Imaging cell interactions with native and crosslinked polyelectrolyte multilayers

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films. Both authors kcontributed equally.     

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.     

Increased Hydrophobicity of Carboxymethyl Starch Film by Conjugation with Zein

A carboxymethyl starch (CMS) film was prepared by a process in which gelatinized CMS was dried, and subsequently treated with water-soluble carbodiimide in the presence of zein in 70% ethanol or 70% acetone to form acid-amide cross-linkages in order to increase the hydrophobicity of the surface of the film. A small amount of zein protein was found to be present on the surface of the zein-CMS conjugate (Zein-CMS) film, resulting in its insolubility in hot water, low water vapor permeability, and resistance to digestion with α-amylase and β-amylase. Digestion of the Zein-CMS film with protease rendered the film readily water-soluble, suggesting conjugation with zein as an effective means of increasing the hydrophobicity of biodegradable starch-based articles.     

Increased hydrophobicity of carboxymethyl starch film by conjugation with zein

A carboxymethyl starch (CMS) film was prepared by a process in which gelatinized CMS was dried, and subsequently treated with water-soluble carbodiimide in the presence of zein in 70% ethanol or 70% acetone to form acid-amide cross-linkages in order to increase the hydrophobicity of the surface of the film. A small amount of zein protein was found to be present on the surface of the zein-CMS conjugate (Zein-CMS) film, resulting in its insolubility in hot water, low water vapor permeability, and resistance to digestion with alpha-amylase and beta-amylase. Digestion of the Zein-CMS film with protease rendered the film readily water-soluble, suggesting conjugation with zein as an effective means of increasing the hydrophobicity of biodegradable starch-based articles.     

The effect of different methods of cross-linking of collagen on its dielectric properties

This paper reports on the effect of different methods of collagen cross-linking on its dielectric properties. In order to obtain collagen-hyaluronic acid (HA) scaffolds, collagen was first dehydrated by a combination of thermal and vacuum drying (DHT) and then treated with the chemical reagent carbodiimide (EDC/NHS) for final cross-linking. The measurements of the relative permittivity varepsilon' and the dielectric loss varepsilon' for all materials were carried over the frequency range of 10 Hz-100 kHz and at temperatures from 22 to 260 degrees C. The results for these samples reveal distinct relaxation processes at low temperatures, below 140 degrees C and at higher temperatures as broad peak around 230 degrees C. The first and second relaxation are associated with changes in the secondary structure of collagen accompanied by the release of water and with the denaturation of dry collagen, respectively. The influence of cross-linking on the permittivity of collagen is significant over the entire temperature range.     

Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450 LM2

Carboxyl groups of NADPH-cytochrome P-450 reductase have been modified with the water-soluble carbodiimide EDC. Although there is no significant loss in DCPIP reduction the activity with cytochrome c and cytochrome P-450 LM2 as electron acceptors was inhibited by about 60 and 85%, respectively (1 h incubation time, 20 mM EDC). The inactivation by EDC was nearly completely prevented in the presence of cytochrome P-450 LM2, but not by bovine serum albumin. These results and crosslinking studies suggest that carboxyl groups of NADPH-cytochrome P-450 reductase are involved in charge-pair interactions to cytochrome c and to at least two amino groups of cytochrome P-450 LM2.     

Resveratrol and Celastrol Loaded Collagen Dental Implants Regulate Periodontal Ligament Fibroblast Growth and Osteoclastogenesis of Bone Marrow Macrophages

Collagen is widely used for dental therapy in several ways such as films, 3D matrix, and composites, besides traditional Chinese medicine (TCM), has been used in tissue regeneration and wound healing application for centuries. Hence, the present study was targeted for the first time to fabricate collagen film with TCM such as resveratrol and celastrol in order to investigate the human periodontal ligament fibroblasts (HPLF) growth and bone marrow macrophages (BMM) derived osteoclastogenesis. Further, the physicochemical, mechanical and biological activities of collagen‐TCM films crosslinked by glycerol and EDC‐NHS (1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide‐N‐hydroxysulfosuccinimide) were investigated. Collagen film characterization was significantly regulated by the nature of plasticizers like hydrophobic and degree of polarity. Interestingly, the collagen film's denaturation temperature was increased by EDC‐NHS than glycerol. FT‐IR data confirmed the functional group changes due to chemical interaction of collagen with TCM. Morphological changes of HPLF cells cultured in control and collagen films were observed by SEM. Importantly, the addition of resveratrol upregulated the proliferation of HPLF cells, while osteoclastogenesis of BMM cells treated with mCSF‐RANKL was significantly downregulated by celastrol. Accordingly, the collagen‐TCM film could be an interesting material for dental regeneration, and especially it is a therapeutic target to restrain the elevated bone resorption during osteoporosis.     

Serum-free cell culture on insulin-immobilized porous collagen beads

Insulin or albumin was immobilized on collagen beads using water-soluble carbodiimide. Adhesion of STO mouse fibroblast cells onto the beads decreased with increasing the amount of immobilized proteins. Growth of the cells was remarkably accelerated on the insulinimmobilized collagen beads, which can be used for serum-free cell culture. The growth acceleration became larger with increasing the amount of immobilized insulin, while it became smaller with increasing the amount of immobilized albumin. In addition, the immobilized insulin more strongly accelerated the cell growth than free insulin plus collagen beads. (c) 1995 John Wiley & Sons, Inc.     

Ⅱ型胶原-硫酸软骨素支架的制备及组织工程软骨的构建

研究经乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC)处理的Ⅱ型胶原-硫酸软骨素支架材料的性能特点,并在体外构建组织工程软骨。从鸡软骨中提取Ⅱ型胶原,以不同浓度的EDC为交联剂通过冷冻干燥的方法制备Ⅱ型胶原与硫酸软骨素复合支架并测定其理化性质。将体外培养的新生兔关节软骨细胞接种在Ⅱ型胶原与硫酸软骨素复合支架上,观察软骨细胞在支架上的生长形态并检测支架上软骨细胞分泌的糖胺聚糖含量及Ⅱ型胶原含量。结果表明:采用EDC与硫酸软骨素交联增加了支架的稳定性,最适的交联剂质量浓度为7 mg/mL。软骨细胞在复合支架上增殖分化良好,并保持软骨细胞特异分化的表型,分泌Ⅱ型胶原与蛋白多糖(GAG)。培养14 d后已有软骨样组织形成。     

The astrocytic ("peripheral-type") benzodiazepine receptor: role in the pathogenesis of portal-systemic encephalopathy

An increasing body of evidence supports the notion that activation of astrocytic (peripheral-type) benzodiazepine receptors contributes to the pathogenesis of the central nervous system symptoms which are characteristic of portal-systemic encephalopathy (PSE). Binding site densities for the PTBR ligand [3H-PK11195] are increased in autopsied brain tissue from PSE patients as well as in the brains of animals with experimental chronic liver failure. In the case of the animal studies, increased PTBR sites resulted from increased PTBR gene expression. Exposure of cultured astrocytes to ammonia or manganese (two neurotoxic agents which under normal circumstances are removed by the hepatobiliary system and which are found to accumulate in brain in PSE) results in increased densities of [3H-PK11195] binding sites. Activation of PTBR is known to result in increased cholesterol uptake and increased synthesis in brain of neurosteroids some of which have potent positive allosteric modulator properties on the GABA-A receptor system. Accumulation of such substances in the brain in chronic liver failure could explain the neural inhibition characteristics of PSE.     

Synthesis and characterization of cellulose/silica hybrid materials with chemical crosslinking

The cellulose/silica hybrid biomaterials are prepared by sol–gel covalent crosslinking process. The tetraethoxysilane (TEOS) as precursor, γ-aminopropyltriethoxylsilane (APTES) as couple agent, and 2,4,6-tri[(2-epihydrin-3-bimethyl-ammonium)propyl]-1,3,5-triazine chloride (Tri-EBAC) as crosslinking agent, are used in the sol–gel crosslinking process. The chemical and morphological structures of cellulose/silica covalent crosslinking hybrids are investigated with micro-FT-IR spectra, nitrogen element analysis, X-ray diffraction, SEM, AFM, and DSC. The results show that the cellulose/silica hybrids form new macromolecular structures. In sol–gel process, inorganic particles are dispersed at the nanometer scale in the cellulose host matrix, bounding to the cellulose through covalent bonds. The cellulose/silica covalent crosslinking hybrid can form good and smooth film on the cellulose. The thermal properties of organic/inorganic hybrids are improved.     

d-Galacturonandigalacturonohydrolase covalently bound on to a polyacrylamide-type support

d -Galacturonandigalacturonohydrolase was immobilized by covalent coupling on to a polyacrylamide-type carrier BIO Gel CM100, activated by water-soluble carbodiimide. Catalytic properties, stability and action pattern of the immobilized enzyme are reported.     

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。     

Interaction of sodium dodecyl sulphate with elastin polypeptides

The interaction that occurs between polypeptidic fragments of elastin and sodium dodecyl sulphate (SDS) as a model of natural amphiphilic substances has been studied by means of thermal concerration of the elastin fragments in the presence of detergents, by solubilization of a lipophilic dye, and by means of gel permeation chromatography. It was found that elastin polypeptides interact with SDS giving mixed micelles. This finding seems to be especially relevant in the tissues, revealing enhanced degradation of elastin and accumulation of lipophilic substances (e.g. in atheromatous plaques). In such tissues, elastin polypeptides formed could interfere with the formation of the normal elastic fibre by means of their interaction with amphiphilic substances.     

Degree of crosslinking of collagen at interfaces: adhesion and shear rheological indicators

Work of adhesion (ΔW) and surface rheology at solid/air and solution/air interface have been used as indicators to study the stabilization of collagen by different crosslinking agents like basic chromium sulfate (BCS), tannic acid, catechin and formaldehyde. The results show that an increase in rate of ΔW would promote adsorption while a decrease leads to hindered adsorption. Shear rheological studies on collagen demonstrate an increase in both shear viscosity and elasticity with time while for collagen with polyphenols like catechin and tannic acid there is an unusual breakdown of these values. A correlation between the rheological properties and the work of adhesion suggests that the time frame in which the viscoelastic behavior is initiated for collagen with different crosslinking agents determines the final macroscopic property of the protein. The study attempts to quantify the degree of crosslinking of collagen through the dynamics and strength of the water molecules in the assembly of hydrated protein and the crosslinking agents.     

Non-enzymatic glycosylation induced changesin vitro in some molecular parameters of collagen

Non-enzymatic glycosylation of rat tail tendon collagen was examined by incubation with D-glucosein vitro. The changes in molecular parameters such as viscosity, thermal stability, electrophoretic mobility and solubility were determined on nonenzymatically glycosylated collagenin vitro. Tendons incubated with 8 and 24 mg glucose/ml showed an increase in dissolution temperature and a l.6-3-fold increase in thermal isometric tension respectively when compared to tendons incubated in the absence of glucose, indicating the formation of new intermolecular bonds. This conclusion was further supported by the decreased solubility of glycosylated collagen in 0.5 N acetic acid and the change in sub-unit composition as measured from the sodium dodecyl sulphate Polyacrylamide gel electrophoresis pattern. Glycosylated collagen gave a characteristic absorption spectra λ_max 248 nm) as distinct from that of control (λ_max 242 nm). Denaturation temperature of glycosylated collagen, as determined from temperature dependent viscosity measurements, was reduced. These studies indicate that glycosylation affects the molecular interactions as well as the crosslinking of collagen.     

Synthesis of a Semi-Interpenetrating Polymer Network as a Bioactive Curcumin Film

This study focused on the synthesis and characterization of a natural polymeric system employing the interpenetrating polymer network (IPN) comprising curcumin as a bioactive. Biopolymers and actives such as chitosan, hypromellose, citric acid, genipin, and curcumin were used to develop an effective, biodegradable, and biocompatible film employed therapeutically as a wound healing platform. The semi-IPN films were investigated for their physicochemical, physicomechanical, and biological properties by quantification by FTIR, DSC, and Young’s modulus. Following characterization, an optimum candidate formulation was produced whereby further in vitro and ex vivo studies were performed. Results revealed a burst release occurring at the first hour with 1.1 mg bioactive released when in contact with the dissolution medium and 2.23 mg due to bioactive permeation through the skin, thus suggesting that the lipophilic nature of skin greatly impacted the bioactive release rate. Furthermore, chemical and mechanical characterization and tensile strength analysis revealed that the degree of crosslinking and concentration of polymeric material used significantly influenced the properties of the film.KEY WORDS: biomaterials, crosslinker, curcumin, films, semi-interpenetrating polymer network, wound healing     

Improved physical and biochemical features of a collagen membrane by conjugating with soluble egg shell membrane protein

Soluble egg shell membrane protein (SEP) prepared from the egg shell membrane by performic acid oxidation and pepsin digestion was applied to prepare a membrane conjugated with pepsin-solubilized collagen (PSC) by cross-linking with water-soluble carbodiimide. SEP enabled a conjugated membrane to be constructed with a well-developed network structure having thicker collagen fibrils and higher stability than the PSC membrane alone, as indicated by reduced solubility and collagenase-digestion, increased denaturation temperature, and superior flexibility. The SEP-PSC membrane (SEP:PSC of 1:1, w/w) had lower sweatiness than the PSC membrane. Fibroblasts and keratinocytes adhered to the SEP-PSC-coated wells (SEP:PSC of 1:10, w/w) as a fundamental model of an artificial skin similar to that to the PSC-coated wells due to similar expression level of several cell-adhesion proteins. An SEP-PSC coating accelerated the cell growth of fibroblasts, whereas the cell growth of keratinocytes did not change, or decreased slightly due to more rapid differentiation indicated by a more substantial expression of differentiation marker proteins than a PSC-coating without SEP.     

Structural transition during thermal denaturation of collagen in the solution and film states

Temperature dependent vibrational circular dichroism (VCD) spectra of type I collagen, in solution and film states, have been measured. These spectra obtained for solution sample suggest that the thermal denaturation of collagen results in transition from poly-L-proline II (PPII) to unordered structure. The PPII structure of collagen is identified by the presence of negative VCD couplet in the amide I region, while the formation of unordered structure is indicated by the disappearance of VCD in the amide I region. The temperature dependent spectra obtained for the supported collagen film indicated a biphasic transition, which is believed to be the first vibrational spectroscopic report to support a biphasic transition during thermal denaturation of collagen film. The temperature dependent spectra of collagen films suggest that the thermal stability of collagen structure depends on its state and decreases in the order: supported film > free standing film > solution state. These observations are believed to be significant in the VCD spectroscopic analysis of secondary structures of proteins and peptides.     

A new, improved technique for automated sequencing of non-polar peptides

A method for the automated sequential degradation of non-polar peptides is reported. Both the polarity and film-forming properties of these peptides are increased by the attachment of 2-amino-1,5 napthalene disulfonic acid to the C-terminal residue via a water-soluble carbodiimide. The sequences of three individual peptides modified by the procedure are reported with high yields while these same peptides could not be successfully sequenced if no modification was made.