Mechanism of crosslinking of proteins by glutaraldehyde I: reaction with model compounds

3H-Glycine and 6-aminohexanoic acid were used as model amine compounds and reacted with glutaraldehyde. Based on the spectral characteristics and the molecular weights obtained from the reaction products, it is concluded that glutaraldehyde can modify amines to form an intermediate which absorbs at 300 nm and has a molecular weight of about 200. In the presence of excess glutaraldehyde, this intermediate is quickly converted to a much larger intermediate which absorb strongly at 265 nm. The larger intermediates are finally altered to yield a strong absorption peak at 325 nm with no apparent change in the molecular weight. These results suggest that a process of polymerization is induced by the initial reaction of glutaraldehyde with amines. The glutaraldehyde-polymer amine complex is self-limiting in size and can undergo internal rearrangement to become chemically inert.     

Mechanism of crosslinking of proteins by glutaraldehyde. IV: In vitro and in vivo stability of a crosslinked collagen matrix

The use of native or reconstituted collagen as a bioprothesis for tissue augmentation requires the introduction of exogenous synthetic crosslinks. The degree of crosslinking determines the rate of resorption or replacement of the implanted materials by the host. Since biophysical and chemical methods to quantify these crosslinks have in general been difficult to evaluate, we have developed in vitro enzymatic approaches which enable us to correlate the degree of crosslinking with the rates of enzymatic degradation. When the number of stable crosslinks formed is large it is essential to partially unfold the collagen fibrils by heating or by exposure to denaturing agents to enhance their susceptibility to hydrolysis. In the present study we demonstrate that increasing the number of reactive amino groups on collagen by coupling 1,6-diaminohexane to carboxyl groups using a water soluble carbodiimide can significantly enhance the number of crosslinks introduced by glutaraldehyde. We also show that the enzymatic method developed correlates well with the biodegradation of radiolabeled crosslinked collagenous tissues implanted subcutaneously in rats.     

Structural mechanism for alteration of collagen gel mechanics by glutaraldehyde crosslinking

Soft collagenous tissues that are loaded in vivo undergo crosslinking during aging and wound healing. Bioprosthetic tissues implanted in vivo are also commonly crosslinked with glutaraldehyde (GA). While crosslinking changes the mechanical properties of the tissue, the nature of the mechanical changes and the underlying microstructural mechanism are poorly understood. In this study, a combined mechanical, biochemical and simulation approach was employed to identify the microstructural mechanism by which crosslinking alters mechanical properties. The model collagenous tissue used was an anisotropic cell-compacted collagen gel, and the model crosslinking agent was monomeric GA. The collagen gels were incrementally crosslinked by either increasing the GA concentration or increasing the crosslinking time. In biaxial loading experiments, increased crosslinking produced (1) decreased strain response to a small equibiaxial preload, with little change in response to subsequent loading and (2) decreased coupling between the fiber and cross-fiber direction. The mechanical trend was found to be better described by the lysine consumption data than by the shrinkage temperature. The biaxial loading of incrementally crosslinked collagen gels was simulated computationally with a previously published network model. Crosslinking was represented by increased fibril stiffness or by increased resistance to fibril rotation. Only the latter produced mechanical trends similar to that observed experimentally. Representing crosslinking as increased fibril stiffness did not reproduce the decreased coupling between the fiber and cross-fiber directions. The study concludes that the mechanical changes in crosslinked collagen gels are caused by the microstructural mechanism of increased resistance to fibril rotation.     

Osteoblastic response to collagen scaffolds varied in freezing temperature and glutaraldehyde crosslinking

Collagen sponges are widely used scaffolds in bone engineering. To form bone, the osteoblastic cells undergo proliferation, differentiation, and mineralization stages in the scaffold. Crosslinking and freezing temperature are two important variables in fabricating collagen sponges. The purpose of this study was to examine the osteoblastic responses to collagen sponges prepared with or without glutaraldehyde crosslinking at different freezing temperatures (-20 degrees C or -80 degrees C). MC3T3-E1 osteoblastic cells were cultured in differently prepared sponges. Osteoblastic responses examined included cell numbers, osteocalcin expression, and calcium deposition. Cell numbers were measured by DNA content. Osteocalcin expression was determined by RT-PCR and real-time RT-PCR. Calcium deposition was assayed by ortho-cresophthalein complexone method and von Kossa stain. The osteoblastic cells grown in all collagen sponges did not show apparent signs of cytotoxicity. Collagen sponges differed in freezing temperatures resulted in similar osteoblastic responses. Glutaraldehyde-crosslinked sponges demonstrated less cell-mediated contraction and more cell numbers at day 7 (p < 0.005). However, they showed lower osteocalcin expression at day 7 (p < 0.05) and less calcium deposition at day 21 (p < 0.001). In summary, different freezing temperatures played a minor role in osteoblastic responses. Glutaraldehyde crosslinking process, though improved the dimensional stability of collagen sponges, might compromise the osteoblastic differentiation and mineralization.     

Lysine-enhanced glutaraldehyde crosslinking of collagenous biomaterials.

Crosslinking of collagenous biomaterials currently employs the use of glutaraldehyde. The putative enhancement of glutaraldehyde crosslinking by lysine was investigated in three model systems: bovine pericardium, collagen membranes, and bovine serum albumin. Repetitive sequential treatment of bovine pericardium with glutaraldehyde and lysine and finally with formaldehyde produced a matrix which, by the two criteria used (shrinkage temperature and urea/SDS soluble collagen), was shown to be more highly crosslinked than pericardium fixed in glutaraldehyde alone. Essentially the same results were obtained when membranes prepared from pepsin-soluble pericardial collagen were subjected to sequential glutaraldehyde and lysine treatments, reaching shrinkage temperatures of more than 90 degrees C. Heart valves prepared from lysine-enhanced glutaraldehyde crosslinked bovine pericardium were tested in vitro in an accelerated fatigue tester and have been shown to behave satisfactorily after 300 million cycles. These additional crosslinks proved to be stable in saline at 37 degrees C. Studies on bovine serum albumin attempted to get an insight into the mechanisms of lysine enhancement of glutaraldehyde crosslinking by treating sequentially albumin with glutaraldehyde and lysine and analysis of the products by gel filtration and SDS-PAGE. These studies suggest that free amino groups exposed by proteins are initially reacted with glutaraldehyde and then bridged by the diamino compound (lysine) producing more extensive intermolecular crosslinking than glutaraldehyde alone.     

L-asparaginase bound to collagen membranes: effect of glutaraldehyde crosslinking on stabilization of catalytic activity.

Reconstituted bovine collagen has been used extensively in our laboratory as a carrier for immobilized E. coli L-asparaginase. The activity and catalytic stability of these collagen-asparaginase membranes can be altered substantially by conditions used in membrane crosslinking with glutaraldehyde. As the concentration of glutaraldehyde used in tanning is increased, the initial specific activity of collagen-asparaginase membranes decreased asymptotically to a limiting value. Similar results occurred when membranes were subjected to increasing time periods of tanning at a constant glutaraldehyde concentration. These observations point to a time-concentration relationship for glutaraldehyde tanning and its effect on the specific activity of collagen-asparaginase membranes. Specific activities of membranes tanned a glutaraldehyde concentrations of 5% or higher appear to be very stable over long periods of alternate storage and assay. This result, however, is not observed with membranes tanned at glutaraldehyde concentrations lower than 5% for short periods of time (approximately 30 sec to 1 min). It is not clear whether the instability of membranes tanned at lower concentrations of glutaraldehyde or shorter intervals of tanning is due to enzyme elution from the membrane or denaturation of the bound enzyme.     

Physicochemical properties of collagen solutions cross-linked by glutaraldehyde

The physicochemical properties of collagen solutions (5?mg/ml) cross-linked by various amounts of glutaraldehyde (GTA) [GTA/collagen (w/w)?=?0–0.5] under acidic condition (pH 4.00) were examined. Based on the results of the determination of residual amino group content, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, dynamic rheological measurements, differential scanning calorimetry and atomic force microscopy (AFM), it was proved that the collagen solutions possessed strikingly different physicochemical properties depending on the amount of GTA. At low GTA amounts [GTA/collagen (w/w)?≤?0.1], the residual amino group contents of the cross-linked collagens decreased largely from 100% to 32.76%, accompanied by an increase in the molecular weight. Additionally, increases of the fiber diameter and the values of G′, G″ and η* were measured, while the thermal denaturation temperature (T_d) did not change visibly and the fluidity of collagen samples was still retained with increasing the GTA amount. When the ratio of GTA to collagen exceeded 0.1, although the residual amino group content only decreased by ～8.2%, the cross-linked collagen solution [GTA/collagen (w/w)?=?0.3] displayed a clear loss of flow and a sudden rise (～2.0?°C) of the T_d value compared to the uncross-linked collagen solution, probably illustrating that the collagen solution was converted into a gel with mature network structure-containing nuclei observed in AFM image. It was conjectured that the physicochemical properties of the collagen solutions might be in connection with the cross-linking between collagen molecules from the same aggregate or different aggregates.     

Inhibition of ectopic calcification of glutaraldehyde crosslinked collagen and collagenous tissues by a covalently bound diphosphonate (APD)

Calcification of collagen-derived prosthesis, such as glutaraldehyde crosslinked porcine heart valves or heart valves assembled out of bovine pericardium, presents a major clinical problem. Their subcutaneous implantation into young rats provides us with a reproducible method of assessing this form of ectopic calcification. Long-term implantation is essential, since some materials which do not calcify within the first month frequently exhibit a delayed calcific response. Crosslinked pericardium is much more likely to calcify than crosslinked tendon or reconstituted crosslinked pepsin extracted bovine type I collagen. The covalent binding of a diphosphonate to collagen and collagen-rich tissues can prevent calcification. The binding of this diphosphonate and its ability to inhibit calcification can be enhanced by increasing the number of amino groups on the collagen molecule. The degree of calcification is inversely related to the number of diphosphonate molecules covalently bound to collagen. Under standard conditions, chemical modifications appear to occur primarily on the surface of the collagen fibrils, as evidenced by the relationship between the number of molecules of APD bound and fibril diameter. The bound diphosphonate seems to interfere with crystal growth and prevent the formation of highly insoluble hydroxyapatite on the surface and interstices of the collagen fibrils.     

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.     

Bioartificial materials based on collagen: 1. Collagen cross-linking with gaseous glutaraldehyde

The effect of exposure time of thin films of soluble collagen to glutaraldehyde (GTA) vapour was studied at 37°C, and was evaluated by examining the thermal and biological stability and the swelling ratio. It was found that the collagen films treated with GTA vapour for 18 h showed the highest denaturation temperature, the lowest swelling ratio, and an enhanced proteolytic stability. This study shows that soluble collagen can be effectively cross-linked with GTA vapour and that the degree of cross-linking can be controlled by varying the exposure time.     

Detection and characterisation of an overmodified type III collagen by analysis of non-cutaneous connective tissues in a patient with Ehlers-Danlos syndrome IV.

The clinical and biochemical observations in a patient with a mild form of Ehlers-Danlos syndrome (EDS) type IV are described. The patient's skin fibroblasts produced markedly diminished amounts of type III collagen. SDS-polyacrylamide gel electrophoresis of collagens produced by cells obtained from other, non-cutaneous tissues showed two forms of collagen alpha 1(III) chains, a normal and a slow migrating, mutant form. Further analysis confirmed that the type III collagen molecules containing mutant alpha chains which were overmodified had a lower thermal stability and were poorly secreted into the extracellular medium. The protein defect was mapped by in situ cyanogen bromide digestion and was located in alpha 1(III) CB9, the C-terminal peptide of the collagen triple helix. This study shows that non-cutaneous connective tissues can be a useful source for the study of type III collagen defects in patients with EDS type IV.     

Comparative distribution of fibronectin and type III collagen in normal human tissues

The distribution of fibronectin (FN) and type III collagen (IIIC) in normal adult human tissues have been directly compared using immunofluorescence and immunoperoxidase techniques on fresh frozen and formalin-fixed paraffin embedded material. Although in many tissues localisation of these two proteins appeared similar, two major differences were identified: (1) Where the ratio of extracellular matrix to cells is high (e.g. in breast, intestinal submucosa), FN was scanty and present predominantly in association with cells, whether of epithelial, endothelial or mesenchymal origin. IIIC was present diffusely throughout interstitial connective tissue. (2) In the highly specialised vascular beds of spleen and renal glomeruli, FN was abundant and accompanied by little or no IIIC. It is postulated that these differences reflect a generalised more intimate association of FN with cell surfaces and basal laminae which is not always discernible by light microscopy. Proximity of FN and IIIC may nevertheless be important for cell-matrix interactions. It was also noted that "reticulin" fibres as defined by silver impregnation do not all have an identical composition.     

Stabilization of collagen tissues by photocrosslinking

Photocrosslinking, using 2 mM Ru(II)(bpy)(3)Cl(2) and various concentrations of sodium persulfate with irradiation by blue light, ～455 nm, has been shown to be a rapid and effective method for crosslinking various tissues: tendon, amnion membrane, pericardium, and heart valve leaflet. The presence of new crosslinking was demonstrated by the increase in the shrinkage temperature of these tissues. In all the cases, increase in the shrinkage temperatures were seen, although at higher sodium persulfate concentrations, for example, 100 mM, both with and without the Ru(II)(bpy)(3)Cl(2) catalyst, some degradation of the collagenous tissues was found. The effectiveness of this photocrosslinking method when used with tissues was also shown through the increase in the break strength of tissues after crosslinking, and by the reduction of protein that could be extracted by urea. In solution studies, dityrosine has been shown to be formed during photocrosslinking. With tissues, Western blotting showed the presence of new dityrosine crosslinked proteins.     

Bioartificial materials based on collagen: 2. Mixtures of soluble collagen and poly(vinylalcohol) cross-linked with gaseous glutaraldehyde

Thin films of both pure soluble collagen (CLG) and poly(vinylalcohol) (PVA) and mixtures of the two, ranging from 20-80% PVA composition were studied to test the effects of PVA content and of glutaraldehyde vapour cross-linking. Both the thermal and mechanical behavior and, in addition, proteolytic stability were clearly influenced by the ratio of CLG/PVA. The experimental results indicate that no thermodynamic compatibility occurs between the two homopolymers. However, there is evidence that strong interactions, probably due to hydrogen bond formation, occur between the biological and synthetic polymers. The interactions appear stronger in those films with a lower PVA content and which were not cross-linked. Both the thermal and biological stability are increased and there is an improvement of the mechanical properties. The mutual intermolecular influence appears to allow the attainment of a good mechanical compatibility between CLG and PVA.     

交联对胶原降解速率的影响

目的:研究交联反应对胶原降解速率的影响.方法:以热交联(DHT)、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)化学交联以及EDC/DHT交联三种方法对胶原海绵材料进行处理,并测定材料在处理前后的降解速率.结果:各种交联反应均不同程度地提高了胶原的生物稳定性,降低了胶原的降解速率.     

瘦素及I和III型胶原蛋白在肝纤维化大鼠肝组织中的变化

 摘要： 目的 研究瘦素（Leptin）及I、 III型胶原蛋白及基因在肝纤维化模型组织中的动态表达水平。方法 四氯化碳（CCl4）皮下注射法制备肝纤维化模型,分别以Western blot及RT-PCR法检测Leptin及I、 III型胶原蛋白及基因在肝纤维化组织中的动态表达。结果 Leptin以及I、 III型胶原蛋白及基因在正常对照组肝脏中均有微量表达,CCl4注射2周后,三者的表达均开始增强,随着纤维化发展呈梯度增加。其mRNA表达水平在模型组明显高于正常组 (P<0.05);在肝纤维化过程中,Leptin与I型胶原(r=0.595,P=0.017)及Leptin与III型胶原(r=0.478,P=0.011) 的动态改变呈显著正相关。结论 Leptin的表达随着纤维化的程度加重而逐步增强,在肝纤维化过程中,Leptin可能参与了细胞外基质成分（ECM）的合成与降解。