The good, the bad and the obvious: 1993 Clemson Award for Basic Research--Keynote Lecture.
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Amino Acid Chloromethyl Ketones↗
Biomedical subjects
M V Sefton
Publications and source records attributed to M V Sefton.
Microencapsulation of viable hepatocytes in HEMA-MMA microcapsules: a preliminary study.
Viable rat hepatocytes were encapsulated in a HEMA-MMA copolymer (80% HEMA). Encapsulated hepatocytes continued to produce urea (a measure of viability) for approximately 2 wk although urea production rates fell steadily over the course of in vitro culture in a pattern similar to those of control hepatocytes in conventional culture. Urea production was slightly higher in 0.01 M Tris buffered glycerol precipitated capsules, relative to phosphate buffered saline precipitated capsules. Hepatocytes were not viable in 0.001 M Tris buffered glycerol precipitated capsules which had a dense wall without the macroporosity seen in the walls of the other capsules. More work is needed to show that HEMA-MMA encapsulated hepatocytes retain some of the differentiated functions of hepatocytes.
Animals↗
Preliminary report on cell culture on a thermally reversible copolymer.
Anchorage-dependent cells (NRK 49F) were grown at 37 degrees C in serum containing medium on glass coated with a N-isopropyl acrylamide/N-t-butyl acrylamide copolymer (NIPAaM/NTBAM) with a lower critical solution temperature (LCST) of 8 degrees C. After incubating the dishes and adherent cells at 4 degrees C for 1 h, the cells had started to detach in 'sheets', without the use of trypsin; by 2 h the cells had fully detached and formed aggregates. We presume that at 4 degrees C, below the LCST of the coating, the polymer swelled and eventually dissolved, leaving the cells unattached to the solid substrate. We speculate that the 'sheet' morphology of the detaching cells implies that the extracellular matrix produced by the cells during culture was left intact, which may then alter the nature of their subsequent behaviour. The polymer was not optimum for cell attachment and optimization would be needed to realize the potential of this novel mode of cell culture and recovery.
Acrylamides↗
Does polyethylene oxide possess a low thrombogenicity?
Because of the 'bland' nature of polyethylene oxide towards proteins and cells, considerable effort has been devoted to preparing surfaces rich in polyethylene oxide, using block copolymers, surface immobilization or other methods. It is clear that these modifications result in reduced levels of cell (including platelet) adhesion and protein adsorption, when compared to unmodified and typically hydrophobic substrates. It is far less clear whether the reduced adhesion or adsorption is due specifically to the thermodynamic effects of polyethylene oxide or to the increase in surface hydrophilicity after its immobilization. Even more so, it is unclear whether the reduction in such parameters is evidence of a reduced thrombogenicity.
Animals↗
Preparation and thrombogenicity of alkylated polyvinyl alcohol coated tubing.
A polyvinyl alcohol hydrogel (PVA) was reacted with a C18 isocyanate at 80 degrees C in dimethyl formamide (DMF) in order to improve the platelet reactivity of the hydrogel through an influence on albumin adsorption or retention. A C4 isocyanate was used as a control. Surface coverage by XPS appeared to be approximately 100% for both C4 and C18 modified surfaces, although the limited solubility of C18 isocyanate in DMF may have resulted in a nonuniform surface. Relative to PVA or the solvent treated control, octadecylation resulted in increased albumin adsorption (from a single protein solution) and increased retention when the adsorbed albumin was exposed to a fibrinogen solution. However, octadecylation did not obviate the platelet reactivity problem in preliminary studies: systemic platelet counts were reduced by about half over 4 days in a canine AV shunt experiment and the initial rate of platelet destruction for C18-PVA was greater (36%/day) even than for the solvent-treated PVA. Surprisingly in preliminary studies butylation of PVA resulted in little or no thrombocytopenia and did not appear to increase significantly the fractional rate of platelet destruction relative to the shunt only blank. It is presumed that the nonspecific effect of alkylation (independent of chain length) was the dominant contribution to the reduced platelet reactivity. A similar effect of C18-PVA presumably would have been observed had the limited solubility of C18 isocyanate not precluded a uniform surface coverage.
Adsorption↗
Measurement of the rate of thrombin production in human plasma in contact with different materials.
Thrombin production in plasma in contact with various materials was consistent with a first-order autocatalytic model (d[T]/dt = kp[T]; [T] = thrombin concentration, t = time, kp = thrombin production rate constant) since the initial portion of a semilogarithmic plot of thrombin concentration against time was linear. Thrombin concentration was measured in clotting plasma (phospholipid enhanced or platelet-rich plasma) using a fluorogenic substrate (BMCA) by aliquot sampling at various intervals or more conveniently by monitoring cumulative fluorescence. The latter was generated by the action, on BMCA incubated in the clotting plasma, of the thrombin as it was generated. The thrombin concentration was determined from the first derivative of the S-shaped cumulative fluorescence curve. kp was greater for glass (7.92 x 10(-3) cm/s) than for the other materials (polypropylene, polystyrene, polyethylene and PVA; kp approximately 3.1 x 10(-3) cm/s) in plasma with cephalin without flow. A kp for heparin-PVA could not be determined since the thrombin concentration was too low to be quantified. A larger difference between polyethylene and PVA was noted with platelet-rich plasma without flow while lower values (1.0 x 10(-3) cm/s) were noted in a flow system but at a higher surface to volume ratio. The first-order rate constant can be used in simple models relating production of thrombin at a wall of a tube to its mass transfer away from the wall in flowing blood. One such model predicts that the concentration of thrombin at the wall should become infinite at the point in the tube when the mass transfer coefficient equals kp. According to this model, kp on the order of 10(-4) cm/s would be a useful target for a nonthrombogenic material.
Amino Acid Sequence↗
Thrombin and albumin adsorption to PVA and heparin-PVA hydrogels. I. Single protein isotherms.
More radiolabeled thrombin was adsorbed to heparin-polyvinyl alcohol (PVA) than to PVA, consistent with a specific interaction with the immobilized heparin. The maximum surface concentration on heparin-PVA was estimated to be approximately 450 nmol/m2 with an apparent affinity constant (Ka) of 2.5 microM-1; on PVA, the plateau concentration was 10 nmol/m2 with a Ka less than 1 nM-1. There was little difference in bovine serum albumin (BSA) adsorption between PVA and heparin-PVA. Interestingly, thrombin adsorption to polyethylene was indistinguishable from that to PVA despite the large difference in surface chemistry. BSA adsorbed to polyethylene with higher affinity than to the hydrogels, although the plateau concentrations were comparable. The adsorbed thrombin was biologically inactive at least towards chromogenic substrate, with the residual activity on PVA unaffected by subsequent incubations with antithrombin III. PVA and heparin-PVA presented a heterogeneous and complex substrate for interaction with proteins. The adsorbed protein was likely present in multiple states depending on the groups with which it interacted.
Adsorption↗
Morphological assessment of hepatoma cells (HepG2) microencapsulated in a HEMA-MMA copolymer with and without Matrigel.
Hepatoma cells (HepG2), an anchorage-dependent cell line, were microencapsulated in a HEMA-MMA polyacrylate membrane to which the cells do not adhere. This environment was altered by the coencapsulation of Matrigel, a reconstituted extracellular matrix derived from the Engelbreth-Holm-Swarm (EHS) mouse tumor basement membrane, to provide sites for cell attachment. The effect on the cells of these two capsule microenvironments during a 2-week in vitro culture period was assessed by examining the spatial arrangement, morphology, and viability of the cells using light microscopy and scanning electron microscopy (SEM). In preparation for microscopy, dissolution of the polymer was prevented by the use of frozen sections embedded in a water-soluble compound. Similarly, freeze cleavage of conductively stained capsules permitted SEM observation of the capsule interior along with ultrastructural detail of the cells. In the absence of Matrigel, cells in HEMA-MMA capsules were found to form aggregates in intracapsular pockets with central necrosis occurring at day 7 in large aggregates. The coencapsulation of HepG2 cells with Matrigel, resulted in an initially uniform distribution of essentially individual cells with aggregates appearing later within the Matrigel. Many cells within these capsules had remained viable when examined up to day 14 with only limited cellular necrosis, implying a favorable environment for microencapsulated HepG2 cells.
Biocompatible Materials↗
FT-IR measurement of emulsified cationic DMAEMA-MMA adsorption to calcium alginate.
The rate of adsorption of aqueous emulsions of DMAEMA-MMA copolymers to calcium alginate substrates was examined using ATR FT-IR spectroscopy. This work was undertaken as part of the development of a hybrid artificial pancreas. Aqueous DMAEMA-MMA copolymer emulsions containing < 25 mol% DMAEMA were slowly adsorbed to calcium alginate substrates; emulsions of 50 mol% DMAEMA-MMA copolymer were adsorbed rapidly to calcium alginate gels. The adsorption of 50 mol% DMAEMA-MMA copolymer emulsions to calcium alginate gels was attributed to electrostatic rather than simple van der Waals' attractive forces.
Adsorption↗
Heparin-poly(ethylene glycol)-poly(vinyl alcohol) hydrogel: preparation and assessment of thrombogenicity.
Heparin was immobilized on to poly(vinyl alcohol) hydrogel (PVA) through the free isocyanate end-group on a poly(ethylene glycol) (PEG2000) which had been previously covalently linked to the hydrogel via a urethane moiety. The intention was to reduce the platelet reactivity of the PVA while also suppressing fibrin formation. Elemental nitrogen analysis revealed that the total amount of bound heparin was 19 +/- 7 mumol/g of dried gel. An increase in the in vitro whole blood clotting time of PVA was observed. This was attributed to bound heparin, as the elution rate of heparin from the gel (23 pmol/m2 min) was too low to produce a significant bulk concentration to interfere with fibrin formation. Ex vivo assessment using a chronic canine A-V shunt showed that the bound heparin hydrogel had no effect on the drop in the number of platelets induced by PVA hydrogel, but increased the fractional rate of platelet destruction from approximately 0.35/d to an average value of 0.42/d.
Animals↗
Platelet consumption by polyvinyl alcohol coated tubing in canines.
Polyvinyl alcohol (PVA)-coated polyethylene tubing, with or without immobilized heparin, caused severe thrombocytopenia and enhanced the production of new platelets when inserted in a chronic arteriovenous shunt in canines. A similar length of uncoated polyethylene tubing neither lead to thrombocytopenia nor significantly enhanced platelet regeneration, relative to the shunt only without a test section. Platelet regeneration was monitored by the malondialdehyde assay, which was assumed to make a distinction between "new" and "old" platelets. This distinction was combined with the platelet count values to enable calculation of the cumulative consumption curve and the initial fractional consumption rate in the presence of a non constant platelet count. The resulting initial fractional consumption rates were: 34%/day for PVA, 20.5%/day for polyethylene, and 18%/day for the shunt only blank.
Animals↗
Platelet consumption by NHLBI reference materials and SILASTIC [corrected].
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Animals↗
Microencapsulation of mammalian cells in a HEMA-MMA copolymer: effects on capsule morphology and permeability.
A new process for preparing uniform microcapsules with a hydroxyethyl methacrylate-methyl methacrylate copolymer (HEMA-MMA) has been devised. Capsule diameters were 900-1000 microns in diameter, (+/- 10-20 microns, +/- SD) depending on the precipitation conditions. The process involved the coextrusion of polymer solution (in PEG 200) and the mammalian cell suspension (here erythrocytes) through a needle assembly which is submerged in a layer of hexadecane which is in turn sitting above a stirred isotonic aqueous solution in a volumetric flask. The needle is repeatedly withdrawn from the hexadecane overlayer shearing a droplet from the needle tip which falls into the water, where the solvent is extracted to precipitate the polymer around the cells to yield the capsules. The morphology of the capsule wall was altered by changing the precipitation bath from phosphate buffered saline (PBS) to 0.3 M glycerol. This resulted in greater macroporosity in the wall, presumably because of the faster precipitation due to the higher solvent/precipitant compatibility with 0.3 M glycerol. The permeability to a series of test solutes (glucose, inulin, albumin, and alcohol dehydrogenase, ADH) increased by a factor of approximately 2, presumably because of the increased macroporosity. Addition of 15% water to the polymer solvent enhanced the macroporosity, presumably by bringing the system closer to the cloud point; however, there was no corresponding increase in permeability. There was a significant decrease in permeability between that of albumin (approximately 69,000 D) and ADH (approximately 150,000 D) suggesting that the molecular weight cut-off of these capsules was on the order of 100,000 D as desired. This process is now being evaluated for the encapsulation of pancreatic islets and other cells of potential clinical interest.
Animals↗
Colorimetric assay for cellular activity in microcapsules.
Cellular activity in microcapsules was determined by a simple colorimetric assay, based on the cellular transformation of a tetrazolium salt, 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide, into an insoluble formazan which was quantified in a spectrophotometer. The results showed that when encapsulated Chinese hamster ovary fibroblasts were exposed to the tetrazolium salt containing tissue culture medium, the formazan crystals were formed inside the poly(hydroxyethyl methacrylate-methyl methacrylate) microcapsules; capsules containing no cells or dead cells formed no formazan. A detectable amount of formazan was readily obtained even from single capsules. Formazan production was dependent on the incubation time, but not on the amount of added reagent. Capsules from a high cell-density encapsulation (4 X 10(6) cells/ml) formed more formazan than capsules from a low cell-density (4 X 10(5) cells/ml) encapsulation, suggesting a positive correlation between the cell density and tetrazolium transformation in microcapsules. The tetrazolium assay indicated the maintenance of cellular activity but slow, if any, proliferation in microcapsules over a 2 wk testing period.
Animals↗
In vitro platelet interactions with a heparin-polyvinyl alcohol hydrogel.
No difference in in vitro platelet reactivity was found between an immobilized heparin containing hydrogel (heparin-PVA) and the hydrogel without heparin (PVA), in a variety of experimental assays. There was no significant difference between the heparin-PVA and PVA coated polyethylene tubing in the number of 51Cr-labeled platelets, the extent of 14C serotonin release by the adherent platelets or in the degree of platelet count decrease after 1 h exposure to citrated canine whole blood in a Chandler loop system. Furthermore, adhesion and release values were lower than those observed with the uncoated polyethylene tubing (e.g., 9.3 +/- 4.3 plt/10(3) microns 2 on PVA; 18.3 +/- 4.6 plt/10(3) microns 2 on polyethylene). There was also no significant difference between heparin-PVA and PVA in bead column retention values with canine blood and with the previously reported washed human platelet adhesion/release values. Thus there appears to be no effect of the immobilized heparin by itself on the in vitro interactions of PVA with platelets, with the reactivity towards platelets dominated by that of the underlying substrate (i.e., PVA).
Animals↗
Effect of heparin-PVA hydrogel on platelets in a chronic canine arterio-venous shunt.
Polyvinyl alcohol (PVA) hydrogel, with or without heparin, was reactive towards canine platelets in a chronic arteriovenous shunt as demonstrated by an increase in platelet regeneration time, a systemic decrease in platelet count and transient decrease in platelet serotonin content. Immobilized heparin (heparin-PVA) had no effect whereas unmodified polyethylene was found to be unreactive despite similar levels of platelet deposition as measured by SEM and a higher in vitro reactivity (J. Biomed. Mater. Res., this issue). Twenty-centimeter lengths of hydrogel coated polyethylene tubing were inserted between the arterial and venous portions of the shunt and left in place for 4-6 days, without the complicating artifacts of anticoagulation, anesthesia, or surgical intervention. Regeneration time was measured as the return to normal platelet cyclooxygenase (co) activity after a single 240-mg dose of aspirin, with co activity measured in vitro as malondialdehyde production. Although measuring new platelet production, regeneration time is an indirect measure of platelet consumption, so that the reduced regeneration time seen here was presumed to reflect enhanced material associated consumption and thromboembolism. Like other hydrogels, PVA does not appear to be "thromboadherent" but it does appear thrombogenic. Immobilized heparin had no additional effect, presumably because the platelet response was dominated by the reactivity of the underlying substrate.
Animals↗
Patency of heparin-PVA coated tubes at low flow rates.
The patency of heparin-PVA coated 1.14 mm i.d. polyethylene tubes was greater than that of control tubes without heparin in a novel test section that enabled ex vivo evaluation in dogs at low flow and shear rates (2-10 ml/min). Low shear rates were achieved by diverting a small portion of a chronic A-V shunt flow through Y-connectors into the small diameter test tubing. For shear rates in the range of 200-650 s-1 half the heparin-PVA tubes remained patent for longer than 72 min whereas half the control tubes (PVA only) only remained patent for longer than 30 min. At higher shear rates (500-1000 s-1), the 50% patency times for heparin-PVA and PVA were 225 and 123 min respectively. The increased patency at the higher shear rates was attributed more to the effect of the connector than just shear rate. The higher shear rates were achieved by changing from a Y-connector in which the small diameter side-branch was cut flush to the larger diameter main line to one in which the small diameter test tubing protruded into the lumen of the main line; the latter 'protruding' connector was presumed to result in less platelet pre-activation than the other. This method has been found useful for assessing the thrombogenicity of heparinized tubes at low flow rates.
Animals↗
Effect of capsule permeability on growth of CHO cells in Eudragit RL microcapsules: use of FITC-dextran as a marker of capsule quality.
Chinese hamster ovary fibroblast cells were encapsulated in Eudragit RL by interfacial precipitation. After encapsulation, they grew to fill the capsules, eventually growing outside the capsules as well. By encapsulating FITC-dextran (MW 150,000) along with the cells, it was possible to show that cell growth was faster in those capsules which lost fluorescence soon after encapsulation. These data were interpreted to signify that the intact Eudragit RL capsule membrane offered significant diffusion resistance to vital nutrients or particular metabolites, slowing or even preventing growth.
Animals↗