Phospholipid polymer surfaces reduce bacteria and leukocyte adhesion under dynamic flow conditions

Persistence of infection can occur when the host immune response is compromised because of the presence of a foreign implant. Surface modification of biomaterials with phospholipid polymers may enhance biocompatibility and reduce incidence of infection by impeding bacterial and leukocyte adhesion. A rotating disk model, which generates shear stress from 0 to 18 dynes/cm(2), was used to characterize adhesion of neutrophils, monocytes, and bacteria in phosphate-buffered saline (PBS) or 25% human serum on polyethylene terephthalate surfaces coated with a phospholipid polymer, poly[omega-methacryloyloxyalkyl phosphorylcholine (MAPC)-co-n-butyl methacrylate (BMA)]. The material designated PMB30 contains a methylene chain length, (CH(2))(n), of n = 2, whereas PMHB30 contains a chain length of n = 6. In PBS, bacterial adhesion was shear stress dependent with the lowest bacterial density observed on PMB30. However, the presence of serum proteins eliminated shear stress and surface chemistry effects in addition to bacterial adhesion reduced to <10% of adhesion in PBS. Trends for leukocyte adhesion in serum demonstrated shear dependence with PMB30 exhibiting the lowest cell density throughout the range of shear stresses. In conclusion, modification of the polyethylene terephthalate surfaces with phospholipid polymers resulted in reduced bacterial and leukocyte adhesion. Furthermore, shortening the methylene chain length of the MAPC copolymer most effectively reduced adhesion.     

Histochemical characteristics of experimental moniliasis under specific sensitization conditions

The role of specific sensitization in the pathogenesis of experimental moniliasis was studied histochemically. Activity of several enzymes of the fungus and of infiltrating inflammatory cells was determined and assessed quantitatively. Tissue changes in visceral moniliasis were found to correspond to changes in the active substances in cells of the fungus and of the focus of inflammatory infiltration. In the early stages of parasitism increased activity of fungal enzymes was observed, followed by a decrease, which coincided with a decrease in the number of vegetative forms. Preliminary administration of monilial allergen induced increased sensitivity to subsequent infection. Changes in specifically sensitized rats developed against the background of marked vascular disorders and followed a hyperergic type of course.Department of Pathological Anatomy and Department of Microbiology and Medical Mycology, S. M. Kirov Leningrad Postgraduate Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 1009–1011, August, 1976.     

Mathematical model of platelet deposition under flow conditions

Platelet deposition in resting blood is well researched and understood. However, the influence of hemodynamic parameters such as wall shear rate is less clear. Clinical experience and experiments show an interaction between flow and platelet deposition. But a complete understanding of the flow influence and hence a quantification has not yet been achieved. A well defined experiment of flow dependant platelet depositions is the stagnation point flow. This kind of flow is ubiquitous in the circulatory system, to be found in every bifurcation and recirculation region. These are the areas where thrombus formations are likely to occur if other conditions are also met. First, experiments were performed in a stagnation point flow chamber. A simplified blood model, platelet rich plasma, was used as a test fluid. With a microscope the platelet deposition was observed and recorded. Platelets deposit in a characteristic pattern showing the influence of the flow. An analysis of this pattern is the objective of this study and is achieved with the help of a numerical model, which is based on a convective diffusive transport. The model results in a platelet deposition pattern, which in its shape and temporal development is very similar to experimental results. Hence it is concluded that the assumed transport processes are causal for platelet depositions and thrombus formation.     

Competitive protein adsorption on biomaterial surface studied with reflectometric interference spectroscopy

Reflectometry interference spectroscopy (RIfS) is known as a highly sensitive and robust technique for direct, label-free detection of the interaction of biomacromolecules in real time and in situ. The aim of the present study was to investigate the competitive protein adsorption on the surface of fluorocarbon end-capped poly(carbonate) urethane (PCUF) and polystyrene (PS) based on the RIfS method. The surface energy and microstructures of PCUF and PS were characterized by contact angle measurement and atomic force microscopy. Interfacial energies between these surfaces and the proteins were then calculated. The protein adsorption experiments were carried out with both single solution and ternary solutions composed of albumin, fibrinogen and immunoglobulin-G (IgG). The results of surface characterization showed that PCUF was more hydrophilic than PS with a smaller surface energy, and micro-phases separation of PCUF was observed. RIfS analysis results revealed that more albumins, less fibrinogen and IgG were detected on the PCUF surface compared with PS after simplex and competitive protein adsorption, which indicated that PCUF had a preferential adsorption for albumin. The special morphology, smaller surface energy and calculated interfacial energies between PCUF and proteins may be responsible for the better blood compatibility of PCUF compared to PS. The results suggest that RIfS could serve as a novel, effective method for studying the competitive protein adsorption on biomaterial surfaces.     

Limitations of flow cytometry for the specific detection of bacteria in mixed populations

Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with fluorescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.     

Tumor cell adhesion under hydrodynamic conditions of fluid flow

Current evidence indicates that tumor cell adhesion to the microvasculature in host organs during formation of distant metastases is a complex process involving various types of cell adhesion molecules. Recent results have shown that stabilization of tumor cell adhesion to the microvascular vessel wall is a very important step for successful tumor cell migration and colonization of host organs. We are beginning to understand the influences of fluid flow and local shear forces on these adhesive interactions and cellular responses within the circulation. Mechanosensory molecules or molecular complexes can transform shear forces acting on circulating tumor cells into intracellular signals and modulate cell signaling pathways, gene expression and other cellular functions. Flowing tumor cells can interact with microvascular endothelial cells mediated mainly by selectins, but the strength of these bonds is relatively low and not sufficient for stable cell adhesions. Integrin-mediated tumor cell adhesion and changes in the binding affinity of these adhesion molecules appear to be required for stabilized tumor cell adhesion and subsequent cell migration into the host organ. Failure of the conformational affinity switch in integrins results in breaking of these bonds and recirculation or mechanical damage of the tumor cells. Various cell signaling molecules, such as focal adhesion kinase, pp60src or paxillin, and cytoskeletal components, such as actin or microtubules, appear to be required for tumor cell adhesion and its stabilization under hydrodynamic conditions of fluid flow.     

Molecular barriers to biomaterial thrombosis by modification of surface proteins with polyethylene glycol

For cardiovascular biomaterials, thrombosis, thromboembolism and vascular graft occlusion are believed to be precipitated by the adsorption of proteins containing adhesive ligands for platelets. Polyethylene-glycol-diisocyanate(PEG-diisocyanate, 3400 MW) may potentially react with protein amines to form molecular barriers on adsorbed proteins on biomaterials, thereby masking adhesive ligands and preventing acute surface thrombosis. To test this notion, PE, PTFE, and glass microconduits were pre-adsorbed with fibrinogen and treated with PEG-diisocyanate, non-reactive PEG-dihydroxyl, or remained untreated. Following perfusion of 111In-labeled platelets in whole human blood for 1 min (wall shear rate = 312 s(-1)), PEG-diisocyanate treated surfaces experienced 96%(PE), 97%(PTFE) and 94% (glass) less platelet deposition than untreated surfaces. Similar reductions were seen for PEG-diisocyanate versus PEG-dihydroxyl treatment. Low shear perfusions of plasma for one hour prior to blood contact did not reduce the inhibitory effect of PEG-diisocyanate. Platelet adhesion onto collagen coated glass coverslips and platelet deposition onto preclotted Dacron was also reduced by treatment with PEG-diisocyanate (93 and 91%, respectively). Protein-reactive PEG may thus have utility in forming molecular barriers on surface associated proteins to inhibit acute thrombosis on cardiovascular biomaterials.     

Isolation of specific antibody under conditions of low ionic strength.

Specific rabbit and goat antibodies to tobacco mosaic virus, bovine serum albumin and human gamma globulin have been isolated by acid dissociation from the antigen in the absence of salt. Maximum recovery of 96-100% of active antibody occurred at pH 2.8-2.9. The amount of antibody recovered within any given pH range was highly reproducible. The average antibody affinity was found to be correlated with the pth of dissociation and to decrease exponentially with the length of exposure to acid conditions.     

Platelets promote eosinophil adhesion of patients with asthma to endothelium under flow conditions

During the late-phase asthmatic response, eosinophils migrate to the bronchial tissue and cause severe damage. In this study we compared in vivo primed eosinophils from patients with allergic asthma with eosinophils from healthy control subjects in their adhesion behavior to tumor necrosis factor-alpha-activated endothelium under flow conditions (0.8 dyn/cm2). More eosinophils from patients with asthma adhered to activated endothelium, compared with cells from healthy control subjects (1,237 +/- 126 versus 887 +/- 94 cells/mm2, respectively). In the presence of blocking antibodies directed against very late antigen-4 and E-selectin, the residual binding of the cells of individuals with allergic asthma was significantly higher than that of the healthy control subjects (353 +/- 64 versus 123 +/- 31 cells/mm2, respectively, P < 0.02). In addition, secondary tethering or formation of clusters of the eosinophils of patients with allergic asthma was significantly increased compared with the healthy control subjects (cluster indices 1.8 +/- 0.3 versus 0.8 +/- 0.2, respectively, P < 0.05). Because patient cells showed an enhanced interaction with platelets during the perfusions, the role of P-selectin on platelets was investigated. Blocking antibodies directed against P-selectin reduced the enhanced binding and clustering of eosinophils of patients with allergic asthma. We conclude that P-selectin-bearing platelets contribute to secondary tethering processes of eosinophils to activated endothelium. Therefore, platelets might play an important role in the chronic inflammatory processes of these patients.     

Hemolysis caused by surface roughness under shear flow

In this study, the relationship between the degree of roughness of blood contact surfaces under laminar shear flow conditions and the level of hemolysis resulting from this roughness was investigated using a rotational shear stressor. Unlike previous in vitro experiments that used a pumped circuit, the level of hemolysis was directly evaluated under a constant shear flow. In total, 1.8% of the blood contact area was roughened to an arithmetic mean roughness (Ra) value of between 0.4 and 9.2 μm by machine processing and a shear load was applied for 30 min at a shear flow rate of 3750 s⁻¹. As a result, the threshold Ra value for the induction of hemolysis was found to be between 0.4 and 0.8 μm. In addition, the results of this experiment suggested that the high shear stress resulting from surface roughness plays a major role in determining the level of hemolysis caused by surface roughness.     

The use of dynamic surface chemistries to control msc isolation and function

Material modifications can be used to induce cell responses, in particular-CH(3) and -NH(2) have shown potential in enhancing the ability of a material to support mesenchymal stem cell (MSC) adhesion and differentiation. Currently this process is variable, due to the lack of definition of controlled contextual presentation of the chemical group of interest across the surface. This paper defines the potential of -CH(3) modified surfaces, with optimised dynamic surface chemistry, to manipulate initial MSC adhesive events, integrin binding, and subsequent cell function. An array of -CH(3) silane modified glass substrates was produced using different -CH(3) chain lengths and mechanisms of bonding to the base substrate. We show that changing the chain length affects the ability of the surfaces to support viable adult MSC adhesion, directly related to induced FGF release, and expression of STRO-1, CD29, 73, 90 and 105. Chlorodimethyloctylsilane (ODMCS) modified surfaces resulted in significant increases of associated adult MSC markers compared to all other -CH(3) modified and control substrates. In contrast Dichlorodimethylsilane (DMDCS) modified surfaces did not support adult MSC adhesion due to high levels of early FGF release, which had an inhibitory effect on adult MSC culture, but enhanced the efficiency and cell selective properties of the substrate in isolation of multi-potent progenitor/MSC from adult human whole blood. Incorporation of optimised -CH(3) groups is a cost effective route for producing substrates that significantly enhance MSC isolation and expansion, highlighting the potential of the optimised substrates to replace RGD and fibronectin modifications in selected applications.     

Cytokine profiling using monocytes/macrophages cultured on common biomaterials with a range of surface chemistries

Cytokines, chemokines, and growth factors were assayed from the supernatants of monocytes and macrophages cultured on common biomaterials with a range of surface chemistries. TNF-alpha, MCP-1, MIP-1alpha, IL-8, IL-6, IL-1beta, VEGF, IL-1ra, and IL-10 were measured from monocyte/macrophage cultures at different stages of activation and differentiation seeded onto polyethylene, polyurethane, expanded polytetrafluoroethylene, polymethyl methacrylate, and a hydrogel copolymer of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol acrylate in tissue culture polystyrene (TCPS) plates. Empty TCPS wells and organo-tin polyvinyl chloride served as "blanks" and positive controls, respectively. Results showed an overall increase in cytokine, chemokine, and growth factor production as monocytes are activated or differentiated into macrophages and that proinflammatory and anti-wound healing cytokines and chemokines dominate this profile. However, cytokine production was only modestly affected by the surface chemistry of these four stable and noncytotoxic biomaterials.     

MRI of cerebral blood flow under hyperbaric conditions in rats

Hyperbaric oxygen (HBO) therapy has a number of clinical applications. However, the effects of acute HBO on basal cerebral blood flow (CBF) and neurovascular coupling are not well understood. This study explored the use of arterial spin labeling MRI to evaluate changes in baseline and forepaw stimulus‐evoked CBF responses in rats (n = 8) during normobaric air (NB), normobaric oxygen (NBO) (100% O ₂ ), 3 atm absolute (ATA) hyperbaric air (HB) and 3 ATA HBO conditions. T ₁ was also measured, and the effects of changes in T ₁ caused by increasing oxygen on the CBF calculation were investigated. The major findings were as follows: (i) increased inhaled oxygen concentrations led to a reduced respiration rate; (ii) increased dissolved paramagnetic oxygen had significant effects on blood and tissue T ₁ , which affected the CBF calculation using the arterial spin labeling method; (iii) the differences in blood T ₁ had a larger effect than the differences in tissue T ₁ on CBF calculation; (iv) if oxygen‐induced changes in blood and tissue T ₁ were not taken into account, CBF was underestimated by 33% at 3 ATA HBO, 10% at NBO and <5% at HB; (v) with correction, CBF values under HBO, HB and NBO were similar (p > 0.05) and all were higher than CBF under NB by ~40% (p < 0.05), indicating that hypercapnia from the reduced respiration rate masks oxygen‐induced vasoconstriction, although blood gas was not measured; and (vi) substantial stimulus‐evoked CBF increases were detected under HBO, similar to NB, supporting the notion that activation‐induced CBF regulation in the brain does not operate through an oxygen‐sensing mechanism. CBF MRI provides valuable insights into the effects of oxygen on basal CBF and neurovascular coupling under hyperbaric conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

Influence of biomaterial surface chemistry on the apoptosis of adherent cells

A common component of the foreign-body response to implanted materials is the presence of adherent macrophages that fuse to form foreign-body giant cells (FBGCs). These multinucleated cells have been shown to concentrate the phagocytic and degradative properties of macrophages at the implant surface and are responsible for the damage and failure of the implant. Therefore, the modulation of the presence or actions of macrophages and FBGCs at the material-tissue interface is an extensive area of recent investigations. A possible mechanism to achieve this is through the induction of the apoptosis of adherent macrophages, which results in no inflammatory consequence. We hypothesize that the induction of the apoptosis of biomaterial adherent cells can be influenced by the chemistry of the surface of adhesion. Herein, we demonstrate that surfaces displaying hydrophilic and anionic chemistries induce apoptosis of adherent macrophages at a higher magnitude than hydrophobic or cationic surfaces. Additionally, the level of apoptosis for a given surface is inversely related to that surface's ability to promote the fusion of macrophages into FBGCs. This suggests that macrophages fuse into FBGCs to escape apoptosis.     

Continuous opacity monitoring of the growth of bacteria under strict anaerobic conditions.

A newly designed instrument is described which generates continuous records of the opacities of six bacterial cultures growing under strict anaerobic conditions. Additions (for example, of antibacterial agents) or withdrawal of culture (for example, for viable counting) can be made at any time without breach of anaerobiosis. Use of the instrument is illustrated by growth curves obtained from small inocula of two strict anaerobes, Bacteroides asaccharolyticus and Peptostreptococcus anaerobius and by the effects on the growth curve of Bacteroides fragilis of adding various concentrations of metronidazole at different times.     

Quantification of the interaction between biomaterial surfaces and bacteria by 3-D modeling

It is general knowledge that bacteria/surface interactions depend on the surface topography. However, this well-known dependence has so far not been included in the modeling efforts. We propose a model for calculating interaction energies between spherical bacteria and arbitrarily structured 3-D surfaces, combining the Derjaguin, Landau, Verwey, Overbeek theory and an extended surface element integration method. The influence of roughness on the interaction (for otherwise constant parameters, e.g. surface chemistry, bacterial hydrophobicity) is quantified, demonstrating that common experimental approaches which consider amplitude parameters of the surface topography but which ignore spacing parameters fail to adequately describe the influence of surface roughness on bacterial adhesion. The statistical roughness profile parameters arithmetic average height (representing an amplitude parameter) and peak density (representing a spacing parameter) both exert a distinct influence on the interaction energy. The influence of peak density on the interaction energy increases with decreasing arithmetic average height and contributes significantly to the total interaction energy with an arithmetic average height below 70 nm. With the aid of the proposed model, different sensitivity ranges of the interaction between rough surfaces and bacteria are identified. On the nanoscale, the spacing parameter of the surface dominates the interaction, whereas on the microscale the amplitude parameter adopts the governing role.     

Chronic ethanol increases fetal cerebral blood flow specific to the ethanol-sensitive cerebellum under normoxaemic, hypercapnic and acidaemic conditions: ovine model

Cerebral hypoxia has been proposed as a mechanism by which prenatal ethanol exposure causes fetal alcohol spectrum disorder (FASD) in children, but no study had tested this hypothesis using a chronic exposure model that mimicks a common human exposure pattern. Pregnant sheep were exposed to ethanol, 0.75 or 1.75 g kg(-1) (to create blood ethanol concentrations of 85 and 185 mg dl(-1), respectively), or saline 3 days per week in succession (a 'binge drinking' model) from gestational day (GD) 109 until GD 132. Fetuses were instrumented on GD 119-120 and studied on GD 132. The 1.75 g kg(-1) dose resulted in a significant increase in fetal biventricular output (measured by radiolabelled microsphere technique) and heart rate, and a reduction of mean arterial pressure and total peripheral resistance at 1 h, the end of ethanol infusion. The arterial partial pressure of CO(2) was increased, arterial pH was decreased and arterial partial pressure of O(2) did not change. Fetal whole-brain blood flow increased by 37% compared with the control group at 1 h, resulting in increased cerebral oxygen delivery. The elevation in brain blood flow was region specific, occurring preferentially in the ethanol-sensitive cerebellum, increasing by 44% compared with the control group at 1 h. There were no changes in the lower dose group. Assessment of regional differences in the teratogenic effects of ethanol by stereological cell-counting technique showed a reduced number of cerebellar Purkinje cells in response to the 1.75 g kg(-1) dose compared with the control brains. However, no such differences in neuronal numbers were observed in the hippocampus or the olfactory bulb. We conclude that repeated exposure to moderate doses of ethanol during the third trimester alters fetal cerebral vascular function and increases blood flow in brain regions that are vulnerable to ethanol in the presence of acidaemia and hypercapnia, and in the absence of hypoxia.     

Osteoblast response to PLGA tissue engineering scaffolds with PEO modified surface chemistries and demonstration of patterned cell response

Because tissues are characterized by a well-defined three-dimensional arrangement of cells, tissue engineering scaffolds that facilitate the organization and differentiation of new tissue will have improved performance in comparison to scaffolds that only provide surfaces for cell attachment and growth. We hypothesize that instructions for cells can be incorporated into tissue engineering scaffolds by patterning the scaffold's architecture and surface chemistry. Our goals for the presented work were to collect data about cell response to three-dimensional, porous scaffolds with uniformly modified surfaces chemistries, and to demonstrate patterning of cell response by patterning surface chemistry. Our system was osteoblast response to poly(l-lactide-co-glycolide) scaffolds modified with poly(ethylene oxide) (PEO). Scaffolds were fabricated using the Three-Dimensional Printing (3DP) process which has control over scaffolds properties to a resolution of approximately 100 microm in all three dimensions. At higher PEO concentrations, adhesion, growth rates, and migration of rat osteoblasts were reduced; alkaline phosphate activity was increased, and cells were less spread and had microvilli. Patterned regions of low and high cell adhesion were demonstrated on scaffolds fabricated with 1 mm thick stripes of PEO and non-PEO regions.     

Interactions of acinar cells on biomaterials with various surface properties

The purpose of this study is to evaluate the interactions of rat parotid acinar cells on biomaterials with different surface properties. The biomaterials used in this study included polyvinyl alcohol (PVA), chitosan, poly (ethylene-co-vinyl alcohol) (EVAL), and polyvinylidene fluoride (PVDF). Cell morphology was observed by photomicroscope. Cell growth and differentiated characteristic function were separately assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity and amylase activity. Results indicated that behaviors of acinar cells on materials might differ to a great extent depending on the surface hydrophilicity and morphology of the materials. On the relatively hydrophobic materials, the abilities of acinar cells to adhere and proliferate increased simultaneously. In addition, porous PVDF had higher cell growth compared with dense PVDF. Therefore, the hydrophobic PVDF with a porous structure was the best substrate for culturing acinar cells. According to our findings, a tubular PVDF scaffold with dense outer surface to prevent saliva leakage and with porous inner surface for the cell growth was proposed to serve as an artificial salivary gland for future use in the treatment of patients with salivary hypofunction.