Grafting of PEO to polymer surfaces using electron beam irradiation.

A new method was developed for binding poly-(ethylene oxide) (PEO) to polymer surfaces that involves the use of electron beam irradiation in two steps. In the first, methacrylic acid was grafted and polymerized to a polymer surface, changing it from hydrophobic to hydrophilic. Exposure of this surface to aqueous PEO solutions resulted in strong hydrogen bonding of the PEO, which was covalently grafted in a second radiation step. The PEO grafts were stable; they could not be removed with extensive washing with water, soaking in basic solution, or gentle mechanical scraping. Both monolayers and multilayers of PEO were formed. The density of the monolayers were found to have little dependence on the molecular weight or concentration of the PEO solution; multilayers could be controlled by varying the viscosity of the PEO solution and the method of application. The PEO-grafted monolayers were tested for their ability to prevent protein adsorption of cytochrome-c, albumin, and fibronectin. Monolayers of star PEO were the most effective, at best showing a 60% decrease in adsorption from untreated controls. One million molecular wight linear PEO monolayers were almost as effective as star monolayers, and 35,000 g/mol linear PEO was bound too closely to the surface, owing to its small size, to have much impact in preventing protein adsorption. The reason for the continued protein adsorption was believed to be due to a close grafting of the PEO to the surface, as well as the grafted methacrylic acid chains being long enough to extend through the PEO monolayer, thus being accessible on the surface.

Gamma sterilization of UHMWPE articular implants: an analysis of the oxidation problem. Ultra High Molecular Weight Poly Ethylene.

Gamma irradiation of Ultrahigh Molecular Weight Polyethylene (UHMWPE) leads to long-lived free radicals which react with oxygen. Diffusion of oxygen, occurring over months or years, controlled by the permeability characteristics of the polymer, results in progressive oxidation, breaking of polymer chains, alteration of the crystalline portion of the polymer, and deterioration of the mechanical properties of the polymer. This paper reviews the observations in the literature on this issue and then presents a conceptual model concerning the interplay of radical diffusion, oxygen diffusion, non-uniform permeability, and free-radically driven chain reactions in order to explain these observations. The suggested model is based on literature that is available on the oxidation of linear polyethylenes during and after irradiation. The model directs the attention of researchers in the field of orthopaedic implants to the complexity of the process and the variety of issues and parameters to be considered while studying the long-term effects of radiation sterilization on UHMWPE.

Hydrogels prepared by electron irradiation of poly(ethylene oxide) in water solution: unexpected dependence of cross-link density and protein diffusion coefficients on initial PEO molecular weight.

Under ionizing radiation aqueous solutions of water-soluble polymers become cross-linked and form hydrogels, primarily by radiolysis of water-generating hydroxyl radicals which attack the polymer chains. The chain radicals thus formed create cross-links by coupling. In particular, hydrogels formed from poly(ethylene oxide) are of interest for biomedical applications, including those in which it is necessary to transport large molecules such as growth factors, in addition to nutrients, to cells attached to the surface. We sought a rapid and simple method for estimating diffusion coefficients by observing the diffusion of two red-coloured proteins: cytochrome C and hemoglobin of respective molecular weights 12,000 and 67,000. In the course of this we discovered a previously unreported effect of the primary molecular weight of the polymer, before cross-linking, on the cross-link density finally achieved and on the diffusion coefficient of the proteins.

Hepatocyte culture on carbohydrate-modified star polyethylene oxide hydrogels.

We describe the synthesis and in vitro biological characterization of a new class of carbohydrate-modified hydrogels based on radiation-cross-linked star polyethylene oxide (PEO). Hydrogels were synthesized from either of two types of PEO star molecules in order to vary the terminal hydroxyl content of the gels while keeping other gel properties such as molecular weight between cross-links and water content constant. The resulting gels were covalently modified with monosaccharide ligands and the behaviour of primary rat hepatocytes on the modified gels was evaluated under culture conditions. Hepatocytes exhibited a sugar-specific adhesion to the modified gels, adhering to gels bearing galactose but not glucose. Cell spreading was observed on both types of galactose-modified PEO star gels; moreover, the gels supported long-term (6 d) culture and differentiated function of primary hepatocytes. Further, on comparing the cell spreading behaviour observed on the PEO star gels with that reported previously for galactose-modified polyacrylamide, we find that our gels elicit spreading at ligand concentrations lower by an order of magnitude. A simple mechanistic analysis indicates that this enhanced ability of PEO star gels to support spreading of primary hepatocytes on low concentrations of immobilized galactose derives from freedom of the immobilized ligands to come within sufficiently close proximity to mimic a high-affinity branched oligosaccharide.

Corneal epithelial wound healing on bilayer composite hydrogels.

We demonstrate that a bilayer composite hydrogel composed of corneal stroma crosslinked to poly(ethylene oxide) provides a substrate suitable for wound healing behavior of corneal epithelial cells and for formation and maintenance of a stable multilayered epithelium. Potential diffusion-limitation of nutrients or regulatory molecules across the hydrogel was investigated experimentally with a new in vitro ocular assay, using epithelial cell migration as an index of molecular diffusion limitations. Corneal epithelial cells explanted on the composite hydrogel in vitro exhibited morphology similar to those in vivo, and migrated effectively over the stromal surface. Importantly, our system yielded multilayered epithelium like that found in normal corneal tissue under conditions that closely simulate the in vivo physiologic arrangement. In addition, our results indicate that molecules of substantially greater molecular weight than glucose appear to control the cell migration rate. Thus, engineering design of this composite hydrogel system may allow it to be useful in corneal wound healing applications.

Partitioning and diffusion of solutes in hydrogels of poly(ethylene oxide).

Hydrogels were created by electron beam irradiation of aqueous solutions of poly(ethylene oxide) (PEO) having a nominal molecular weight of 35,000. The molecular weight between cross-links Mc varied from 3000 to 15,000, and the equilibrium volume fractions of polymer V2,s from 0.01 to 0.08. These hydrogels were exposed to aqueous solutions of solutes: tricyclic antidepressants, cyanocobalamin, four globular proteins and three linear species of PEO. Partition coefficients and diffusion coefficients were determined. For each solute the ratio diffusion coefficient in hydrogel/diffusion coefficient in free solution was determined, and related to the hydrogel parameters Mc and v2,s and to the solute effective radius rE (Einstein radius). The diffusion coefficient ratio is greater for the flexible random coiling PEO than for the 'rigid' solutes at a given set of Mc, v2s and rE, and the disparity increases rapidly as rE increases. Among the globular proteins the diffusion coefficient ratio decreases by orders of magnitude with small changes in rE (20.6-27.6 A) and was found to be nearly zero for albumin (rE = 36.1 A). The tricyclic antidepressants had partition coefficients of around 2, whereas the other solutes had partition coefficients of about unity. By reason of the partition coefficient of around 2, the diffusion coefficient ratio of a tricyclic antidepressant having a value of rE = 5.5 A is half that of the larger cyanocobalamin, for which rE = 8.5 A.

Challenge to the concept that UHMWPE acetabular components oxidize in vivo.

Severe wear of the ultra-high molecular weight polyethylene (UHMWPE) components of total joint replacements limits their long-term success. In previous studies, the infrared spectra obtained on retrieved UHMWPE components were interpreted as evidence that the UHMWPE oxidizes in vivo. In direct contrast, infrared spectroscopy of the retrieved UHMWPE acetabular components examined in this study demonstrated adsorbed esterified fatty acids, readily extractable by hexane, and no substantial evidence of in vivo oxidation. This emphasizes that special care must be taken when using infrared spectroscopy to assess retrieved components.

PEO enhancement of platelet deposition, fibrinogen deposition, and complement C3 activation.

Whereas it has been commonly thought that adding polyethylene oxide PEO to a surface would diminish the capacity of the surface to cause deposition of platelets and of fibrinogen, and to activate complement C3, we present data showing exactly the opposite. These unexpected results are obtained with low molecular weight (2000) PEO, and are not found with higher molecular weight (20,000) PEO.

Whole blood viscosity in beta thalassemia minor.

Patients with heterozygous beta-thalassemia minor have a decreased hematocrit (HCT). Since the HCT is a primary determinant of whole blood viscosity, the known reduction in HCT in beta-thalassemia minor should lead to a measurable reduction of whole blood viscosity. The influence of the relatively lower mean corpuscular volume and consequent higher red blood cell count and beta-thalassemia minor on whole blood viscosity using a microporous viscometer has not previously been the subject of investigation. Accordingly, the blood of a group of normal and beta-thalassemia minor subjects was examined with a microporous viscometer to elucidate further the relations between whole blood viscosity, HCT, and red blood cell count. The data show that for normal and beta-thalassemia minor subjects a significant positive correlation (r = 0.65, p less than 0.01) exists between HCT and whole blood viscosity. However, the slope of the regression of whole blood viscosity and HCT of beta-thalassemia minor subjects was significantly higher z = 3.14, p less than 0.001) than that of normals. Thus, for any given HCT their whole blood viscosity was higher than that of normals. Studies of the relation of red blood cell counts to whole blood viscosity indicate the higher whole blood viscosity at a given HCT was related to the increased red blood cell counts in beta-thalassemia minor subjects. Because of the opposing interactions of HCT and red blood cell counts, the mean whole blood viscosity of the group of beta-thalassemia minor subjects examined was not significantly lower than the normal whole blood viscosity.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug partitioning and release characteristics of tricyclic antidepressant drugs using a series of related hydrophilic-hydrophobic copolymers.

A series of crosslinked polymer networks formed from hydrophilic polyethylene oxide (PEO) and a hydrophobic polysiloxane (PGPMDMS) were studied with respect to the partitioning and release of five tricyclic antidepressants (TCAs) at pH 7.4. The TCAs, chemical analogues of one another, have both nonpolar and ionic characteristics at pH 7.4, but differ considerably in hydrophobicity. In PEO-PGPMDMS copolymer networks, the partition coefficient of protriptyline (the TCA studied most extensively) was observed to be higher than in networks of PEO or PGPMDMS singly. This finding, which may represent adsorption of the amphiphilic drug at interfacial sites between hydrophilic and hydrophobic phases within the copolymeric network, shows that in some cases, higher drug loadings of amphiphilic drugs can be obtained with a hydrophilic-hydrophobic copolymer compared with a material made of only one polymer. As the PEO content in PEO-PGPMDMS networks was increased from 20 to 100%, the release rate of protriptyline increased by greater than 1000-fold. Thus, a key variable in achieving a desired release rate is the PEO content of the copolymer. On the other hand, release rates of the five TCAs from PEO-PGPMDMS networks containing 50% PEO varied by a factor of less than 3. Thus, minimal effect on drug release rates was obtained by using a different TCA analogue.

Scanning electron microscopy analysis of polyethylene oxide hydrogels for blood contact.

Hydrogels are a class of synthetic material, composed of a polymer-water matrix and have been proposed as tissue substitutes and drug delivery vehicles. Polyethylene oxide (PEO) hydrogels were synthesized and used to produce coated wires and conduits for baboon blood compatibility studies. Blood-material interactions were studied both by Scanning Electron Microscopy (SEM) and 111In labeled platelet deposition. SEM processing modifications were first evaluated in order to reduce shrinkage and surface distortion incurred during sample preparation of these high water content materials. Pretreatment with 1% tannic acid reduced bulk shrinkage associated with critical point drying by 10-20%. This effect is small, nevertheless, it prevented major sample disruption. Coated guidewires were exposed to baboon blood for one hour in the inferior vena cava and conduits were placed for either 30 or 60 minutes in an ex vivo femoral arteriovenous shunt. Reference materials included Gore-tex, polyethylene and silica-free polydimethyl siloxane (PDMS). In the guidewire studies, 111In labeled platelet levels were highest on Gore-tex (6568.97 platelets/1000 microns 2) and large thrombotic deposits were well visualized by SEM. Formulations containing PEO had low levels of platelet deposition and little evidence of platelet activation was noted by SEM. Shunt studies demonstrated that materials of high PEO content and molecular weight had the lowest levels of platelet deposition. After 60 minutes of blood flow, mean platelet deposition on PDMS and Gore-tex was 50 and 1000 fold higher than on a network composed of 65% PEO 20,000 (p less than 0.05). SEM confirmed these findings.

Development and evaluation of a new polymeric material for small caliber vascular prostheses.

Polyethylene oxide (PEO), because of its low levels of protein and cellular adsorption, may provide a suitable coating for synthetic small caliber vascular prostheses. PEO/polysiloxane networks were synthesized via an acid-catalyzed epoxy/hydroxyl crosslinking reaction and used to produce conduits with a 4-mm internal diameter. Three networks with nominal PEO molecular weights of 2000, 8000, and 20,000 and all 65% PEO by weight were studied. Blood compatibility was assessed by measuring 111In-platelet and 125I-fibrinogen deposition in a baboon ex vivo shunt, over a 1-hr time period and at a flow rate of 50 ml/min. Differences in material performance were noted particularly after the initial 30-min blood contact period. Materials in the mid and high PEO molecular weight range (8000 and 20,000) had significantly lower levels of platelet adsorption than networks of low PEO molecular weight (2000) at 30 min (P less than 0.005) and 60 min (P less than 0.05). The lowest level of platelet deposition was noted on networks of high PEO molecular weight (20,000). During the observation period, platelet accumulation on this surface was less than one platelet per 1000 microns. Platelet deposition on Gore-Tex was two and three orders of magnitude greater than that on the high molecular weight PEO material at 30 and 60 min, respectively (P less than 0.001). Fibrinogen adsorption was also lower on materials of mid and high PEO molecular weights, when compared with low molecular weight networks (P less than 0.05) and Gore-Tex (P less than 0.05). Scanning electron micrographs confirmed these observations. Overall, platelet and fibrinogen depositions are low for PEO networks, particularly for materials of high PEO molecular weight. This latter observation may be related to increased surface molecular mobility and a relative enhancement of PEO content at the blood-material interface.

Activity toward thrombin-antithrombin of heparin immobilized on two hydrogels.

Commercially obtained (Diosynth) heparin was covalently bonded to poly (vinyl alcohol) (PVA) hydrogels and to polyethylene oxide (PEO) hydrogels activated by tresyl chloride. We found that as tresyl chloride activation of PVA increased, the specific activity of the bound heparin toward thrombin and antithrombin decreased by nearly a factor of 10 and that commercial heparin bound to PEO had nearly ten-fold greater activity than when bound to PVA at comparable concentrations. These findings suggest that the long 'leash' provided by PEO hydrogels may give the heparin more access to the thrombin-antithrombin pair than the tight bond to PVA, and that crowding of heparin units on a surface limits access of the thrombin-antithrombin pair.

Hemodynamic effects of different preparations of stroma free hemolysates in the isolated perfused rat kidney.

We have examined the effects of Stroma Free Hemolysate (SFH) solutions in the isolated perfused rat kidney. Three types of SFH, stored for 6 to 8 months at 4 degrees C, were tested: 1) unmodified, 2) glyoxalated and lightly cross linked and 3) pyridoxalated and polymerized. All three SFH solutions, added to the perfusate at a concentration of approximately 420 mg/100ml, increased renal vascular resistance (RVR) and reduced glomerular filtration rate (GFR). Unmodified, glyoxalated and lightly cross linked and pyridoxalated polymerized SFH resulted in a rise in RVR of 55%, 38% and 33% respectively and a fall in GFR of 42%, 57% and 83% respectively. In order to determine whether storage had altered the effect of SFH on renal function, one of the forms of SFH (glyoxalated and lightly cross linked) was studied only 4-6 weeks after preparation. While this preparation caused an increase in RVR of 41% it did not alter GFR; filtration fraction (FF) rose. However, after further storage of this preparation for 6-7 months, the solution resulted in a marked decrease in GFR of 47% as well as a rise in RVR of 23%. We conclude that three different SFH preparations resulted in marked vasoconstriction and reductions in GFR. These deleterious effects on renal hemodynamics were noted at a concentration of hemoglobin well below that necessary to effectively improve oxygen content. Storage of the SFH solutions may cause or contribute to their effects on renal function. SFH solutions intended for use as blood substitutes should be tested for vasoconstrictor activity.

Crosslinked polyether/polysiloxane networks for blood-interfacing applications.

The interaction of blood with new artificial surfaces is an area of continual medical interest. In this study, a series of polyether/polysiloxane networks were synthesized, characterized in terms of both bulk and surface compositions, and evaluated for blood compatibility. The crosslinked networks were produced by reacting the epoxy groups of polyglycidoxy propyl methyl siloxane (PGPMS) with the hydroxyl end groups of polypropylene glycol (PPG). Blood compatibility was evaluated using an in vitro platelet retention test and fibrinogen adsorption experiments from human plasma and buffered saline. The PPG/PGPMS networks exhibit low fibrinogen adsorption and low platelet activation. Such properties make the networks potentially attractive as materials for blood-interfacing applications.

Fibrinogen adsorption and platelet adhesion at the surface of modified polypropylene glycol/polysiloxane networks.

The protein film adsorbed at an artificial surface ultimately affects platelet adhesion and activation. This study examines the role of fibrinogen in platelet adhesion at the surface of crosslinked polypropylene glycol (PPG)/polyglycidoxy propyl methyl siloxane (PGPMS) networks which contain polyethylene glycol monomethyl ether (PEGME) chains. These crosslinked networks were produced by reacting the epoxy groups of PGPMS with the hydroxyl groups of the polyethers. PEGME chains were attached covalently to the network at only one end while PPG chains were attached at both ends. The incorporation of PEGME resulted in a substantial reduction in fibrinogen adsorption as compared to the model network (PPG + PGPMS only), but the expected concomitant decrease in platelet adhesion was not observed.

Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces?

Platelet activation by polymer surfaces is thought to require preliminary adsorption of fibrinogen and perhaps changes in fibrinogen conformation. We measured fibrinogen adsorption by a series of polymers by two methods, using either 125I-labeled fibrinogen or 125I-labeled antifibrinogen antibodies, and correlated the results with platelet reactivity (retention and secretion) in columns of beads coated with the polymers. For polyalkyl methacrylates with 1 to 4 carbon side chains, platelet reactivity varied directly with increasing length of the alkyl side chain and with the quantity of bound fibrinogen recognizable by antifibrinogen antibody but not with the total quantity of fibrinogen adsorbed. The same pattern of results was seen with five antibody preparations, including affinity-purified Fab fragments against the D or E domain of fibrinogen. Tests of platelet retention and fibrinogen binding to four polyalkyl acrylates and to three unrelated polymers (polystyrene, polymethyl methacrylate, and a polyether polyurethane) indicated that platelet retention correlated positively with both total fibrinogen binding and with the amount of antibody-recognizable fibrinogen bound. Drugs that block platelet aggregation, but not adhesion, did not alter the hierarchy of platelet retention to the polyalkyl methacrylates. These data suggest that, contrary to previous views, platelet adhesion to artificial surfaces increases with increasing surface coverage of adsorbed fibrinogen if the bound fibrinogen maintains a conformation such that its functional domains remain recognizable by antibody probes.