The combined effect of surface chemistry and flow conditions on Staphylococcus epidermidis adhesion and ica operon expression.

The assessment of biomaterial susceptibility to infection relies mainly on the analysis of macroscopic bacterial responses to material interactions, usually under static conditions. However, new technologies permit a more profound understanding of the molecular basis of bacteria-biomaterial interactions. In this study, we combine both conventional phenotypic analysis - using confocal microscopy - and genotypic analysis - using the relative reverse transcription polymerase chain reaction (RT-PCR) - to examine the interaction of bacteria with OH- and CH3-terminated glass surfaces, under dynamic flow conditions. Bacterial adhesion, as well as slime production and biofilm formation, was much higher on the CH3-terminated than on the OH-terminated glass - for four Staphylococcus epidermidis strains. This was in agreement with the icaA and icaD gene expression results that showed increased expression for the bacteria adhering to the CH3-terminated substrate, especially under the higher shear rate. Therefore, the combined effect of the surface chemistry and shear significantly influence the adhesion and phenotype of interacting bacterial cells, while there are putative links between phenotypic responses to bacteria-material interactions and gene-expression profile alterations. This indicates that analysis of gene expression not only can greatly refine our knowledge of bacteria-material interactions, but also yield novel biomarkers for potential use in biocompatibility assessment.

Slow flow of passive neutrophils and sequestered nucleus into micropipette.

In the present study, the role of the nucleus and its contribution to the deformability of the passive neutrophils was investigated. To determine the rheological properties of the nucleus and of the neutrophil itself, deformation tests on single neutrophil and sequestered nucleus have been performed by micropipette under low aspiration pressure (80 Pa = 2-3 Pcr). The stiffness of the nucleus was found to be larger than that of the neutrophil, and its viscosity was found almost ten-fold higher. A subpopulation of neutrophils (Sub-A) showed two phases of deformation, a first rapid phase and a second phase with a constant deformation rate up to their full entrance, with an apparent viscosity mu app-second-Phase(N Sub-A) = 286 +/- 123 Pa x s, calculated by the liquid drop model. Another subpopulation (Sub-B) of the tested neutrophils displayed three deformation phases: a first one reflecting the rapid entry of cell into the micropipette, a second with constant deformation rate, and a third phase, with a slower, also constant, deformation rate were recorded. The corresponding apparent viscosities were found as mu app-second-Phase(N Sub-B) = 341 +/- 94 Pa x s and mu app-third-Phase(N Sub-B) = 1651 +/- 734 Pa x s. The apparent viscosity values of the neutrophilic nucleus, mu app (N nucl) = 2468 +/- 1345 Pa x s and of the whole neutrophil calculated in the third phase of deformation, mu app-third-Phase(N Sub-B) = 1651 +/- 734 Pa.s were comparable. These results support our hypothesis that the nucleus plays a significant role in the mechanical and rheological behavior of the neutrophil, especially when it has to pass through openings much smaller than its size.

Bacterial adhesion onto materials with specific surface chemistries under flow conditions.

Staphylococcus epidermidis adhesion onto materials with specific chemical functionalities, under flow, was investigated by using surfaces prepared by self-assembly of alkyl silane monolayers on glass. Terminal methyl (CH(3)) and amino (NH(2)) groups were formed by chemical vapor deposition of silanes, at elevated temperature. Carboxyl (COOH) terminated groups were prepared by further modification of NH(2) groups with succide anhydride and positively charged NH(2) groups by adsorption of poly-L: -lysine hydrobromide. Hydroxyl (OH) terminated glass was used as control. Surface modification was verified by contact angle measurements, atomic force microscopy and X-ray photoelectron spectroscopy. A parallel plate flow chamber was used to evaluate bacterial adhesion at various shear rates. Adhesion was found to be depended on the monolayer's terminal functionality. It was higher on the CH(3) followed by the positively charged NH(2), the non-charged NH(2) groups, the COOH and minimal on the OH-terminated glass. The increase in the material surface free energy significantly reduced the adhesion of a hydrophilic bacterial strain, and this is in accordance with the predictions of the thermodynamic theory. However, the increase in the shear rate restricted the predictability of the theory and revealed macromolecular interactions between bacteria and NH(2)- and COOH-terminated surfaces.

Interactions of bacteria with specific biomaterial surface chemistries under flow conditions.

The effect of specific chemical functionalities on the adhesion of two Staphylococcus epidermidis strains under flow was investigated by using surfaces prepared by self-assembly of alkyl silane monolayers on glass. Terminal methyl (CH(3)) and amino (NH(2)) groups were formed in solution and by chemical vapor deposition of silanes, at elevated temperature. Hydroxyl (OH)-terminated glass was used as control. Surface modification was verified by contact angle and zeta potential measurements, atomic force microscopy and X-ray photoelectron spectroscopy. A parallel plate flow chamber was used to evaluate bacterial adhesion at various shear rates. The effect of the solution's ionic strength on adhesion was also studied. Adhesion was found to be dependent on the monolayer's terminal functionality. It was higher on the CH(3) followed by the NH(2) and minimal on the OH-terminated glass for both strains. The increase in the ionic strength significantly enhanced adhesion to the various substrates, in accordance with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The extended DLVO theory explained well the combined effects of surface and solution properties on bacterial adhesion under low shear rates. However, the increase in the shear rate restricted the predictability of the theory and revealed macromolecular interactions between bacteria and NH(2)-terminated surfaces.

Staphylococcus epidermidis adhesion to He, He/O(2) plasma treated PET films and aged materials: contributions of surface free energy and shear rate.

Adhesion studies of bacteria (Staphylococcus epidermidis) to plasma modified PET films were conducted in order to determine the role of the surface free energy under static and dynamic conditions. In particular, we investigated the effect of the ageing time on the physicochemical surface properties of helium (He) and 20% of oxygen in helium (He/O(2)) plasma treated polyethylene terephthalate (PET) as well as on the bacterial adhesion. Treatment conditions especially known to result in ageing sensitive hydrophilicity (hydrophobic recovery) were intentionally chosen in an effort to obtain the widest possible range of surface energy specimens and also to avoid strong changes in the morphological properties of the surface. Both plasma treatments are shown to significantly reduce bacterial adhesion in comparison to the untreated PET. However, the ageing effect and the subsequent decrease in the surface free energy of the substratum surfaces with time - especially in the case of He treated samples - seem to favor bacterial adhesion and aggregation. The dispersion-polar and the Lifshitz-van der Waals (LW) acid-base (AB) thermodynamic approaches were applied to calculate the Gibbs free energy changes of adhesion (DeltaG(adh)) of S. epidermidis interacting with the substrates. There was a strong correlation between the thermodynamic predictions and the measured values of bacterial adhesion, when adhesion was performed under static conditions. By decoupling the (DeltaG(adh)) values into their components, we observed that polar/acid-base interactions dominated the interactions of bacteria with the substrates in aqueous media. However, under flow conditions, the increase in the shear rate restricted the predictability of the thermodynamic models.

Liposome coated stents: a method to deliver drugs to the site of action and improve stent blood-compatibility.

A method to correct stent related complications non-invasively, is the local delivery of therapeutic agents. Different drugs have been delivered on stents, after being either dispersed or encapsulated in polymeric materials, and placed on stents to form drug-eluting-stents (DE-stents). Investigation of possibility to cover polymer - coated metallic stents, with liposomal drugs, for preparation of novel DE-liposome-coated-stents, has been initiated few years ago. In this context our research has been focused on answering the following questions: (i) Can liposomes be applied as coatings on polymer covered stents? (ii) Can drug release from liposome coated-stents be controlled? And: (iii) how is haemo-compatibility of stents affected? The results of the experiments carried out demonstrate that liposomal formulations of drugs can be used as coating systems of polymer covered stents for achieving sustained release of drugs at the site of interest. By modifying liposome characteristics, different amounts of drugs may be placed on the stents and their release rates can be adjusted for maximum therapeutic benefit. Finally, haemocompatibility of stents is highly improved (mainly in terms of cell adhesion and activation of coagulation system), when stents are coated with heparin-encapsulating -DRV liposomes.

Adhesion of slime producing Staphylococcus epidermidis strains to PVC and diamond-like carbon/silver/fluorinated coatings.

Staphylococcus epidermidis has emerged as a pathogen associated with infections of implanted medical devices. Bacterial adhesion is a crucial step in infection on biomaterial surfaces. To quantitatively determine the relationship between poly (vinyl chloride) (PVC) surface properties and bacterial adhesion, we have compared attachment of slime-producing S. epidermidis strains on PVC and various coatings under flow conditions. Bacterial adhesion and colonization was quantified by counting the viable organisms on the adherent surface as well as by scanning electron microscopy, epifluorescence microscopy and atomic force microscopy. Fluorination of the PVC surface encourages S. epidermidis adhesion whereas; diamond-like carbon (DLC) and especially silver (Ag) coatings seem to inhibit its adhesion. In most materials, the number of adherent bacteria decreased with the increase of shear rate. These results indicate that bacterial adhesion is influenced by the chemical properties of the polymeric surfaces, the surface roughness and the associated flow conditions.

Development of polymer membranes with improved haemocompatibility for biohybrid organ technology.

Biomedical technology has opened up possibilities of treating the failure of internal organs like kidney and liver by artificial organ therapy. Most of these techniques are based on polymer membranes, which allow the removal of excess of water, salts and toxins from the circulation. However, haemodialysis for the replacement of kidney function results in an increased morbidity and mortality of patients after long-term application. Conventional therapy, such as haemofiltration for the treatment of acute liver failure does not improve significantly the survival rate of patients. Biohybrid organ support as a combination of the artificial organ therapy with the functional activity of immobilised cells seems to be a solution of the problem. Membranes applied in these devices have to face both tissue cells and blood. Organ cells in biohybrid organs have to make intimate contact with the surface of membrane but must also develop close cell-cell-connections as a prerequisite for their survival and high functional activity. Blood to be detoxified will contact the other side of membrane and may not become activated by the synthetic material. New polymer membranes based on acrylonitrile were developed to address these requirements by tailoring the composition of copolymers and to be applied in a specific hollow fibre bioreactor with an outer fibre for blood contact, and an inner fibre for tissue contact or vice versa.

Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions.

This article reviews the mechanisms of bacterial adhesion to biomaterial surfaces, the factors affecting the adhesion, the techniques used in estimating bacteria-material interactions and the models that have been developed in order to predict adhesion. The process of bacterial adhesion includes an initial physicochemical interaction phase and a late molecular and cellular one. It is a complicated process influenced by many factors, including the bacterial properties, the material surface characteristics, the environmental factors, such as the presence of serum proteins and the associated flow conditions. Two categories of techniques used in estimating bacteria-material interactions are described: those that utilize fluid flowing against the adhered bacteria and counting the percentage of bacteria that detach, and those that manipulate single bacteria in various configurations which lend themselves to more specific force application and provide the basis for theoretical analysis of the receptor-ligand interactions. The theories that are reviewed are the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the thermodynamic approach and the extended DLVO theory. Over the years, significant work has been done to investigate the process of bacterial adhesion to biomaterial surfaces, however a lot of questions still remain unanswered.

Comparison of haemocompatibility improvement of four polymeric biomaterials by two heparinization techniques.

Two surface heparinization procedures, introduced by Bamford and Al-Lamee (Polymer 22 (1996) 4885; 13 (1994) 2844) and Seifert et al. (J. Mater. Sci.: Mater. Med. 7 (1996) 465), respectively, were applied to four commercially available biomaterials (silicone rubber, polyethylene, polypropylene and polyvinylchloride) in order to compare their efficiency in improving haemocompatibility. The indirect method (Bamford and Al-Lamee) produced a much better heparinization yield-10.5% maximum, compared to the direct one (Seifert et al.), of only 0.20% maximum. Both methods provided a better response of the heparinized biomaterials compared to the uncoated ones in terms of platelet retention and a significantly better response in terms of activation of the coagulation system, suggesting that heparin molecules remained biologically functional in both cases. The results were particularly interesting in the case of polyvinylchloride where the maximum immobilization yield was obtained by the indirect method resulting also to a pronounced haemocompatibility improvement. Scanning electron microscopy studies confirmed adhered platelet morphology whereas atomic force microscopy was used to examine surface morphology of heparinized and reference materials surface.

Assessment of techniques used in calculating cell-material interactions.

A review of the techniques used in measuring the forces of deadhesion of cells that have been adhering on substrate surfaces is presented. Two categories of techniques are described, those that utilize fluid flowing against the adhered cells and counting the percentage of cells that detach (global tests) and the manipulation of single cells in various configurations which lend themselves to more specific force application and provide the basis for theoretical analysis of the receptor-ligand mechanics.

Polyetherimide: a new membrane-forming polymer for biomedical applications.

Membranes for biohybrid organs such as the biohybrid liver support system have to face 2 different environments, namely blood and tissue cells. Accordingly, the respective membrane surfaces must have optimal properties in terms of biocompatibility for blood or tissue. Flat membranes prepared by a phase inversion process from polyetherimide were modified by binding of tris-(hydroxymethyl)-aminomethane to obtain a surface with hydroxyl groups by binding of polyethylene imine to attach a hydrophilic macromolecule with amine groups useful as a spacer for later bonding of further ligands and by attachment of heparin. The binding of the different ligands was successful as monitored by different physicochemical methods. The blood response of plain polyetherimide was comparable to that of polyacrylonitrile, and it could be further improved by the binding of heparin. The tissue compatibility of polyetherimide and its different modifications was compared with commercial cell culture substrate membranes (Millicell) and found to be comparable for polyetherimide and even better after the modification with tris-(hydroxymethyl)-aminomethane. In conclusion, polyetherimide seems to be an interesting material for the production of membranes for application in biohybrid organ systems.

Influence of test protocol in determining the blood response to model polymers.

A multi-parametric, multi-center evaluation of three polymers was performed measuring their response to blood contact. The purpose of this study was to pinpoint differences in tests performed for assessing "basic" hemocompatibility on identical materials at different centers and attempt to rationalize. Assays for platelet adhesion, activation, aggregability and activation of the coagulation system in addition to an ex vivo patency assay were performed at four centers across Europe, using protocols favored by each center for determining the blood-contacting performance of a biomaterial. Three polymers were chosen for their expected blood response spanning the range of undesirable to desirable: ethylenevinylacetate (EVA), polyvinylchloride (PVC) and PVC modified with polyethylene oxide (PEO). The assays were ranked in terms of their efficacy compared to cost and simplicity. A correlation between assays was calculated, indicating the ability of one test to correctly determine the blood response compared to another. Some assays were unable to distinguish between materials, but of the assays which could, the materials were ranked in the following order: EVA; PVC; PVC-PEO, EVA producing the most undesirable response. It is concluded that many commonly used assays for determining hemocompatibility are inappropriate, but there are simple and reliable test methods available which correlate well with the more sophisticated protocols.

Impairment of erythrocyte viscoelasticity is correlated with levels of glycosylated haemoglobin in diabetic patients.

Erythrocytes from diabetic patients exhibit impaired viscoelastic properties when estimated by various methods. We determined erythrocyte filterability through 5-microm pores, in 51 patients with non-insulin-dependent diabetes mellitus, 18 healthy controls, 15 patients with homozygous beta-thalassemia and 15 with beta-thalassemia traits. The filtration measurements were made with a Hemorheometer, which uses the "initial flow rate" principle. To determine the Index of Rigidity (IR) of the red blood cells, we measured the passage time of white blood cell-free erythrocyte suspensions, 8% per volume, through the filter. Diabetic patients had significantly increased IR in comparison to healthy controls and to patients with beta-thalassemia trait, but not at the level found in patients with homozygous beta-thalassemia. In diabetic patients, a strong correlation between IR and the percentage of glycosylated haemoglobin was found (r=0.737, P < 0.0001), and a weaker one with serum unconjugated bilirubin (r=0.363, P=0.0097) and serum total lipids (r=0.321, P=0.0286). Patients with severe retinopathy also had significantly increased IR, in comparison to those with or without mild retinopathy. Anaemic diabetic patients, especially those with the anaemia of chronic disease, also had significantly increased IR in comparison to non-anaemic diabetics. No correlation between IR, MCV, MCH, MCHC, RDW, RBC morphology, serum LDH or the presence of erythrocyte inclusions after incubation with nitrous sodium solution was found. Our findings suggest that glycosylation of skeletal proteins probably contributes significantly to the increased membrane rigidity of diabetic erythrocytes.

Effect of surface roughness of the titanium alloy Ti-6Al-4V on human bone marrow cell response and on protein adsorption.

The effect of surface roughness of the titanium alloy Ti-6Al-4V (Ti alloy) on the short- and long-term response of human bone marrow cells in vitro and on protein adsorption was investigated. Three different values in a narrow range of surface roughness were used for the substrata (R(alpha): 0.320, 0.490 and 0.874 microm). Cell attachment, cell proliferation and differentiation (alkaline phosphatase specific activity) were determined past various incubation periods. The protein adsorption of bovine serum albumin and fibronectin, from single protein solutions, on rough and smooth Ti alloy surfaces was examined with two methods, X-ray photoelectron spectroscopy (XPS) and radiolabeling. Cell attachment and proliferation were surface roughness sensitive and increased as the roughness of Ti alloy increased. No statistically significant difference was observed in the expression of ALP activity on all three Ti alloy surfaces and culture plastic. Both methods, XPS and protein radiolabeling, showed that human serum albumin was adsorbed preferentially onto the smooth substratum. XPS technique showed that the rough substratum bound a higher amount of total protein (from culture medium supplied with 10% serum) and fibronectin (10-fold) than did the smooth one. The cell attachment may be explained by the differential adsorption of the two proteins onto smooth and rough Ti alloy surfaces.

Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength.

Initial attachment of osteoblast cells and mineralization phenomena are generally enhanced on rough, sandblasted substrata. In the present work the effect of surface roughness of hydroxyapatite (HA) on human bone marrow cell response was investigated. Human bone marrow cells were plated onto HA disc-shaped pellets, prepared from synthetic HA powder. The pellets were sintered and polished with SiC paper 180-, 600- and 1200-grit, resulting in three surface roughness grades. Cell adhesion, proliferation and differentiation (evaluated with the expression of ALP activity) were determined following various incubation periods. Cell detachment strength was determined as the shear stress required to detach a given quantity of the adherent cells from the different substrata, using a rotating disc device that applied a linear range of shear stresses to the cells. The cells attached and grew faster on culture plastic in comparison with HA. No statistically significant differences were observed in the expression of ALP activity on all three HA surfaces and culture plastic. Cell adhesion, proliferation and detachment strength were surface roughness sensitive and increased as the roughness of HA increased. The percentage of the adherent cells decreased in a sigmoidal mode as a function of the applied shear stress. In conclusion, surface roughness of HA generally improved the short- and longer-term response of bone marrow cells in vitro. This behavior could be explained by the selective adsorption of serum proteins.

Polymorphonuclear leukocyte rigidity is defective in patients with chronic renal failure.

BACKGROUND: The purpose of the study was to investigate the rigidity of polymorphonuclear leukocytes (PMNs) in non-dialysed chronic renal failure (CRF) and haemodialysis (HD) patients. METHODS: PMN rigidity as well as tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) plasma levels were assessed in 10 early-stage CRF, 10 late-stage non-HD, and 10 HD patients, before and during dialysis. In HD patients both cellulose acetate and polysulphone membranes were used. Ten healthy subjects served as controls. Rigidity was tested by counting the deformability in morphologically passive PMNs by the micropipette method. Cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: PMN rigidity was significantly increased in end-stage CRF patients regardless of HD but not in early-stage CRF. In HD patients PMN rigidity increased significantly 60 min after initiation of HD. There was an increase of TNF-alpha and IL-1beta levels in end-stage non-HD and HD patients and a further increase at 60 min after initiation of HD. The percentage of morphologically activated PMNs was increased only during dialysis. The nature of the HD membrane had no influence on rigidity, PMN activation, or cytokine production. CONCLUSIONS: The results indicate that PMN rigidity is defective in end-stage chronic CRF patients and is further increased 60 min after initiation of HD, regardless of the nature of the HD membrane used. PMN activation, increased TNF-alpha and IL-1beta levels, or a direct PMN impairment may cause the observed cell rigidity.

Effect of cytokines and colony-stimulating factors on passive polymorphonuclear leukocyte deformability in vitro.

Cytokines are potent polymorphonuclear leukocyte (PMN) activators and can decrease their deformability. We evaluated passive PMN deformability using the micropipette method after incubation with different concentrations of lipopolysaccharide (LPS), interleukins (IL-) 1, 6, 8 and 10, tumour necrosis factor (TNF), granulocyte (G) and granulocyte-macrophage (GM) colony-stimulating factors (CSF). TNF, IL-1, G-CSF, GM-CSF and, to a lesser degree, IL-6 significantly and in a dose-dependent fashion decrease PMN deformability. LPS had no direct effect on PMN deformability. When cytokines at concentrations with no effect on deformability were combined they increased PMN rigidity. The findings suggest that several cytokines and CSF impair directly, and not by activation alone, PMN deformability.

Neutrophil deformability in patients with sepsis, septic shock, and adult respiratory distress syndrome.

OBJECTIVE: To investigate the deformability of morphologically active and passive neutrophils in patients with sepsis (SP), septic shock (SS), and adult respiratory distress syndrome (ARDS). DESIGN: Prospective, observational study. SETTING: A university hospital intensive care unit and research laboratory. PATIENTS: Six patients with sepsis, six patients with septic shock, and six patients with ARDS. Eight healthy volunteers and eight ventilated but noninfected patients served as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Morphologically passive and active neutrophil deformability as defined by the micropipette method was significantly decreased in patients with SP, SS, and ARDS associated with sepsis as compared with both control groups. Neutrophils from SS and ARDS patients were significantly more rigid as compared with neutrophils from SP patients but they did not differ from each other. The percentage of activated neutrophils was significantly higher in SP, SS, and ARDS patients. Increased passive neutrophil rigidity was significantly attenuated after coincubation with cytochalasin D. Tumor necrosis factor-alpha and interleukin-1beta serum levels were significantly higher in SP, SS, and ARDS patients. CONCLUSIONS: The entire neutrophil population is less deformable in SP, SS, and ARDS patients. The decreased deformability of passive neutrophils suggests that a direct mechanism involving actin polymerization, distinct from cell activation, is involved. These observations may be important in the mechanism of impaired vascular flow in patients with sepsis.

Deformability and filterability of white blood cell subpopulations. Evaluation of these parameters in the cell line HL-60 and in type II diabetes mellitus.

The rheological properties of human leukocytes (WBCs) have been studied using the micropipette aspiration and the filtration technique. Partial micropipette (i.d. 2.8-4.5 microm) aspiration of individual leukocytes under constant aspiration pressure of 8 mm H20 and measurement of the aspirated length as a function of time (creep experiments) according to the Evans model have been carried out and the apparent viscosity mu(app) was estimated. In the filtration experiments, using the Hemorheometer, the Index Rigidity of Leukocytes, ILR, was also estimated. The apparent viscosity mu(app) of normal PMN and MNC was significantly different p < 0.05), while the LYM and PMN had no statistical difference (p < 0.5). The leukocytes of the cell line HL-60 were more rigid than the normal PMN (p < 0.01), while the PMN from patients with type II diabetes mellitus were more rigid than the normal PMN (p < 0.005). The results of IRL showed similar differences among all of the leukocyte subpopulations. Comparison of these findings suggests a possible relationship between ILR and mu(app) which in this case is: ILR = 598 + 0.54 mu(app) (r = 0.986, p-value 0.0003).