Water activity effect on lipase immobilized onto different compositions of poly(HEMA-MMA).
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The organization of encapsidated herpes simplex viral DNA in situ was examined by use of the osmium-amine stain specific for DNA. After either formaldehyde or glutaraldehyde fixation the DNA is packaged in a compact toroid without inner structure with Epon or GMA embedment but revealed a complex inner structure with Lowicryl K4M embedment. In the latter there was an inner cylindrical core, 50 X 80 nm, around which were apposed one or more thick filaments of 5-8 nm diameter. Thinner DNA filaments of 3-4 nm diameter form a cage of loose coils around the core with an intervening space of approximately 15 nm. Lowicryl embedding may be considered as a tool to investigate the packaging of viral DNA in virions.
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The interaction of the four most commonly used preservatives in contact lens solutions (chlorbutol, thiomersal, chlorhexidine gluconate and benzalkonium chloride) with polyhydroxyethylmethacrylate (polyHEMA), has been examined. Benzalkonium chloride and chlorhexidine gluconate show typical high affinity type isotherms. The interaction of benzalkonium chloride with polyHEMA from aqueous solution was reversible whereas that of chlorhexidine was only reversible in the presence of electrolyte or surfactant. Chlorbutol showed a typical reversible linear isotherm. Thiomersal does not interact with polyHEMA above pH 5.0. The extent of chlorhexidine--polyHEMA interactions in increased by the presence of formulatory adjuvants such as electrolyte and hydrophilic polymers. PolyHEMA lenses that apparently have been equilibrated with chlorhexidine gluconate will, on the addition of fresh preservative solution, bind further quantities of chlorhexidine above that which would be predicted from the sorption isotherm.
The influence of solute concentration, gel hydration, and crosslinking density on diffusion rates in polyHEMA hydrogels has been investigated using a radio-tracer technique. At hydrations above 31% water, diffusion is by pore flow, and increases in the crosslinking density of gels bring about a decrease in the diffusion coefficient, D. Below 31% hydration, diffusion is predominantly by solution diffusion and changes in crosslinking density have little effect on the diffusion coefficient. The diffusion coefficient is invariant with solute concentration within the range reported, although a very high solute concentration, for the gels of lower hydration, the diffusion coefficient is higher than expected. A high value for D may be due to saturation of the binding sites of the diffusant on the polymer chains, leaving a greater proportion of diffusant available for transport.
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Cultured neurons become attached to hydrogel substrates prepared from 2-hydroxyethylmethacrylate but grow few nerve fibers unless fibronectin, collagen, or nerve growth factor is incorporated into the hydrogel. Antibodies to fibronectin inhibit nerve fiber growth on hydrogels containing fibronectin, which suggests that growing neurons interact directly with proteins trapped in the hydrogel. The adhesive requirements for attachment of neurons appear distinct and possibly less specific than those for fiber growth. Defined hydrogel substrates offer a controlled method for analyzing complex substrates that support nerve fiber growth and neuronal differentiation.
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A soft material (poly-HEMA) has been implanted into a number of rabbit eyes in order to evaluate its use in lens implantation following cataract surgery. No reaction to the implant was seen on light microscopy of the sectioned enucleated eyes.
A group of pseudophakic patients was investigated to determine whether their implants shift along an anteroposterior axis under different conditions of ciliary muscle stimulation. There was no statistically significant change in refraction after either pilocarpine or cyclopentolate administration. A change in anterior chamber depth between the position after pilocarpine and that after cyclopentolate was found. It appears that rigid posterior chamber implants do move backwards on ciliary muscle relaxation, but by a maximum 0.25 mm. This is not thought to represent a mechanical threat to ocular health. It is also not enough to account for the apparent accommodative ability of some pseudophakic patients. The possible causes for this phenomenon are discussed.
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Polyhydroxyethylmethacrylate polymers (poly-HEMA) form hydrogels that provide an excellent alternative to agar in the production of salt bridges for use in bioelectrochemical experiments. A method for the simple production of poly-HEMA salt bridges is described. The poly-HEMA bridges were compared with agar bridges of similar geometry. Whereas poly-HEMA salt bridges have a conductivity that is 20 times lower than that of agar bridges of a similar geometry, poly-HEMA bridges are capable of dissipating twice the power compared with agar bridges. The mechanical properties of the poly-HEMA bridges make them easy to manufacture, store, and sterilize. When agar bridges were compared with poly-HEMA bridges in long-term cell culture experiments, the failure rate of the agar bridges was found to be approximately 10% per week vs. a virtually nonexistent failure rate for the poly-HEMA bridges. Because poly-HEMA salt bridges are reliable, durable, and nontoxic to cells, they are a practical alternative to agar salt bridges in certain experimental designs.
The study was designed to investigate the feasibility of using an acidic gelatin hydrogel as a biodegradable vehicle for basic fibroblast growth factor (bFGF). bFGF was incorporated by polyion complexation into a biodegradable hydrogel prepared by cross-linking acidic gelatin with the isoelectric point of 4.9. The dried hydrogel (sized to 2x1 mm) was hydrated with bFGF aqueous solution including different doses of bFGF (20, 50, 125, 250 and 500 ng) and implanted into a rabbit corneal pocket (2.5x2 mm). As a control group, the gelatin hydrogel without bFGF or bFGF alone (500 ng) was used. Corneal angiogenesis was evaluated by biomicroscopy, corneal fluorescein angiography and histology for 21 days. Photographs were taken and corneal angiogenesis was evaluated by image analysis. The hydrogel degraded with time after its implantation into the corneal pocket. Experimental eyes receiving the hydrogel containing more than 50 ng of bFGF demonstrated significant corneal angiogenesis. Control eyes and eyes receiving the hydrogel containing 20 ng of bFGF showed no corneal angiogenesis. Corneal angiogenesis, which occurred on the 3rd or 4th day after implantation, reached maximal growth on about day 7 and regressed from day 10 after implantation. The area of angiogenesis showed a dose-dependency on bFGF. The gelatin hydrogel itself induced neither angiogenesis nor inflammation. These results suggested that acidic gelatin hydrogel releases bioactive bFGF with its biodegradation, resulting in corneal neovascularization.
PURPOSE: To evaluate the interaction of various intraocular lens (IOL) materials with commonly used dyes. MATERIALS AND METHODS: One IOL of each of the five groups (polymethylmethacrylate, silicone, three-piece hydrophobic acrylic, single-piece hydrophobic acrylic and single-piece hydrophilic acrylic) was bathed in trypan blue 0.1%, fluorescein sodium 2% and indocyanine green (ICG) 0.5% for 15 min. Digital photographs of the IOLs were obtained prior to bathing and after the 15-min bath in each dye. The same IOLs were immersed in the same dye for another 15 min and digital images were reobtained to depict the 30-min dye uptake. New IOLs from the group that exhibited visible colour change after the 15-min bath were then bathed in twofold dilution and if there was still visible dye uptake, fourfold dilution was performed, repeating the 15-min bath and digital photography. The images were then processed using Adobe Photoshop 5.1 to get mean luminosity and red-green-blue values. These values were compared between the groups and the undyed control. Also, dye washout was observed in serum-containing vials. RESULTS: None of the polymethylmethacrylate, silicone and hydrophobic acrylic IOLs were stained with the dyes used. The only IOL material that changed colour was hydrophilic acrylic and did so with all dyes. The most marked colour change was with fluorescein sodium 2%, the least was with trypan blue 0.05%. Twofold dilution of trypan blue and fourfold dilutions of fluorescein sodium and ICG still stained the hydrophilic acrylic IOL; however, fourfold dilution of trypan blue did not cause a significant colour change. Trypan blue washed out within 6 h, while the IOL stained with ICG remained stained for longer than 24 h. COMMENT: Hydrophilic acrylic IOLs should be used with caution together with dyes since this material demonstrates marked dye uptake and washout may also take some time.
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Microporous membranes made of poly(2-hydroxyethylmethacrylate) [poly(HEMA)] carrying protein A were used for selective sorption of human-IgG from human plasma. Poly(HEMA) membranes were prepared by a photo-polymerization technique, and activated by cyanogen bromide (CNBr) in an alkaline medium (pH 11.5). Bioligand protein A was then immobilized by covalent binding onto these CNBr-activated membranes. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The non-specific adsorption of protein A on the plain poly(HEMA) membranes was 2.9 microg cm(-2). Maximum protein A immobilization was observed at pH 9.5. Up to 186 microg cm(-2) was immobilized on the CNBr-activated poly(HEMA) membranes. The maximum adsorption of human-IgG on the protein A-immobilized poly(HEMA) membranes was observed at pH 8.0. The non-specific adsorption of human-IgG onto the plain poly(HEMA) membranes was low (about 4.4 microg cm(-2)). Higher human-IgG adsorption values (up to 394 microg cm(-2)) were obtained in which the protein A-immobilized poly(HEMA) membranes were used. Much higher amounts of human-IgG (up to 489 microg cm(-2)) were adsorbed from human plasma. Up to 91% of the adsorbed human-IgG was desorbed by using 0.1 M aminoacetic acid as elution agent. The adsorption-desorption cycle was repeated ten times using the same polymeric membranes. There was no remarkable reduction in the adsorption capacity of the protein A-immobilized poly(HEMA) membranes.
This study employed two water-soluble and nontoxic molecules, sucrose and glycerol, to enhance the permeability of PEG-PHEMA polymer gels coated onto 100 kDa molecular weight cutoff polyethersulfone (PES) microdialysis probes. Sucrose precoating of the probes prior to prepolymer coating prevented penetration of the prepolymer into the microdialysis membrane. Glycerol mixed with the prepolymer introduced porosity in the polymer coating upon curing. The sucrose and glycerol were completely removed by soaking in PBS after curing of the polymer coat on the probe tip. Polymer coated probe glucose permeability was tested by measuring glucose recovery from PBS solutions. Biocompatibility was assessed by measuring glucose recovery of polymer coated probes from heparanized whole porcine blood. Results show that the sucrose and glycerol treatments yielded polymer coated probes with glucose permeability nearly equal to bare probes when tested in PBS solution, but that this increased permeability was not observed when tested in whole blood. This suggests that the thickness of the polymer films (10-100 microm), while not a limiting factor in PBS solution, may have presented a diffusion barrier to glucose recovered from blood. Surprisingly, however, the polymer coated probes exhibited less thrombus formation that did the bare probes after blood exposure.
Bioaffinity chromatography has a unique and powerful role that is used as a purification tool in the production of therapeutic plasma protein derivatives. In this study, a bioaffinity-ligand, i.e. gelatin, was covalently immobilized with PHEMA microspheres (150-200 microm in diameter). The affinity sorbent carrying 7.5 mg gelatin g(-1) polymer was then used to separate fibronectin from human plasma in a packed-bed column system. Fibronectin separation from human plasma on unmodified PHEMA microspheres was 0.45 mg g(-1), while much higher adsorption values, up to 21.8 mg g(-1), were obtained with gelatin-immobilized microspheres. The fibronectin adsorption capacity of the microspheres decreased with an increase in the recirculation rate of plasma. Fibronectin adsorption increased with decreasing temperature, and the maximum adsorption achieved at 4 degrees C (26.3 mg fibronectin g(-1)). Up to 94.7% of the adsorbed fibronectin was desorbed by using 2 M urea in the presence of 1 M sodium chloride as elution agent. The adsorption-desorption cycle was repeated ten times using the same affinity column. There was no remarkable reduction in the adsorption capacity of the gelatin-immobilized PHEMA microspheres.