two-step process analogous to Pinner's method of 1877. The amidine groups were hypothesized to reduce platelet adhesion and activation through the inhibition of the classical complement pathway, as was noted for benzamidine and pentamidine in earlier studies. A slightly higher nitrogen content was detected on the amidine-surface modified (ASM) samples by X-ray photoelectron spectroscopy relative to the solvent-treated and unmodified controls. Reaction with pentafluoroaldehyde resulted in increased fluorine and decreased nitrogen contents (relative), consistent with the formation of a Schiff base with the primary nitrogen of the amidine on ASM PAN-VC surface. The corresponding yield was 17.2% based on nitrile groups and 9.6% based on all repeating units of the base polymer. Despite this low degree of amidine modification, platelet adhesion was approximately 40% lower on the amidine-modified film compared to the solvent-treated and unmodified controls. The scanning electron micrographs also showed less activation in the adherent platelets. A small reduction in C1s binding was also noted on the amidine-modified surface. Taken together, these results support the notion that amidine modification may be a useful approach to improve the nonthrombogenicity of a biomaterial. However, much effort is required to improve the degree of surface modification and to provide unequivocal evidence linking amidines to the improved thrombogenicity.