Showing papers on "Glutaraldehyde published in 1986"

An examination of the biologic response to injectable, glutaraldehyde cross-linked collagen implants

Abstract: The biologic response to injectable, glutaraldehyde cross-linked, fibrillar collagen implants in the rat subcutaneous model was shown to be a function of the concentration of glutaraldehyde used for cross-linking. The collagen was prepared from bovine hide by pepsin solubilization and reconstituted as a fibrillar suspension of 35 mg collagen/mL. Fibrillar collagen implants cross-linked with glutaraldehyde concentrations equal to or less than 0.01% exhibited a response characterized by fibroblast invasion, neovascularization and little, if any, evidence of inflammation. Implants cross-linked with 0.1 and 1.0% glutaraldehyde elicited a foreign body/giant cell reaction and varying degrees of implant erosion. The interaction of human skin fibroblasts with 0.01% glutaraldehyde cross-linked collagen in vitro was found to be dependent on the culture conditions utilized to evaluate the interaction. When the ratio of cell culture media to collagen was 20:1, cell invasion of the cross-linked preparations was observed, whereas, when this ratio was reduced to 1:1, such interactions could not be detected. Noncross-linked preparations were colonized by cells regardless of the experimental conditions used. Studies of implants in both the rat and guinea pig subcutaneous models indicated that glutaraldehyde cross-linking concentrations as low as 0.0075% provided enhanced wet weight recovery (wet weight persistence) and resistance to biologic degradation (collagen persistence) as compared to noncross-linked fibrillar collagen preparations. These cross-linked implants also exhibited a greater degree of fibroblast infiltration and vascularization. Between 30 and 60 days, some degree of calcification developed in both collagen formulations implanted in rats and guinea pigs; however, the reaction occurred with greater frequency and intensity in cross-linked preparations in guinea pigs. Calcification in the guinea pig was followed by the appearance of focal areas of ossification.

Calcification of subcutaneously implanted type I collagen sponges. Effects of formaldehyde and glutaraldehyde pretreatments.

Abstract: Although collagen-containing implants are widely used in various surgical applications, there has been relatively little attention paid to the possibility that this type of biomaterial may undergo pathologic calcification which could compromise its function. The present study reports for the first time the calcification of a series of implants of purified collagen sponges prepared with graded degrees of aldehyde-induced cross-linkages (assessed by shrinkage-temperature, wetting time, and collagenase digestibility). Type I collagen sponges were pretreated with either glutaraldehyde (0.1% to 2.0% aqueous solution, for 5-180 minutes) or formaldehyde (as vapors for 15 minutes to 15 hours), and implanted subcutaneously for 21 days in weanling rats. Although specimens not pretreated with either aldehyde reagent and the formaldehyde sponges pretreated for 15 minutes were resorbed without evidence of calcification, all other aldehyde-pretreated implants mineralized. The degree of calcification did not correlate with extent of cross-linking. Formaldehyde-pretreated implants calcified more extensively (Ca2+ = 87.8 +/- 2.8 micrograms/mg, mean +/- standard error of the mean; n = 58) than did glutaraldehyde-pretreated implants (Ca2+ = 40.9 +/- 1.4 micrograms/mg; n = 52). It is concluded that both glutaraldehyde- and formaldehyde-pretreated Type I collagen sponges calcify after subdermal implantation in young rats. Although aldehyde pretreatment of Type I collagen sponge implants is a prerequisite for their eventual mineralization, the threshold level of aldehyde-induced cross-linking required to potentiate their maximal pathologic calcification is low.

Evaluation of the immunocytochemical method for amino acids.

Abstract: Free amino acids can be coupled to proteins by glutaraldehyde. Rabbits immunised with a bovine serum albumin-glutaraldehyde-amino acid conjugate form antibodies that recognise similar conjugates with brain proteins in glutaraldehyde-fixed tissue. Antisera raised against conjugated GABA (gamma-aminobutyrate), glutamate, aspartate, taurine, glutamine, or glycine were tested against a variety of small molecular compounds that had been fixed by glutaraldehyde to brain protein and immobilised on cellulose ester filters for processing together with the brain sections. This system permitted closely similar conditions for testing and immunocytochemistry. After removing antibodies against the carrier used for immunisation and against cross reacting amino acid conjugates the antisera showed a high specificity. The specific nature of the antisera was corroborated by solid phase adsorption to the homologous antigens and by inhibition experiments with free amino acids and amino acid-glutaraldehyde fixation complexes. After transection of the striatonigral pathway the ipsilateral substantia nigra was almost depleted of GABA-like immunoreactivity; this observation lends additional support to the selectivity of the GABA antiserum. A semiquantitative relation was established between the concentration of amino acid before fixation in a model system and the subsequent intensity of immunostaining. Similar model experiments suggested that the conjugation of an amino acid to brain protein with glutaraldehyde, and the immunoreactivity of the conjugates, may be significantly inhibited in the presence of high concentrations of other amino compounds.

The preparation and physicochemical characterization of an injectable form of reconstituted, glutaraldehyde cross-linked, bovine corium collagen.

Abstract: Pepsin-solubilized bovine corium collagen was purified, reconstituted, and treated with various levels of glutaraldehyde. Treatment of suspensions of fibrillar collagen with low concentrations of glutaraldehyde appeared to have little effect on the gross morphology of fibrils, as judged by electron microscopy, but did have a significant impact on their physicochemical stability. Fibrillar collagen treated with glutaraldehyde at a concentration equal to or greater than 0.0075% demonstrated significant decreases in neutral solubility at elevated temperatures as compared to noncross-linked controls. Differential scanning calorimetry provided a convenient and quantitative means to correlate increases in melting temperature with increases in glutaraldehyde treatment concentration. Fibrillar collagen cross-linked with glutaraldehyde concentrations as low as 0.0075% demonstrated a significantly greater resistance to proteolytic degradation than did noncross-linked fibrillar collagen samples. The residual, extractable aldehyde content of such preparations was between 1 and 3 ppm. Rheological measurements on such cross-linked suspensions demonstrated that they were non-Newtonian, shear-thinning fluids, and that they were two- to threefold more viscous than corresponding preparations of noncross-linked collagen.

Postembedding immunogold labeling for electron microscopy using “LR White” resin

Abstract: A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White-embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high-resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.

Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy.

Abstract: SUMMARY The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde-fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction. The cytoplasmic membrane of sectioned Acholeplasma-cclls dehydrated and infiltrated as described above appeared more diffuse than that of cells fixed with glutaraldehyde and osmium tetroxide in epoxy resin at room temperature. Glutaraldehyde-fixed erythrocyte ghosts retained 85% of their phospholipid content when dehydrated with ethanol between 277 and 223 K and infiltrated with methacrylate at 223 K. Spinach chloroplasts and thylakoid vesicles retained 61% and 35%, respectively, of their cholorophyll content.

Studies on chitin. IX. Crosslinking of water‐soluble chitin and evaluation of the products as adsorbents for cupric ion

Abstract: Water-soluble chitin was successfully crosslinked to varying extents with glutaraldehyde in homogeneous aqueous solutions to improve the properties as an adsorbent for metal cations, and the effects of crosslinking were discussed. Complete insolubilization was achieved with the fivefold excess aldehyde, but, in terms of adsorptivity of Cu2+, the chitin crosslinked at an aldehyde/amino group ratio of 1.0 was found to exhibit remarkable capacity and was much superior to others. The desorption of Cu2+ from the adsorption complex was also attained effectively at pH 2.0. These results indicated that the loose crosslinking was quite simple and efficient to produce high capacity adsorbents for practical use. Thermal behavior of the crosslinked chitin was examined by TMA and TGA; a softening phenomenon was observed at 145°C.

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

Abstract: The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.

Immobilized enzyme membrane for a semiconductor sensor

Abstract: A membrane containing an immobilized enzyme for a semiconductor sensor is prepared containing a water soluble photosensitive resin including a high molecular weight polyvinyl pyrrolidone crosslinked to 2, 5-bis (4'-azide-2'-sulfobenzal) cyclopentanone sodium salt, and an enzyme. Glutaraldehyde and bovine serum albumin, polyamino acid or polyamino amino acid copolymer may also be present to provide chemical crosslinking. The enzyme may be glucose oxidase, urease or lipase. The membrane can be directly formed on ion-sensitive protions of a pH-ion sensitive field effect transistor to form a semiconductor sensor by coating an aqueous solution of the resin and enzyme on the ion-sensitive portion, drying and irradiating with light such as ultraviolet light to provide photo crosslinking.

Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina.

Abstract: Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.

Kinetics of the tuberculocidal response by alkaline glutaraldehyde in solution and on an inert surface

Abstract: Single cell suspensions of BCG and Mycobacterium tuberculosis were exposed to 2% alkaline glutaraldehyde solution (pH 8.0) and the rate of kill measured at intervals up to 30 min. Residual glutaraldehyde was neutralized with freshly prepared 1% sodium bisulphite. The rate of kill was directly proportional to the temperature and independent of the inoculum size whether the organism was tested in suspension or attached to an inert surface. Glutaraldehyde was slightly more bactericidal for the virulent M. tuberculosis than for the attenuated BCG. A substantial proportion of the mycobacterial population on an inert surface floated off during its exposure to the glutaraldehyde solution but the 'floaters' were killed at an equivalent rate to the attached bacilli. Complete sterility of a standardized suspension of M. tuberculosis could not be achieved within the 10 min period specified by the tuberculocidal assay, although it was usually attained within 20 min.

Effect of glutaraldehyde on hemoglobin: functional aspects and Mössbauer parameters.

Abstract: Glutaraldehyde is widely used for the cross-linking of hemoglobin for blood substitute research or for technological purposes. The effects of this reagent on the biochemical properties of hemoglobin were correlated with Mossbauer data. Human hemoglobin was cross-linked by glutaraldehyde as soluble polymers and insoluble particles. Effects of cross-linking on oxygen affinity, oxidation-reduction potential, autoxidation kinetics, and thermal stability were studied. Stability of cross-linked hemoglobin was specifically studied by Mossbauer spectroscopy. Oxygen affinity is increased, redox potential is decreased, autoxidation rates are increased, and stability towards thermal denaturation is increased. The regeneration of partially denatured hemoglobin by glutaraldehyde cross-linking is shown. Effects of cross-linking on biochemical properties are explained by the hypothesis of the opening of the heme pocket on the distal-histidine side and the concomitant charge transfer from the iron to the oxygen.

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

Abstract: We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

Systemic absorption of 14C-glutaraldehyde from glutaraldehyde-treated pulpotomy sites.

Abstract: The purpose of this research was to measure the. systemic absorption and distribution of glutaraldehyde from pulpotomy sites in dogs. Pulpotomies were performed on the incisors and canines of 5 mongrel dogs. A cotton pellet containing 20 ~1 of 2.5% "4C-(1,5) glutaraldehyde with an activity 6.25 x 105 dpm/~l was placed in each pulpotomy site for 5 rain. Whole blood samples, urine, and expired air were collected up to 90 rain when the animals were sacrificed and tissue samples removed from various organs. The tissue samples were prepared and counted in a scintillation counter to determine 14C-activity. The results demonstrate that glutaraldehyde is absorbed into the systemic circulation following 5-rain application of the agent to vital pulpotomy sites. Tissue binding of absorbed glutaraldehyde was relatively low and the remaining portion of the absorbed glutaraldehyde was metabolized and excreted in the urine or exhaled as carbon dioxide. Gradual impairment of the microcirculation of the pulp occurred following glutaraldehyde application.

The osmotic effect of glutaraldehyde-based fixatives on plant storage tissue

Abstract: SUMMARY Mass changes were used to monitor water gain or loss during fixation of plant tissue. Mannitol was used as reference osmoticum. Glutaraldehyde in Na-Na-phosphate, Na-cacodylate or PIPES buffer exerts an osmotic effect on plant tissue. This effect is in addition to the osmotic effect of the buffer. The effective osmolarity of the fixative depends on the glutaraldehyde concentration, the buffer type, the buffer concentration and the tonicity of the tissue. Relationships among these parameters are non-linear. Graphs are given to find the effective osmotic pressure of a buffered or unbuffered fixative containing 2% glutaraldehyde, for use with plant storage tissue with tonicity between 350 and 850 mOsm.

Entrapment of bioactive compounds within native albumin beads: II. Effects of rate and extent of crosslinking on microbead properties.

Abstract: The rate of crosslinking of serum albumin is affected by several parameters such as pH, buffer concentration, and the ratio of serum albumin to glutaraldehyde concentration. Selection of experimentally controllable variables makes it possible to control crosslinking rate at room temperature to form a serum albumin microbead with an appropriate extent of crosslinking that provides both structural integrity and biodegradability. It was confirmed that serum albumin concentrations of 200 mg/ml of sodium phosphate buffer solution (pH = 7.5, 10 mM) and 1% glutaraldehyde is a very favorable combination using a mixing time of around 20 sec before adding to oil phase to form an emulsion. A curing time of around 30 min is then used. Drug loading efficiency is crosslink rate-controlled. Mixing time also affects drug loading efficiency since it fixes the time for potential partitioning of drug out of the aqueous phase. The biodegradable character of the microbeads has been shown and it appears to be a function of the amount of serum albumin and glutaraldehyde used.

Ethanol production by yeast cells immobilized in open-pore agar

Abstract: An open-pore agar matrix has been shown to be suitable for the entrapment of microbial whole cells required for use in reactions that involve cell growth and gas evolution. Beads of porous agar with entrapped yeast cells have been used for the continuous fermentation of sugar cane molasses to ethanol, without apparent bead rupture, even after periods of 3 mo of use. The agar gel does not erode during prolonged operation, unlike porous gelatin cross-linked with glutaraldehyde.

Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry. Existence of cyclic secondary amino groups in the immunogen.

Abstract: The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro-beta-carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.

The use of malachite green during fixation of plant tissues for the preservation of lipids

Abstract: SUMMARY Osmiophilic droplets are shown in chloroplasts of leaves of ryegrass after the addition of malachite green to glutaraldehyde during primary fixation. Similar droplets are shown in leaves of bean and maize but not in the roots of these plants. These droplets are not shown in tissue fixed in glutaraldehyde in the absence of malachite green. Evidence is presented which indicates that these droplets contain a high proportion of lipids, particularly phospholipids. This evidence includes the use of other fixation methods which retain lipids, e.g. formal calcium, potassium dichromate. The droplets give a positive reaction with the silver hydroxylamine reaction for ester linkages. Tests for aldehydes by treatment with thiocarbohydrazide and silver proteinate, and proteins by digestion with protease were ambiguous. Digestion with solvents before fixation indicate that the droplets are lipids. TLC of chloroform: methanol extracts of fresh and fixed tissue and of the fixative solvents support the idea that malachite green aids retention of lipids during fixation. Phospholipids are also demonstrated by silver hydroxylamine in plant cell nuclei, myelin of rat brain and vesicles in bean root tips.

Immobilization of enzymes

Abstract: An enzyme producing microorganism or an enzyme produced thereby is immobilized by contacting the microorganism or enzyme with polyethylenimine and adding chitosan and glutaraldehyde to form a reaction product. In a preferred embodiment, the reaction product is extruded onto the revolving plate of a spheronizing device to form spherical enzyme aggregates having enhanced physical and biocatalytic properties.