Water and the acute biological response to surfaces.

Molecular self association in water through hydrogen bonding is a powerful organizational force leading to a three-dimensional hydrogen-bonded network (water structure) that profoundly influences solvent properties. Localized perturbations in the chemical potential of water as by, for example, contacting with a solid surface, induces compensating changes in water structure that can be sensed tens of nanometers from the point of origin using the surface force apparatus (SFA) and ancillary techniques. These instruments reveal attractive or repulsive forces between opposing surfaces immersed in water, over-and-above that anticipated by continuum theory (DLVO), that are attributed to a variable density (partial molar volume) of a more-or-less ordered water structure, depending on the water wettability (surface energy) of the water-contacting surfaces. Water structure at surfaces is thus found to be a manifestation of hydrophobicity and, while mechanistic/theoretical interpretation of experimental results remains the subject of some debate in the literature, convergence of experimental observations permit a quantitative definition of the heretofore relative terms 'hydrophobic' and 'hydrophilic'. In particular, long-range attractive forces (< 100 nm) are detected only between surfaces exhibiting a water contact angle theta > 65 deg (defined as hydrophobic surfaces with pure water adhesion tension tau0 = gamma0 cos theta < 30 dyn cm(-1) where gamma0 is water interfacial tension = 72.8 dyn cm(-1)). Short range repulsive forces (< 5 nm) are detected between surfaces exhibiting theta < 65 deg (hydrophilic surfaces, tau0 > 30 dyn cm(-1)). These findings together with other lines of chemical evidence suggest at least two distinct kinds of water structure and reactivity: a relatively less-dense water region against hydrophobic surfaces with an open hydrogen-bonded network and a relatively more-dense water region against hydrophilic surfaces with a collapsed hydrogen-bonded network. Solvent properties of interfacial water profoundly influence the biological response to materials in a surprisingly straightforward manner when key measures of biological activity sensitive to interfacial phenomenon are scaled against water adhesion tension tau0 of contacting surfaces. Protein adsorption, activation of blood coagulation, and bioadhesion are offered as examples in point, illustrating that the hydrophobic/hydrophilic contrast in the biological response to materials, often disputed in biomaterials science, is very clear when viewed from the perspective of water structure and reactivity at surfaces.

Structure and reactivity of water at biomaterial surfaces.

Molecular self association in liquids is a physical process that can dominate cohesion (interfacial tension) and miscibility. In water, self association is a powerful organizational force leading to a three-dimensional hydrogen-bonded network (water structure). Localized perturbations in the chemical potential of water as by, for example, contact with a solid surface, induces compensating changes in water structure that can be sensed tens of nanometers from the point of origin using the surface force apparatus (SFA) and ancillary techniques. These instruments reveal attractive or repulsive forces between opposing surfaces immersed in water, over and above that anticipated by continuum theory (DLVO), that are attributed to a variable density (partial molar volume) of a more-or-less ordered water structure, depending on the water wettability (surface energy) of the water-contacting surfaces. Water structure at surfaces is thus found to be a manifestation of hydrophobicity and, while mechanistic/theoretical interpretation of experimental results remain the subject of some debate in the literature, convergence of experimental observations permit, for the first time, quantitative definition of the relative terms 'hydrophobic' and 'hydrophilic'. In particular, long-range attractive forces are detected only between surfaces exhibiting a water contact angle theta > 65 degrees (herein defined as hydrophobic surfaces with pure water adhesion tension tau O = gamma O cos theta < 30 dyn/cm where gamma O is water interfacial tension = 72.8 dyn/cm). Repulsive forces are detected between surfaces exhibiting theta < 65 degrees (hydrophilic surfaces, tau O > 30 dyn/cm). These findings suggest at least two distinct kinds of water structure and reactivity: a relatively less-dense water region against hydrophobic surfaces with an open hydrogen-bonded network and a relatively more-dense water region against hydrophilic surfaces with a collapsed hydrogen-bonded network. Importantly, membrane and SFA studies reveal a discrimination between biologically-important ions that preferentially solubilizes divalent ions in more-dense water regions relative to less-dense water regions in which monovalent ions are enriched. Thus, the compelling conclusion to be drawn from the collective scientific evidence gleaned from over a century of experimental and theoretical investigation is that solvent properties of water within the interphase separating a solid surface from bulk water solution vary with contacting surface chemistry. This interphase can extend tens of nanometers from a water-contacting surface due to a propagation of differences in self association between vicinal water and bulk-phase water. Physicochemical properties of interfacial water profoundly influence the biological response to materials in a surprisingly straightforward manner when key measures of biological activity sensitive to interfacial phenomena are scaled against water adhesion tension tau O of contacting surfaces. As examples, hydrophobic surfaces (tau O < 30 dyn/cm) support adsorption of various surfactants and proteins from water because expulsion of solute from solution into the interphase between bulk solid and solution phases is energetically favorable. Adsorption to hydrophobic surfaces is driven by the reduction of interfacial energetics concomitant with replacement of water molecules at the surface by adsorbed solute (surface dehydration). Hydrophilic surfaces (tau O > 30 dyn/cm) do not support adsorption because this mechanism is energetically unfavorable. Protein-adsorbing hydrophobic surfaces are inefficient contact activators of the blood coagulation cascade whereas protein-repellent hydrophilic surfaces are efficient activators of blood coagulation. Mammalian cell attachment is a process distinct from protein adsorption that occurs efficiently to hydrophilic surfaces but inefficiently to hydrophobic surfaces. (ABSTRACT TRUNCATED)

Contact activation of the plasma coagulation cascade. III. Biophysical aspects of thrombin-binding anticoagulants.

A biophysical model linking fibrin polymerization kinetics (following release from a thrombin-fibrinogen complex), coagulation time, and competitive inhibition of thrombin illustrates the utility of thrombin-binding ligands as anticoagulants in blood collection applications. The resulting mathematical relationship connecting fibrinogen, ligand, and thrombin concentrations was tested against experimentally observed anticoagulation of whole, platelet-poor porcine plasma induced by short, single-stranded DNA oligonucleotides originally found to bind thrombin by screening combinatorial libraries. The thrombin-fibrinogen dissociation constant Ks served as the single adjustable parameter in a least-squares fitting of the model to experimental anticoagulation data. Best-fit Ks values corroborated microM values measured in plasma-free systems, and application of the model to a ligand challenge to the intrinsic pathway of plasma coagulation corroborated nM endogenous thrombin concentrations measured in porcine blood activated by endotoxin insult in vivo. The model fit to data suggests that only about 20% conversion of blood fibrinogen to fibrin is required to coagulate (gel) porcine plasma. This prediction is consistent with the common clinical laboratory observation of latent fibrin formation in "serum" separated from blood before fibrinogen is fully converted to fibrin. It was concluded that the thrombin-binding anticoagulation model was a reasonable simulation of the situation in which an initial bolus of either exogenous or endogenous thrombin is rapidly partitioned between fibrinogen-bound and ligand-bound forms with little or no additional free thrombin created over time.

Contact activation of the plasma coagulation cascade. I. Procoagulant surface chemistry and energy.

Contact activation of the intrinsic pathway of porcine blood plasma coagulation is shown to be a steep exponential-like function of procoagulant surface energy, with low activation observed for poorly water-wettable surfaces and very high activation for fully water-wettable surfaces. Test procoagulants studied were a system of oxidized polystyrene films with varying wettability (surface energy) and glass discs bearing close-packed self-assembled silane monolayers (SAMs) with well-defined chemistry consisting of 12 different terminating chemical functionalities. A monotonic trend of increasing coagulation activation with increasing water wettability was observed for the oxidized polystyrene system whereas results with SAM procoagulants suggested a level of chemical specificity over and above the surface energy trend. In particular, it was noted that coagulation activation by SAMs terminated with--CO2H was much higher than anticipated based on surface wettability whereas--NH3(+)-terminated SAMs exhibited very low procoagulant activity. SAMs terminated in--(CH2)2(CF2)7CF3 behaved as anticipated based on surface energy with very low procoagulant activity and did not exhibit special properties sometimes attributed to perfluorinated compounds. Quantitative ranking of the inherent coagulation activation properties of procoagulant surfaces was obtained by application of a straightforward phenomenological model expressed in a closed-form mathematical equation relating coagulation time to procoagulant surface area. Fit of the model with a single adjustable parameter to experimental measurements of porcine platelet-poor plasma coagulation time was very good, implying that assertions and simplifications of the model adequately simulated reality. Two important propositions of the model were that (1) the number of putative "activating sites" scaled linearly with procoagulant surface area, and (2) contact activation of the plasma coagulation cascade was catalytic in the sense that these activating sites were not consumed or "poisoned" by irreversible or slowly reversible protein adsorption during coagulation. An extension of the coagulation model proposed that procoagulant activation properties scale exponentially with the surface density of polar (acid-base) sites, which, in turn, was related to procoagulant wettability.

Contact activation of the plasma coagulation cascade. II. Protein adsorption to procoagulant surfaces.

A study of blood protein adsorption to procoagulant surfaces utilizing a coagulation time assay, contact angles, Wilhelmy balance tensiometry, and electron spectroscopy (ESCA) is presented. Using a new contact angle method of measuring protein adsorption termed "adsorption mapping" it was demonstrated that protein-adsorbent surfaces were inefficient activators of the intrinsic pathway of the plasma coagulation cascade whereas water-wettable, protein-repellent surfaces were efficient procoagulants. Repeated use of fully water-wettable (spreading) glass procoagulants in the coagulation time assay demonstrated that putative "activating sites" were not consumed in the coagulation of platelet-poor porcine plasma. Furthermore, these procoagulant surfaces retained water-wettable surface properties after incubation with blood proteins and saline rinse. The interpretation of these observations was that plasma and serum proteins were not adsorbed to water-wettable surfaces. However, ESCA of these same surfaces revealed the presence of a thin protein layer. Wilhelmy balance tensiometry resolved these seemingly divergent observations by demonstrating that protein was "associated" with a bound hydration layer, but not formally adsorbed through a surface dehydration or ionic interaction mechanism.

A frame-relay approach for a state-wide health information network.

An approach for a frame-relay implementation is described which is intended to establish connectivity between the health care providers in the state of Connecticut with a gateway to the internet. While other health care networking efforts have based the interconnectivity efforts on direct connections to the internet for each institution, our design takes a more cost effective approach by establishing a private health care network with a single entry point to the internet. This will not only provide the advantages of internet connections to all participating providers, but it will also isolate intrastate patient care traffic from the internet, reserving internet traffic for those information needs not available within the statewide network. In addition to the network solution, an extensive user support infrastructure is also presented.

Peptide, protein, and cellular interactions with self-assembled monolayer model surfaces.

Model biomaterial surfaces with well defined chemistry were prepared from close-packed alkyltrichlorosilane monolayers on polished silicon and glass. The outermost molecular groups which come in direct contact with the biological environment were varied across a wide range of oxidation states by employing -CF3, -CH3, -CO2CH3, and -CH2OH terminal functionalities. Characterization by contact angles, surface spectroscopy, and ellipsometry verified that these model surfaces could be repeatedly prepared with good consistency for routine use to study biomolecule adsorption onto model surfaces. Adhesion of canine endothelial cells and the adsorption of proteins (human serum albumin and human fibrinogen) as well as series of synthetic defined oligopeptides to these model surfaces have been studied. Endothelial cells attachment and growth were in the rank order of: -CH2OH > -CO2Me > -CH3 > -CF3. The peptides were comprised of different alternating sequences of lysine, leucine, and tryptophan residues. These structural differences imparted different amphiphilic characters that led to measurable differences in the adsorption of these peptides to liquid-vapor interfaces. The adsorption to model surfaces was studied using ESCA, radiometry, and concentration-dependent contact angles. ESCA and radiometry measured irreversible biomolecules adsorption whereas the contact angle method measured steady-state adsorption. Radiometric results were inconsistent with ESCA, possibly due to artifacts associated with protein radiolabeling.

A compartmentalized device for the culture of animal cells.

A simply-constructed device for the batch culture of animal cells is described that takes advantage of gas-permeable polymer films and continuous dialysis of nutrient fluids. The design separates a cell-growth chamber from a nutrient-medium reservoir with a dialysis membrane, effectively compartmentalizing growth and feeding functions. Resulting culture environment is extraordinarily stable since frequent medium exchanges are not required and macromolecules biosynthesized by cells are retained within the cell-growth compartment. Culture experiments with a variety of mammalian cells (epithelioid, fibroblastic, hybridoma, primary murine) are described that demonstrate unattended culture for up to 30 days. Immunoglobulin (IgG) secreted by cultured hybridoma cells concentrated in the growth compartment more than 25-fold over levels attained at confluence in conventional flasks. Applications for the device in various areas using animal cell culture and potential advantages in scale are discussed.

Thermodynamics of short-term cell adhesion in vitro.

A thermodynamic theory of short-term (less than 2 hr) in vitro cell adhesion has been developed which allows calculation of reversible work of adhesion and estimation of a term proportional to cell-substrate contact area. The theory provides a means of determining a parameter related to membrane wetting tension for microscopic cells that does not require special manipulations which might desiccate or denature delicate cell membranes. Semiquantitative agreement between predicted and experimentally-measured cell adhesion obtained for three different cell types (MDCK, RBL-1, and HCT-15) in two different liquid phase compositions of surfactants (Tween-80 and fetal bovine serum) supports concepts and approximations utilized in development of theory. Cell-substrate contact areas were largest for wettable surfaces treated with ionizing corona or plasma discharges and smallest for hydrophobic materials for each cell type studied. Contact area for the continuous dog-kidney cell line MDCK was larger than that of either the leukemic blood cell RBL-1 or the anaplastic human colon cell HCT-15.

[Quality criteria - urography (author's transl)]. / Qualitätskriterien - Urographie.

The technique of urography is discussed on the basis of the pharmacokinetics of contrast media with easy renal passage. The quality characteristics of a technically good urogram are defined. The factors influencing the quality of this method of examination are discussed.

[Methodical optimization of redioiodine uptake experiments in living rats]. / Methodische Optimierung von Radiojodaufnahme-Versuchen an der Iebenden Ratte.

The paper deals with the estimation of thyroidal uptake of 131I in living rats. The animals are fixed in a specially marked glass tube. This tube is discontinuously moved over a scintillation counter within a lead collimator. Counts of 131I are estimated segmentically. The highest counts ratio with the geometrical factor of the appropriate segment is used to calculate the thyroidal radioiodine uptake. Similar results, obtained with an 131I-source placed in various segments of the top side of glass tube, indicate that the uptake values obtained in this manner exact. Thyroidal uptake values, which were received on living rats with this method (in vivo) and compared with values obtained with the prepared and plated thyroid of the same rats (in vitro) show a very high correlation (r = 0,99; p greater than 0,001). In repeated estimations of the thyroidal 131I-uptake on one animal a variation coefficient of 1.5% (n = 13) was obtained. The advantage of this in vivo method is the possibility to determine the thyroidal activity at various times after 131I-application (2 phase test) and by repeated 131I-applications under different conditions (diet, age, for instance).