HMDC crosslinking of bovine pericardial tissue: a potential role of the solvent environment in the design of bioprosthetic materials

The need for alternative crosslinking techniques in the processing of bioprosthetic materials is widely recognized. While glutaraldehyde remains the most commonly used crosslinking agent in biomaterial applications there is increasing concern as to its biocompatibility-principally due to its association with enhanced calcification, cytotoxicity, and undesirable changes in the mechanical properties of bioprosthetic materials. Hexamethylene diisocyanate (HMDC), like glutaraldehyde, is a bifunctional molecule which covalently bonds with amino groups of lysine residues to form covalent crosslinks. Evidence within the literature indicates HMDC-treated materials are less cytotoxic than glutaraldehyde-treated materials; however, there is limited characterization of the material properties of HMDC-treated tissue. This study uses a multi-disciplined approach to characterize the mechanical, thermal, and biochemical properties of HMDC-treated bovine pericardial tissue. Further, to facilitate stabilization of the HMDC reagent, non-aqueous solvent environments were investigated. HMDC treatment produced changes in mechanical properties, denaturation temperature, and enzymatic resistance consistent with crosslinking similar to that seen in glutaraldehyde treated tissue. The significantly lower extensibility and stiffness observed under low stresses may be attributed to the effect of the 2-propanol solvent environment during crosslinking. While the overall acceptability of HMDC as a crosslinking agent for biomaterial applications remains unclear, it appears to be an interesting alternative to glutaraldehyde with many similar features.     

γ辐射和EDC/NHS改性对胶原壳聚糖支架性能的影响EI北大核心CSCD

以5%梯度制备11组壳聚糖质量分数为0%~50%的胶原壳聚糖支架。使用γ辐射和EDC/NHS分别改性处理各组胶原壳聚糖支架,采用傅里叶变换红外光谱仪(FTIR)和扫描电镜(SEM)分析支架内部结构,利用吸水率、孔隙率、降解率和力学性能等指标对其性能进行检测,研究γ辐射和EDC/NHS改性对胶原壳聚糖支架性能的影响。结果表明:γ辐射和EDC/NHS改性均能使胶原与壳聚糖产生交联,壳聚糖的加入改善了γ辐射对支架分子结构的损伤;EDC/NHS改性支架的微结构好于γ辐射支架;两种改性支架壳聚糖较优,质量分数均为25%;γ辐射和EDC/NHS改性均能使支架产生取向结构。     

Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility

To construct a novel scaffold for nucleus pulposus (NP) tissue engineering, The porous type II collagen (CII)/hyaluronate (HyA)–chondroitin-6-sulfate (6-CS) scaffold was prepared using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking system. The physico-chemical properties and biocompatibility of CII/HyA–CS scaffolds were evaluated. The results suggested CII/HyA–CS scaffolds have a highly porous structure (porosity: 94.8 ± 1.5%), high water-binding capacity (79.2 ± 2.8%) and significantly improved mechanical stability by EDC/NHS crosslinking (denaturation temperature: 74.6 ± 1.8 and 58.1 ± 2.6°C, respectively, for the crosslinked scaffolds and the non-crosslinked; collagenase degradation rate: 39.5 ± 3.4 and 63.5 ± 2.0%, respectively, for the crosslinked scaffolds and the non-crosslinked). The CII/HyA–CS scaffolds also showed satisfactory cytocompatibility and histocompatibility as well as low immunogenicity. These results indicate CII/HyA–CS scaffolds may be an alternative material for NP tissue engineering due to the similarity of its composition and physico-chemical properties to those of the extracellular matrices (ECM) of native NP.     

Evaluation of different chemical methods for cros-linking collagen gel, films and sponges

Collagen-based films and sponges are widely used as biomaterials. The rate of their biodegradation can be reduced by treating them with different cross-linking agents. The efficiency of different reticulation procedures on thermal stability (measured by differential scanning calorimetry) and susceptibility to bacterial collagenase digestion of the final material (films or sponges) is compared. The chemical agents used on collagen gels or directly on collagen sponges and films were glutaraldehyde (GTA), hexamethylene diisocyanate (HMDC), cyanamide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and the two acyl azide methods (with hydrazine or diphenylphosphorylazide (DPPA)) developed in the authors' laboratory. Under these experimental conditions, collagen stabilization by the different agents increased in the following order: cyanamide T _d=75.8°C; after 3 months, T _d=75.6°C). Direct treatment of collagen films with DPPA gave similar thermal stability (T _d=72.6°C) and collagenase resistance when compared with treatment with 0.6% GTA for 96 h (T _d=74.6°C). It is demonstrated that collagen sponges and films can be prepared with a wide range of thermal stability (49–75.8°C) and collagenase digestion resistance (10–100%). Recent biocompatibility studies show, however, that DPPA and EDC are the best choices for preparing cross-linked collagen sponges and films.     

新型壳聚糖/PBLG-co-PGA复合膜的研究

采用壳聚糖和聚谷氨酸苄酯-co-聚谷氨酸(PBLG-co-PGA)通过1-乙基-3-(3-二甲基氨基丙基)碳化二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)交联得到复合膜.采用FTIR和XRD对复合膜的结构进行了表征,同时对复合膜的吸水率进行了研究.研究结果表明,复合膜中壳聚糖和PBLG-co-PGA分子之间成功地发生了交联反应,二者之间的交联与氢键作用改变了二者原有的结晶结构,这对复合膜的性能有着重要的影响.以复合膜为载体培养软骨细胞发现,复合膜与壳聚糖膜相比,具有更高的细胞粘附率,预示着该复合膜是一种具有良好应用前景的生物医用材料.     

Enhanced physicochemical properties of collagen by using EDC/NHS-crosslinking

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Crosslinking density, matrix morphology, swelling ratio shrinkage temperature and resistance against collagenase digestion were determined to evaluate the physicochemical properties of the collagen matrices with and without crosslinking. The results conformed that the porous structure of collagen was largely preserved and adjusted by crosslinking treatment. Furthermore, crosslinked collagen samples showed significantly reduced swelling ratio and increased resistance against thermal treatment and enzymatic degradation compared to non-crosslinked samples. An in vitro evaluation of MC3T3-E1 cells seeded onto the crosslinked and non-crosslinked collagen matrix indicated that crosslinked collagen was nontoxic and improved cell proliferation. Through this work, it was shown that an osteoconductive collagen matrix with optimized properties used as bioactive and bioresorbable scaffolds in bone tissue engineering could be fabricated through the EDC/NHS-crosslinking method.     

Heparin-functionalized collagen matrices with controlled release of basic fibroblast growth factor

Tissue engineering scaffolds with controlled long-term release of growth factors are constructed in an attempt to mimic the intelligent ability of the extracellular matrix (ECM) to release endogenous growth factors. In this study, collagen sponges (Collagen group) were modified by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) crosslinking (EDC/NHS group) and heparin immobilization (EDC/NHS-H group), and subsequently seeded with human umbilical vein endothelial cells (HUVECs). Native and modified sponges were pre-adsorbed with basic fibroblast growth factor (bFGF) to evaluate the sustained release and bioactive maintenance of bFGF from the sponges. We found that modified collagen matrices permitted HUVECs to proliferate and migrate well and to distribute uniformly. The EDC/NHS-H group exhibited an excellent sustained-release profile and bioactive maintenance of the pre-adsorbed bFGF as compared with the Collagen and EDC/NHS groups. These results suggest that heparin-functionalized collagen matrices can support a controlled release of bFGF and thus, have potential as a tissue engineering scaffold.     

Glutaraldehyde as a crosslinking agent for collagen-based biomaterials

The formation of Schiff bases during crosslinking of dermal sheep collagen (DSC) with glutaraldehyde (GA), their stability and their reactivity towards GA was studied. All available free amine groups had reacted with GA to form a Schiff base within 5 min after the start of the reaction under the conditions studied (0.5% (w/w) GA). Before crosslinks are formed the hydrolysable Schiff bases initially present were stabilized by further reaction with GA molecules. An increase in shrinkage temperature (T _s) from 56°C for non-crosslinked DSC (N-DSC) to 78°C for GA crosslinked DSC (G-DSC) was achieved after crosslinking for 1 h. From the relationship between the free amine group content and the T _s during crosslinking it was concluded that higher GA concentrations and longer reaction times will result in the introduction of pendant-GA-related molecules rather than crosslinks. After 24 h crosslinking an average uptake of 3 GA molecules per reacted amine group was found. No increase in the tensile strength of the materials was observed after crosslinking, which may be a result of formation of crosslinks within the fibres rather than in between fibres. Aligning of the fibres by applying a pre-strain to the samples and subsequent crosslinking yielded materials with an increased tensile strength.     

X-ray diffraction and leaching of CsAlSi5O12 and CsZr2(PO4)3 irradiated by argon (3 MeV) ions

Metamict damage cross-sections for the candidate nuclear fuel waste immobilization materials CsAlSi₅O₁₂ and CsZr₂(PO₄)₃ irradiated with 3 MeV argon ions were found to be 0.6±0.2 nm² and 0.7±0.3 nm², respectively, from powder X-ray diffraction measurements. Based on caesium extraction into solution, heavy irradiation produced increases of up to a factor of 25 in the dissolution rates of the materials in deionized water and a brine at 100° C.     

Low temperature synthesis of multi-walled carbon nanotubes via a sonochemical/hydrothermal method

In this investigation, multi-walled carbon nanotubes (MWCNTs) have been prepared by a facile sonochemical/hydrothermal method. MWCNTs have been hydrothermally fabricated with using dichloromethane, cobalt chloride and metallic lithium as starting materials in 5 mol/lit NaOH aqueous solution. Ultrasonic pre-treatment of the solution mixture had an important step prior to the hydrothermal condition, which could generate a considerable amount of multi-walled carbon nanotubes for the subsequent hydrothermal growth. Finally, high pure MWCNTs with lengths of 2-5 μm and diameters of 60 ± 20 nm could be synthesized at as low temperature as 160 °C. As a matter of fact, the method of sonochemical/hydrothermal guarantees the production of multi-walled carbon nanotubes (MWCNTs) for different applications, especially reinforcement materials.     

Low temperature hydrothermal synthesis of Ba(Mg1/3Ta2/3)O3 sol-derived powders

Powders of the microwave dielectric material barium magnesium tantalate Ba(Mg1/3Ta2/3)O3 have been produced by hydrothermal synthesis at moderately low temperatures (160 to 350°C). It was found that while it is relatively straightforward to produce the material in the desired perovskite phase at or below 200°C, the powder particles tend to be highly irregular in morphology with extremely small dimensions (of the order of 10 nm) and deficient in magnesium (with some precipitation of the excess magnesium as the hydroxide). The effects of both higher synthesis temperatures and different feedstock preparation were thus investigated with the aim of improving the precipitation of magnesium under hydrothermal conditions in order to produce a more homogeneous, stoichiometric powder and significant progress was made. It was found that when near-stoichiometric particles are formed, they adopt rounded morphologies and exhibit larger particle sizes (around 30–50 nm). These results show that the hydrothermal feedstock and the synthesis temperature used have a profound effect on particle stoichiometry, which in turn affects the growth morphology of the particles.     

Cross-linking by 1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC) of a collagen/elastin membrane meant to be used as a dermal substitute: effects on physical, biochemical and biological features in vitro

Next to in vitro-cultured autogeneic keratinocytes for the restoration of epidermis, a suitable dermal matrix is a mandatory component of an artificial skin substitute for the permanent covering of full thickness skin defects. In our model a xenogeneic membrane, consisting of processed native collagen and elastin of porcine origin is meant to serve as a template for the formation of a neo-dermis. In order to improve the resistance of this matrix against enzymatical degradation, we cross-linked it by using the carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) together with N-hydroxysuccinimide. Chemical cross-linking by these agents at two different degrees (shrinkage temperatures 63 °C and 81 °C) had no relevant effect on mechanical features or water-uptake capacity. The time needed for enzymatic digestion was increased by cross-linking. Concerning growth and spreading of fibroblasts and keratinocytes on and within the structure of this membrane, we did not observe a difference between cross-linked and non-cross-linked material (shrinkage temperature 48 °C). We therefore expect that cross-linking by EDC is an effective means to control the degradation of the collagen/elastin membranes in vivo without a significant influence on their biocompatibility. © 2001 Kluwer Academic Publishers     

Kinetic characterization and comparison of various protein crosslinking reagents for matrix modification

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; l-threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP ? LT. Most of the crosslinkers displayed a pH dependence and were more effective at more alkaline pH. At optimal pH and saturating conditions, the relative reaction rates of the crosslinkers were: PA = GA > EDC > GP > MG ? LT.     

The immobilization of trypsin on soap-free P(MMA-EA-AA) latex particles

Poly(methyl methacrylate-ethyl acrylate-acrylic acid) latex particles with narrow size distribution and with surface carboxyl groups were produced by soap-free emulsion polymerization, and covalent immobilization of trypsin onto these particles was carried out by using the water-soluble carbodiimide (EDC) as an activating agent under various conditions. Different immobilization methods were employed and the factors affecting the efficiency and activity of the immobilized enzyme, such as the amount of trypsin and EDC, pH and temperature of the immobilization reaction were investigated. Results showed that both relatively high immobilization efficiency and high enzyme activity were achieved when pre-adsorption method was employed. The immobilization efficiency decreased as the trypsin amount increased, and increased as pH and temperature increased. When the EDC amount varied, the immobilization efficiency first increased significantly and then decreased slowly. A maximum of enzyme activity can be obtained at the optimum value of 958.0 mg trypsin/g dried particles and 372.5 mg EDC/g dried particles at 25 °C and pH 5.0. The immobilized trypsin exhibited much higher relative activity than its free counterpart.     

碳化二亚胺改性胶原纤维支架材料的特性表征

用碳化二亚胺(EDC)对胶原纤维支架材料(CFSM)进行改性,评价了改性前后的胶原纤维支架材料的理化及生物降解性能.结果表明:胶原的羧基与EDC的氨基发生了酰胺化反应,且EDC改性未破坏胶原的三股螺旋结构.随着EDC用量增加,EDC-CFSM的热变性温度升高明显,耐降解性显著提高.用浓度为14mmol/L的EDC改性得到的EDC-CFSM表现出较优的生物学性能,其热变性温度达96.7℃,耐降解性最优,孔隙率为80.91%,孔径约为70～180μm.     

Attachment of functionalized single-walled carbon nanotubes (SWNTs) to silicon surfaces

Single-walled carbon nanotubes (SWNTs) were functionalized by direct fluorination and subsequent reaction with 6-aminohexanoic acid for water-soluble carboxylic acid functionalized SWNTs (AHA-SWNTs). Both of the compounds were used as precursors to attach SWNTs to APTES coated silicon surfaces. AHA-SWNTs in aqueous solution were reacted with APTES self-assembled monolayers (SAMs) with coupling reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). The surface coverage is a function of concentration of AHA-SWNTs, solvent and coupling method. While for the fluorinated SWNTs (F-SWNTs), direct addition of F-SWNTs to preformed APTES SAMs at 90 degrees C shows essentially no reaction, in contrast to the one-pot reaction of F-SWNTs with APTES molecules in the presence of SWNTs on a silicon substrate. This reaction route provides a convenient method to attach SWNTs to silicon surfaces.     

Melting behaviour and metastability of yttrium aluminium garnet (YAG) and YAlO3 determined by optical differential thermal analysis

The melting point of yttrium aluminium garnet (YAG), reinvestigated by optical differential thermal analysis (ODTA), was found to be 1940±7° C. Above this temperature YAG liquids are opaque, suggesting the presence of two immiscible liquids. In the composition range 10.0 to 47.5 mol% Y₂O₃, crystallization of the equilibrium phases can only occur in the presence of YAG nuclei; otherwise solidification of YAlO₃ and Al₂O₃ will take place. A metastable phase diagram has been defined with a metastable eutectic at 23 mol% Y₂O₃-77 mol% Al₂O₃ and 1702±7° C. YAlO₃ (perovskite) was found to melt incongruently with a peritectic temperature of 1916±7° C and a liquidus temperature of 1934±7° C. YAlO₃ formed during metastable solidification transforms to YAG in the presence of Al₂O₃ at 1418±7° C. It is suggested that the metastability arises from the difficulty of the aluminium to attain four-fold co-ordination in the YAG structure.     

Synthesis of Nanotubular Mg3Si2O5(OH)4-Ni3Si2O5(OH)4 Silicates at Elevated Temperatures and Pressures

Mg₃Si₂O₅(OH)₄-Ni₃Si₂O₅(OH)₄ nanotubes with the chrysotile structure and MgO : NiO molar ratios of 1 : 2 and 2 : 1 are synthesized by hydrothermal reactions at temperatures from 250 to 450°C and pressures from 30 to 100 MPa. The reaction path and kinetics, as well as the dimensions and morphology of the resulting nanotubes, are shown to depend on the nature of the starting reagents, chemical composition of the reaction system, and hydrothermal synthesis conditions. At higher nickel concentrations in the hydrous silicates, nanotube formation requires higher temperatures, longer hydrothermal treatment times, and higher NaOH concentrations in the reaction system.__________Translated from Neorganicheskie Materialy, Vol. 41, No. 7, 2005, pp. 849–855.Original Russian Text Copyright © 2005 by Korytkova, Maslov, Pivovarova, Polegotchenkova, Povinich, Gusarov.     

Cross-linkage of hydroxyapatite/gelatin nanocomposite using imide-based zero-length cross-linker

Hydroxyapatite [HAp]/Gelatin [GEL] nanocomposite was prepared at 37 and 48 °C through coprecipitation process. The HAp/GEL nanocomposite slurries were cross-linked by imide-based zero-length cross-linking agent such as N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). The chemical bond formation and microstructure in HAp/GEL nanocomposite was investigated as a function of cross-linking agents and temperature. The single addition of EDC into the composite slurries resulted in a tougher microstructure in both samples prepared at 37 and 48 °C. However, in the case of the simultaneous addition of EDC and NHS the sample prepared at 48 °C showed a coarse microstructure. These results were consistent with the fact that the chemical reactivity of NHS is degraded at 48 °C whereas the reactivity of EDC increases up to 80 °C.     

Hydrothermal dissolution of perovskite (CaTiO3)

Perovskite (CaTiO₃) has been exposed to hydrothermal chemical attack in aqueous solution. Dependences on temperature (150 to 250°C) and on duration of attack (1 to 35 days) have been investigated. It was found that a precipitate surface layer was formed. The thickness of this layer ranged from a few monolayers to several hundred nanometres for the least and the most severe hydrothermal conditions, respectively. The composition of the layer, as deduced by surface analytical techniques, suggests that (i) there is congruent dissolution of the perov skite surface, (ii) a TiO₂ precipitate layer is formed, and (iii) the presence of silica and CO₂ in solution do not appear to affect substantially the mechanism or the rate of dissolution.