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PubMed1988

Actin filament morphology in living and nonliving cultured mesangial cells: formation and dissolution.

Mesangial cells are contractile and are believed to play a role in the regulation of glomerular filtration. Actin filaments constitute an integral part of the cytoskeleton of these cells. To visualize actin filaments in both living and fixed acetone-extracted (nonliving) mesangial cells, we have used the fluorophore nitrobenzoxadiazole-phallacidin. Under the fluorescence microscope, mesangial cells displayed continuous staining. Distinct linear structures running from one end to the other were actin filaments or stress fibers (SF). SF were rather feathery and of slightly curvilinear appearance in living versus fixed, acetone-extracted cells. Formation of SF started a 4 h, and the majority of the cells had well developed SF within 24 h. To find out whether adhesion to a substrate was necessary to SF formation, we plated mesangial cells on variable thickness of poly-HEMA(2-hydroxyethyl methacrylate). This plastic surface at a high concentration (10(-1) M) produces poor adherence of mesangial cells. At 10(-1) M of poly-HEMA, the cells were rounded even after 48 h and did not develop any SF. In contrast, at a lower concentration (10(-4) M), mesangial cells were well spread, and SF were readily observed. These data indicated that spreading as well as formation of SF were directly related to the adhesion properties of the substrate. To evaluate the role of SF in contraction and relaxation of mesangial cells, we have first treated these cells with angiotensin II (5 x 10(-7) M) or dibutyryl cyclic adenosine monophosphate (5 x 10(-4) M) and then labeled them with nitrobenzoxadiazole phallacidin. Dibutyryl cyclic adenosine monophosphate caused dissolution of SF.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed2000

Single-cell analysis using capillary electrophoresis: influence of surface support properties on cell injection into the capillary.

Capillary electrophoresis (CE) is an important tool of chemical cytometry. Whole-cell analysis using CE starts with cell injection into the capillary by either siphoning or electroosmosis. However, strong adherence of the cell to the support surface can prevent efficient cell injection and lead to irreproducible analysis. Here we evaluated several surfaces as potential cell supports for HT29 cells (human colon adenocarcinoma). These cells strongly adhered to the surface of untreated glass or polystyrene. Hydrophobic coating with dimethyldichlorosilane (DMS) or Sigmacote did not significantly reduce cell adhesion. In contrast, cell adhesion was reduced significantly when the surface was modified with hydrophilic polymers (hydrogels) such as poly(2-hydrohyethyl methacrylate) (PHEMA) and polyvinyl alcohol (PVA). In addition to their pronounced antiadhesive properties, PHEMA and PVA coatings were the most biocompatible (had highest survival of cells in contact with surface). Hydrogel-coated polystyrene plates were tested as a commercial alternative to hydrogel-coated glass slides. The cell adhesive properties of such plates were similar to those of PHEMA and PVA. However, the biocompatibility of the plates was lower than that of the other surfaces tested. Moreover, in contrast to PHEMA- and PVA-coated glass slides, the plates were sensitive to UV light and therefore should not be used when fluorescent image microscopy with UV excitation precedes CE. The analyses of the data obtained showed that PHEMA- and PVA-coated glass slides were the most suitable cell supports for cell injection into the capillary.

Adenocarcinoma↗
PubMed1993

Bacterial adhesion to poly(HEMA)-based hydrogels.

The effects of water content and comonomer chemistry upon the adhesion of Pseudomonas aeruginosa to poly(hydroxyethyl methacrylate)-based hydrogels were studied. Hydrogels which varied in swollen water content from 33-69 wt% were polymerized onto glass microscope slides pretreated with a vinyl silane. The hydrogel water content was varied by adding methacrylic acid (1-5 wt%) or N-vinyl pyrrolidone (NVP, 10-25 wt%) or combinations of the two comonomers. The resulting hydrogel surfaces, which were 0.1 mm thick, transparent, and adherent to the glass slide, formed the test surfaces of laminar flow cells (Re = 1.3, wall shear rate = 1.6/s). The bacteria were grown for 8 h in tryptic soy broth (TSB), washed by filtration, and collected on 0.45-microns filters, resuspended in phosphate buffered saline (PBS) at pH = 7.2, and recirculated through the flow cell and across the test surface at 0.85 mL/min for 2 h. Results show that P. aeruginosa adhered less to hydrogels with higher water contents. In the presence of TSB and possible poly(NVP) contamination, the concentration of adherent bacteria was reduced to low and uniform levels independent of the hydrogel chemistry.

Bacterial Adhesion↗
PubMed1981

Progestin permeation through polymer membrane V: Progesterone release from monolithic hydrogel devices.

Progesterone release from monolithic devices prepared from various copolymers of poly(hydroxyethyl methacrylate) and poly(methoxyethoxyethyl methacrylate) or poly(methoxyethyl methacrylate) was examined. In general, plots of the fraction of drug released versus (time)1/2 were linear during the early stages of drug release. This behavior is similar to that found for drugs released from hydrophobic polymers such as polydimethyl siloxane. However, for some release curves using the hydrogels, a breakpoint appeared during the early stages of drug release. These breakpoints were due to the effects of water absorption by these polymers. From analyses of permeability coefficients, it was demonstrated that release rates also were dependent on the initial drug load and the equilibrium water content of the polymer. These conclusions were verified from cross-membrane diffusion studies on films depleted of their initial drug load. In conjunction with this work, the aqueous solubility of progesterone was determined by several methods; an average value of 38 micrograms/ml at 23 degrees was obtained.

Kinetics↗
PubMedDate not supplied

Evaluating prostate cancer cell culturing methods: a comparison of cell morphologies and metabolic activity.

LNCaP prostate cancer cells were grown under four unique cultivation conditions. Two types of bioreactor systems were used to observe the influence of low-shear culture conditions allowing for three-dimensional growth: a) a perfusion rotating wall vessel (RWV) bioreactor; and b) a high aspect ratio vessel (HARV) RWV bioreactor, with periodic medium exchanges (fed-batch). In addition, two growth methods utilized tissue culture flasks (TCFs): a) unaltered or conventional TCFs; and b) poly(2-hydroxyethyl methacrylate) [poly(HEMA)] coated TCFs, to inhibit cell attachment. Comparisons were drawn based on qualitative observation of cell morphology and quantitative metabolic data. Similar cellular metabolism was demonstrated for cells grown under each condition. The degree of aggregation, however, varies considerably. Spherical shaped aggregates with diameters of 1 to 3 mm were produced when cells were grown within the perfusion-RWV bioreactor. All other growth conditions produced irregular shaped aggregates of various sizes. Quantitative results demonstrated the expected glucose utilization concomitant with lactate accumulation. Immunohistochemical evaluations were unremarkable for all four cultivation conditions. Results demonstrate that use of the perfusion-RWV bioreactor is advantageous in obtaining spherical aggregates, while grown in a controlled environment.

Bioreactors↗
PubMed1990

Preparation and evaluation of radiopaque hydrogel microspheres based on PHEMA/iothalamic acid and PHEMA/iopanoic acid as particulate emboli.

Highly porous poly(2-hydroxyethyl methacrylate) (PHEMA) microspheres prepared by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) in presence of polymeric diluents such as poly(methyl methacrylate) (PMMA) in toluene and poly(tetramethylene glycol) (PTMG) were made radiopaque by esterification of the reactive hydroxyl groups with iothalamic acid and iopanoic acid, two radiopaque substances clinically used. Of the various solvents and catalysts examined, tetrahydrofuran (THF) and N,N'-dimethyl paratoluidine (DMPT) were found to be best for obtaining a high degree of conversion. More than 30 wt% iodine could be bound to the microspheres which made them sufficiently radiopaque to be imaged radiographically. Microspheres retained their porosity, swelling ability, hydrophilicity, and surface morphology to a significant extent after iodination. Preliminary implantation studies of such microspheres subcutaneously in rats have shown no adverse tissue reactions over a 6-month period. It is suggested that these microspheres would prove to be useful as particulate emboli in endovascular embolization.

Animals↗
PubMed2005

Detection of DNA point mutation by atom transfer radical polymerization.

We report here a new DNA detection method in which polymer growth in atom transfer radical polymerization (ATRP) is used as a means to amplify detection signals. In this method, DNA hybridization and ligation reactions led to the attachment of ATRP initiators on a solid surface where specific DNA sequences were located. These initiators subsequently triggered the growth of poly(hydroxyethyl methacrylate) (PHEMA) at the end of immobilized DNA molecules and formed polymer brushes. The formation of PHEMA altered substrate opacity, rendering the corresponding spots readily distinguishable to the naked eye. A second ATRP reaction to form branched polymers on the surface drastically improved the visibility of DNA hybridization and significantly shortened the detection time. The resulting polymer film was characterized using infrared spectroscopy, ellipsometry, contact angle measurements, and atomic force microscopy. Direct visualization of 1 fmol of target DNA molecules of interest was demonstrated. A proof-of-principle experiment to detect DNA point mutation was conducted. The perfectly matched DNA targets were distinctively differentiated from those with mutations. The demonstrated capability to detect DNA mutation with direct visualization laid the groundwork for the future development of detector-free testing kits in single-nucleotide polymorphism screenings.

DNA↗
PubMed2004

Ultraviolet radiation absorption of intraocular lenses.

OBJECTIVES: To record and compare the spectral transmittance curves of intraocular lenses (IOLs) made out of polymethyl methacrylate (PMMA), acrylic, hydrogel, and silicone from different manufacturers; to evaluate their ultraviolet radiation absorption capacities; and to contrast the recorded transmittance curves with that of the natural lens. DESIGN: Experimental study. METHODS: We studied 17 different 21-diopter IOLs. A high-performance spectrophotometer with a diffuse transmittance accessory was employed to measure the transmittance of wavelengths from 200 nm to 800 nm through a 1.5-mm aperture. MAIN OUTCOME MEASURES: Transmittance percentage and 10% transmittance cutoff wavelength. RESULTS: All studied IOLs offered good ultraviolet radiation protection in the ultraviolet C (200-280 nm) and ultraviolet B (280-315 nm) ranges. A number of silicone, PMMA, and acrylic lenses showed different and, at times, only low degrees of absorption in the ultraviolet A (315-400 nm) range. CONCLUSIONS: Intraocular lenses of different compositions have ultraviolet radiation absorption characteristics different from that of the crystalline lens.

Absorption↗
PubMed1997

Characterization of protein release through glucose-sensitive hydrogel membranes.

Glucose-sensitive phase-reversible hydrogels have been prepared based on the specific interaction between polymer-bound glucose and concanavalin A (Con-A). The main goal of this study was to characterize the release of model proteins (insulin and lysozyme) through the hydrogel membrane as the free glucose concentration in the environment was changed. The diffusion of the model proteins through the hydrogel membrane was examined using a diffusion cell. Porous poly(hydroxyethyl methacrylate) (PHEMA) membranes were used to sandwich the mixture of glucose-containing polymers and Con-A in between the donor and receptor chambers. The porous PHEMA membranes allowed diffusion of glucose, insulin and lysozyme, while preventing loss of glucose-containing polymers and Con-A in the sol state. The release rate of model proteins through the glucose-sensitive hydrogel membrane was dependent on the concentration of free glucose. The release rate of the proteins did not remain constant, however, due to the change in free glucose concentration resulting from diffusion of glucose from the receptor chamber to the donor chamber. This study demonstrated the possibility that the glucose-sensitive phase-reversible hydrogels can be used to regulate the insulin release as a function of the free glucose concentration in the environment.

Concanavalin A↗
PubMed1994

The effects of transcervical monofilament insertion on the microbial status of the uterus in guinea-pigs.

The monofilament marker tail attached to intra-uterine contraceptive devices (IUCDs) has been implicated in the development of pelvic infection because it acts as a substrate for bacterial adhesion, and facilitates the transmission of vaginal bacteria into the uterus. A guinea-pig model was used to investigate the role of transcervical monofilaments in the transmission of vaginal bacteria into the uterus. By 21 days the degree of uterine contamination was significantly lower than after 24 h (P < 0.05), but was still significantly greater than control values (P < 0.01). Coating the monofilaments with poly (2-hydroxyethyl-methacrylate) (polyHEMA) had no effect on uterine bacterial counts at 24 h. After 21 days, however, contamination within the uteri fitted with polyHEMA-coated monofilaments had significantly increased from levels observed at 24 h (P < 0.01). These counts were also significantly greater than those recorded for the uncoated and control groups at the same time interval. In animals fitted with monofilaments, in which chlorhexidine had been incorporated into the polyHEMA coating, the level of uterine contamination after 24 h was significantly lower than that observed with uncoated threads at the same time interval (P < 0.02). After 21 days there was no significant difference between uterine bacterial counts from the chlorhexidine group and control animals. It is concluded that the use of such drug-loaded monofilaments offers the potential to minimize infections associated with the transcervical insertion of IUCDs.

Animals↗
PubMed1978

The influence of glycol methacrylate (GMA) and paraffin embedding on freeze substituted and fixed tissues for enzyme histochemistry.

The influences of paraffin and GMA-embedding on acid phosphatase, esterase and beta-glucuronidase activity of differently fixed or freeze substituted rat livers were studied. 1. Embedding generally causes a reduction of the enzyme activities but improves considerably the quality of the microscopical pictures when compared with appropriate cryostat sections. Embedding therefore may serve as a very useful tool for detail studies on the cytological level. 2. The embedding media act differently on the reactive sites: a. Paraffin causes a heavy denaturation of the enzyme activity in lysosomes but preserves the activities of the ergastoplasmic (= "microsomal") enzymes. The degree of denaturation increases with increasing embedding temperature. b. GMA-embedding delivered opposite effects by preserving lysosomal activities and quenching endoplasmic enzymes. UV-polymerization of GMA causes a general inactivation of enzymes. 3. The histochemical reactivity of substrates such as glycogen was not influenced by the embedding. However, its most natural localization is achieved by freeze drying or isopropanol freeze substitution followed by GMA-embedding.

Acid Phosphatase↗
PubMed1988

Immunoaffinity chromatography of lymphocyte subpopulations using tert-amine derived matrices with adsorbed antibodies.

New polymeric solid-phase matrices for cell affinity chromatography were prepared and their advantageous characteristics compared with conventional matrices were highlighted. These new matrices are derivatives of poly(2-hydroxyethyl methacrylate) (PHEMA) containing a slight quantity of amino compounds as a co-monomer. They were applied to immunoaffinity selection between IgG+ and IgG- lymphocytes of the rat mesenteric lymph node. Simple physical adsorption was sufficient for anti-rat IgG antibodies to be immobilized on these matrices, allowing us to omit the laborious procedure of covalent-linking of antibodies on a matrix. As these matrices themselves showed extremely low non-specific adsorption of lymphocytes, a very dilute solution of antibody (0.02-0.08 mg/ml) was enough for column conditioning. This separation method gave IgG- lymphocytes of more than 90% purity and almost 95% yield within as short a time as 7 min. Further, IgG+ lymphocytes were obtained in good yield (80-90% of loaded number) by recovering the adsorbing cell fraction from the column by gentle pipetting of the matrix.

Amines↗
PubMed2005

Involvement of fractal geometry on solute permeation through porous poly (2-hydroxyethyl methacrylate) membranes.

Fractal geometry was applied to quantify the complexity of an internal structure of porous membranes prepared with poly(2-hydroxyethyl methacrylate) (pHEMA). The porous pHEMA membranes were synthesized by means of free-radical solution polymerization. Boundary lines of the porous structures in the pHEMA membrane were taken by a scanning electron microscope as image data, and these images were fed into a computer to estimate the fractal dimension. The boundary images of porous pHEMA membranes were observed to be a typical fractal and their complexity was quantified as a non-integral fractal dimension. The permeation of fluorescein isothiocyanate-labeled dextran, molecular weight 4400 (FD-4) as a model penetrant through the porous pHEMA membrane was determined using water-jacket type two-chamber diffusion cells. A fairly good negative relationship between the permeability coefficient of FD-4 and the fractal dimension was observed, suggesting the usefulness of the fractal dimension as a novel means for evaluating solute permeation through the porous membranes.

Algorithms↗
PubMed2003

Development of in situ thermosensitive drug vehicles for glaucoma therapy.

The goal of this research was to design thermosensitive drug vehicles for glaucoma therapy. Thermosensitive ophthalmic drop was prepared by mixing linear poly(N-isopropylacrylamide-g-2-hydroxyethyl methacrylate) (PNIPAAm-g-PHEMA), PNIPAAm-g-PHEMA gel particles and antiglaucoma drug. This produced polymeric eyedrop containing the drug epinephrine was a clear solution at room temperature which became a soft film after contacting the surface of cornea. The drug entrapped within the tangled polymer chains was therefore released progressively after topical application. Evaluation of the drug release responded as a function of crosslinking density and PHEMA macromer contents. The in vivo studies indicated that the intraocular pressure (IOP)-lowering effect for a polymeric eyedrop lasted for 26 h, which is significantly better than the effect of traditional eyedrop (8 h). Hence our investigations successfully prove that the thermosensitive polymeric eyedrop with ability of controlled drug release exhibits a greater potential for glaucoma therapy.

Acrylic Resins↗
PubMed1981

A hematoxylin and eosin-like stain for glycol methacrylate embedded tissue sections.

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5% ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075% ponceau de xylidine and 0.025% acid fuchsin in 10% acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.

Animals↗
PubMed2006

Isolation of microbial DNA by newly designed magnetic particles.

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) microspheres and cobalt ferrite nanoparticles modified with alginic acid (natural carboxylic polysaccharide) were used for isolation of microbial DNA of lactic acid bacteria (LAB) from dairy products, lyophilised cell cultures, and bacterial colonies grown on hard media, and Trichophyton fungi DNA from lyophilised cells. DNA from the samples with lysed cells was reversibly adsorbed to the particles in the presence of high poly(ethylene glycol) (PEG 6000) and sodium chloride concentrations. The optimal final PEG and NaCl concentrations were 9.1 wt.% and 2.0 M, respectively. The adsorbed DNA was released from the particles in low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification. Moreover, PCR amplicons were isolated on cobalt ferrite nanoparticles modified with alginic acid and checked by restriction analysis.

Bifidobacterium↗
PubMed2004

Surface friction of hydrogels with well-defined polyelectrolyte brushes.

Hydrogels of poly(2-hydroxyethyl methacrylate) (PHEMA) with well-defined polyelectrolyte brushes of poly(sodium 4-styrenesulfonate) (PNaSS) of various molecular weights were synthesized, keeping the distance between the polymer brushes constant at ca. 20 nm. The effect of polyelectrolyte brush length on the sliding friction against a glass plate, an electrorepulsive solid substrate, was investigated in water in a velocity range of 7.5 x 10(-5) to 7.5 x 10(-2) m/s. It is found that the presence of polymer brush can dramatically reduce the friction when the polymer brushes are short. With an increase in the length of the polymer brush, this drag reduction effect only works at a low sliding velocity, and the gel with long polymer brushes even shows a higher friction than that of a normal network gel at a high sliding velocity. The strong polymer length and sliding velocity dependence indicate a dynamic mechanism of the polymer brush effect.

Electrolytes↗
PubMed2002

Tissue-engineered cartilage on biodegradable macroporous scaffolds: cell shape and phenotypic expression.

OBJECTIVE: The purpose of the study was to establish in vitro culture of chondrocytes on biodegradable, poly(D,L-lactic-co-glycolic acid) [PLGA] scaffolds. STUDY DESIGN: Laboratory experiment using cartilage of rat rib and biodegradable scaffolds. METHODS: Chondrocytes were cultured on a poly-hydroxyethyl methacrylate (poly-HEMA)-coated dish, proliferated, and transferred into the PLGA scaffolds. Phenotypic expression of cells was examined according to the condition of poly-HEMA coating. Morphological, biochemical, and immunohistochemical characteristics of cells cultured within PLGA scaffolds were also examined. RESULTS: Chondrocytes cultured on a poly-HEMA-coated dish aggregated into distinct nodules containing large clusters of spherical cells and showed cartilage-specific phenotype, collagen type II. The results of immunostaining andreverse transcriptase-polymerase chain reaction of cells cultured within PLGA scaffolds showed cartilage-specific morphological appearance and structural characteristics such as lacunae and expression of collagen type II. CONCLUSION: The chondrocytes cultured on a poly-HEMA-coated dish and PLGA scaffolds showed chondrocyte-specific phenotypes and morphological appearance.

Animals↗