The Chemical Protecting Group Concept Applied in Crosslinking of Natural Tissues with Glutaraldehyde Acetals

This work describes the results of the controlled crosslinking of collagen matrices by glutaraldehyde based on a double protection strategy, glutaraldehyde acetals and lysine protonation due to the acidic conditions of acetal formation. Materials crosslinked by this approach were characterized by thermal stability comparable to those obtained by conventional procedures with mechanical properties expected for bioprosthesis manufacture and with a higher stability toward collagenase hydrolysis. The integrity of the microfibrillar structure was confirmed by optical and scanning electronic microscopy. The results indicate that the glutaraldehyde acetals procedure may be of potential use for the crosslinking of bovine pericardium used in the manufacture of bioprosthetic devices. Advantages may be related to the production of materials with homogeneous crosslinking distributions, associated with better definition in the nature of the chemical link that they introduce, due to a better distribution of glutaraldehyde within the tissue matrix before the crosslinking reaction is allowed to occur. As a result, materials with improved biological and mechanical properties are expected.     

不同处理方法的牛心包生物力学特性测试分析

目的　探索一种理想的牛心包处理方法。方法　观察戊二醛及 2 ,3 -丁二醇改性戊二醛处理后的牛心包热挛缩温度、生物力学特性及超微结构变化。结果　鞣制后心包厚度和僵硬度增加 ,柔韧性降低。丁二醇组的弹性模量 (8.75± 1 .71 )较戊二醛组 (6.5 9± 1 .3 7) (P <0 .0 5 )更近似于正常瓣膜组织 ;且其极限拉伸强度优于戊二醛组。超微结构显示丁二醇组胶原纤维与弹性纤维结构完整 ;戊二醛组胶原纤维和弹性纤维均明显变性 ,结构模糊。结论　 2 ,3 -丁二醇改性处理后的牛心包具有更好的生物力学特性 ,该方法是一种较为理想的牛心包处理方法     

FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde

FT-IR analysis was performed for the hydroxyapatite (HAp)/collagen (COL) nanocomposite cross-linked by glutaraldehyde (GA). The amide bands I, II and III from COL matrix, and phosphate and carbonate bands from HAp were identified. The amide B band arising from C–H stretching mode showed a sensitive conformation by the degree of cross-linking. The amide I band showed a complicate conformational change by the degree of cross-linking. The characteristic amide I band at 1685 cm⁻¹, which is known as an aging parameter in the biological bone, did not show a monotonous tendency by the degree of cross-linking. The relative contents of the organics in the cross-linked HAp/COL nanocomposite were evaluated as an integration ratio between the amide I band at 1600–1700 cm⁻¹ and PO₄³⁻ band at 900–1200 cm⁻¹. The increase of the organics content by the cross-linking is enabled by the further organization of Ca²⁺ ions of HAp crystals in HAp/COL nanocomposite. The complicate conformational behavior in the amide I, II and III bands seems to be affected by the cross-linking induced directional arrangement of HAp/COL nanocomposite fibrils.     

Preservation of the non-rectangular cuticular plate/cell axis angle of outer hair cells

Motile properties of outer hair cells (OHCs) may contribute to sharp tuning and amplification in the mammalian cochlea. Shape changes of isolated OHCs in response to various physical and chemical influences have been investigated intensively. However, determinations of shape may have been influenced by unanticipated effects of preparation and preservation of the OHCs investigated. Thus, in a first step, lengths of freshly isolated OHCs from the guinea pig cochlea were determined using a video-enhancing magnification system. The cuticular plate/cell axis angle (CP/CA angle) was then measured in native cells and under the influence of potassium chloride and potassium gluconate incubation. To show the influence of glutaraldehyde (GA) fixation on the isolated OHCs, fixative dependent changes on cell length and CP/CA angle were recorded in native and preincubated OHCs. In these experiments, the cell length of vital isolated OHCs was between 41.5 m, in the basal turn, and 103.7 m, in the apical turn. The average CP/CA angle was 106° ± 4.2° (n = 324 cells, turns 1–4) with no statistically significant differences for the four turns. Under the influence of potassium chloride, cell length was reduced by 8.1%. Potassium gluconate incubation led to a shortening of cell length, followed by a 5.3% increase after 5 min. The CP/CA angle under potassium chloride was decreased (97.0°) and was then increased under the influence of potassium gluconate (110.7°) as a result of cuticular plate tilting. Cell shrinkage after fixation depended on the fixative's osmolarity and on the GA concentration. Increased GA levels amplified cell shrinkage from 34% for hypo-osmolar solutions to 15% in iso-osmolar and 29% in hyperosmolar solutions. The CP/CA angle of native and incubated OHCs was not different from those fixed with GA. The present data provide a rational basis for isolated OHC shape parameters. Moreover, functionally induced changes can be better interpreted when OHCs are influenced by fixatives, as shown in the GA experiments.     

Enrichment of the antibodies against the C-terminus of Taiwan cobra cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen.

The repertoire of antibodies producing by immunizing rabbits with cobrotoxin and dimeric glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward cobrotoxin, GA-cobrotoxin and recombinant cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-cobrotoxin and recombinant cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant cobrotoxin toward anti-cobrotoxin antibodies compared to anti-dGA-cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-cobrotoxin antibodies and the total anti-cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native cobrotoxin.     

不同消毒灭菌方案对纤维支气管镜活检钳的消毒效果分析

目的：探讨不同灭菌方案对纤维支气管镜活检钳的消毒效果。方法：对环氧乙烷消毒、戊二醛浸泡消毒及高压蒸汽灭菌3种消毒灭菌方案对纤维支气管镜活检钳的灭菌合格率、污染率和使用年限进行效果比较。结果：环氧乙烷消毒法的合格率高于戊二醛浸泡消毒和高压蒸汽灭菌消毒法,且污染率也显著低于另外两组,器械使用寿命显著延长。结论：低温环氧乙烷灭菌法对纤维支气管镜活检钳的灭菌效果好,且污染率较低,使用寿命更长。     

Effect of ethylene glycol dimethacrylate on swelling and on metformin hydrochloride release behavior of chemically crosslinked pH–sensitive acrylic acid–polyvinyl alcohol hydrogel

Background

The present work objective was to prepare and to observe the effect of ethylene glycol dimethacrylate on swelling and on drug release behavior of pH-sensitive acrylic acid–polyvinyl alcohol hydrogel.

Methods

In the present work, pH sensitive acrylic acid–polyvinyl alcohol hydrogels have been prepared by free radical polymerization technique in the presence of benzoyl peroxide as an initiator. Different crosslinker contents were used to observe its effect on swelling and on drug release. Dynamic and equilibrium swelling studies of prepared hydrogels were investigated in USP phosphate buffer solutions of pH 1.2, 5.5, 6.5 and 7.5 with constant ionic strengths. Hydrogels were evaluated for polymer volume fraction, solvent interaction parameter, molecular weight between crosslinks, number of links per polymer chain, diffusion coefficient, sol–gel fraction and porosity. To demonstrate the release pattern of the drug, zero-order, first-order, higuchi and korsmeyer-peppas models were applied. Quality and consistency of hydrogels was examined by FTIR and surface morphology of hydrogels was examined by SEM.

Results

Decrease in swelling and in drug release was seen by increasing content of ethylene glycol dimethacrylate. A remarkable high swelling was observed at high pH indicating the potential of this hydrogel for delivery of drugs to intestine. By increasing the concentration of ethylene glycol dimethacrylate, porosity decreased. Order of release was observed first order in all cases and the mechanism was non–fickian diffusion. FTIR confirmed the formation of network. SEM results showed the incorporation of drug.

Conclusion

The prepared hydrogels can be suitably used for targeted drug delivery to the intestine.     

Fixation strategies for retinal immunohistochemistry

Immunohistochemical and ex vivo anatomical studies have provided many glimpses of the variety, distribution, and signaling components of vertebrate retinal neurons. The beauty of numerous images published to date, and the qualitative and quantitative information they provide, indicate that these approaches are fundamentally useful. However, obtaining these images entailed tissue handling and exposure to chemical solutions that differ from normal extracellular fluid in composition, temperature, and osmolarity. Because the differences are large enough to alter intercellular and intracellular signaling in neurons, and because retinae are susceptible to crush, shear, and fray, it is natural to wonder if immunohistochemical and anatomical methods disturb or damage the cells they are designed to examine. Tissue fixation is typically incorporated to guard against this damage and is therefore critically important to the quality and significance of the harvested data. Here, we describe mechanisms of fixation; advantages and disadvantages of using formaldehyde and glutaraldehyde as fixatives during immunohistochemistry; and modifications of widely used protocols that have recently been found to improve cell shape preservation and immunostaining patterns, especially in proximal retinal neurons.     

内镜细胞刷几种低温灭菌方法的实验研究

目的：探讨几种低温灭菌方法对内镜细胞刷的灭菌效果。方法：内镜细胞刷浸染枯草杆菌黑色变种芽孢（ATCC9372），干燥后分别经戊二醛浸泡3h、4h、5h，戊二醛熏蒸3h、5h和环氧乙烷灭菌处理．采样培养7d。结果：内镜细胞刷经戊二醛浸泡3h、4h不能完全杀灭细胞刷污染的枯草杆菌黑色变种芽孢，浸泡5h，则可完全杀灭芽孢，达到灭菌合格；经戊二醛熏蒸3h、5h，不能完全杀灭细胞刷污染的枯草杆菌黑色变种芽孢；经环氧乙烷灭菌，能完全杀灭细胞刷污染的枯草杆菌黑色变种芽孢，达到灭菌合格。结论：戊二醛浸泡5h灭菌和环氧乙烷气体灭菌可有效杀灭内镜细胞刷污染的细菌芽孢，达到灭菌效果。