Effect of aqueous ethanol on the triple helical structure of collagen

Collagen, the most abundant protein in mammals, is widely used for making biomaterials. Recently, organic solvents have been used to fabricate collagen-based biomaterials for biological applications. It is therefore necessary to understand the behavior of collagen in the presence of organic solvents at low (≤50 %, v/v) and high (≥90 %, v/v) concentrations. This study was conducted to examine how collagen reacts when exposed to low and high concentrations of ethanol, one of the solvents used to make collagen-based biomaterials. Solubility testing indicated that collagen remains in solution at low concentrations (≤50 %, v/v) of ethanol but precipitates (gel-like) thereafter, irrespective of the method of addition of ethanol (single shot or gradual addition); this behavior is different from that observed recently with acetonitrile. Collagen retains its triple helix in the presence of ethanol but becomes thermodynamically unstable, with substantially reduced melting temperature, with increasing concentration of ethanol. It was also found that the CD ellipticity at 222 nm, characteristic of the triple-helical structure, does not correlate with the thermal stability of collagen. Time-dependent experiments reveal that the collagen triple helix is kinetically stable in the presence of 0–40 % (v/v) ethanol at low temperature (5 °C) but highly unstable in the presence of ethanol at elevated temperature (~34 °C). These results indicate that when ethanol is used to process collagen-based biomaterials, such factors as temperature and duration should be done taking into account, to prevent extensive damage to the triple-helical structure of collagen .     

Recombinant expression of hydroxylated human collagen in Escherichia coli

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.     

Gel beads composed of collagen reconstituted in alginate

Spherical gel beads of collagen/alginate were prepared by discharging droplets of a mixture containing collagen (1.07-1.9 mg/ml) and alginate (1.2-1.5% w/v) into 1.5% w/v CaCl2 solution at 4°C. Collagen in the gel beads was reconstituted by raising the temperature to 37°C after alginate was liquefied by citrate. Scanning electron microscopy of the beads revealed the characteristic fibrous structure of collagen. To demonstrate the application of this new technique in cell culture, GH3 rat pituitary tumor cells were entrapped and grown in the gel beads. The immobilized cells proliferated to a density of 1.95 x 106 cell/ml which is about an order of magnitude higher than that grown in the alginate beads.     

胶原／壳聚糖复合膜的制备及止血效果的研究

目的 以胶原和壳聚糖制备复合膜,检验其止血效果,并探讨其止血原因。材料与方法：以酸解法从牛腱中提取胶原,用甲壳素制得壳聚糖,以胶原和壳聚制成复合膜,通过动物实验测不同配比的复合膜对出血创面的止血时间,并与其它止血材料做对比。结果：各种配比的复合膜的止血效果均比明胶等一般止血材料好。结论：胶原/壳聚糖复合膜有良好的止血作用,可望在外科手术上得到广泛应用。     

Partial purification and characterization of collagen galactosyltransferase from chick embryos.

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.     

Effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone tissue

The effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone in guinea pig was studied. Heterotopic bone formation was induced by transplantation of allogenic urinary bladder epithelium. Starting from the day of transplantation the animals were treated subcutaneously with HEBP and Cl2MBP with a dose of 12.5 mg P/kg/day during 35 days. The control group was injected with 0.9% NaCl solution. The advantage of heterotopic bone induction as an experimental model is the fact that the applied drugs act on de novo bone formation. Collagen fibers were treated as markers of bone because their size and spatial arrangement reflect the structure and maturity of organic matrix of this tissue. Decalcified histological sections of induced bone, taken 35 days after implantation of inductor, were stained by the picrosirius method. This staining enhances the natural birefringency of collagen fibers and allows for better and specific visualization of collagen fibers bundles under polarizing microscope. In this way the amount of information in the analysed image is increased. Thirty five microphotographs were analysed from each of the investigated groups with the use of optical diffractometry. The radial distribution of light intensity in diffraction patterns was analysed what allowed to evaluate spatial frequencies connected with the width of collagen bundles in induced bone tissue. Since the spatial arrangement of collagen fibers in newly formed bone is random, analysis of angular distribution of light intensity in diffractograms was not performed. Using discriminant analysis the significant differences between all three studied groups of animals were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomimetic CoUagen/Hydroxyapatite Composite Scaffolds： Fabrication and Characterizations

Biomimetic collagen/hydroxyapatite scaffolds have been prepared by microwave assisted co-titration of phosphorous acid-containing collagen solution and calcium hydroxide-containing solution. The resultant scaffolds have been characterised with respect to their mechanical properties, composition and microstructures. It was observed that the in situ precipitation process could combine collagen fibril formation and hydroxyapatite （HAp） formation in one process step. Collagen fibrils guided hydroxyapatite precipitation to form bone-mimic collagen/hydroxyapatite composite containing both intrafibrillar and interfibrillar hydroxyapatites. The mineral phase was determined as low crystalline calcium-deficient hydroxyapatite with calcium to phosphorus ratio （Ca/P） of 1.4. The obtained 1% （collagen/HAp = 75/25） scaffold has a porosity of 72% and a mean pore size of 69.4 ~tm. The incorporation of hydroxyapatite into collagen matrix improved the mechanical modulus of the scaffold significantly. This could be attributed to hydroxyapatite crystallites in collagen fibrils which restricted the deformation of the collagen fibril network, and the load transfer of the collagen to the higher modulus mineral component of the composite.     

Characterization of a Novel Subtilisin-like Protease Myroicolsin from Deep Sea Bacterium Myroides profundi D25 and Molecular Insight into Its Collagenolytic Mechanism

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal β-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.     

Fibrils of different collagen types containing immobilised proteoglycans (PGs) as coatings: characterisation and influence on osteoblast behaviour

Collagen, the main organic component of bone, is used as a coating on titanium implants and as a scaffold material in bone tissue engineering. Surface modifications of titanium which promote osteoblast adhesion, proliferation and synthesis of collagen by osteoblasts are desirable. One biomimetic approach is the coating of titanium with collagen in fibrillar form. Other organic components of bone may be bound to fibrils and exert additional effects. In this study, the collagen types I-III were compared regarding their ability to bind the proteoglycans decorin and biglycan, which are found in bone. More collagen was bound to collagen II fibrils than to those of types I and III. Therefore, titanium surfaces were coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the effect of the proteoglycans on human primary osteoblast behaviour. In addition, the growth factor TGF-beta1 was adsorbed onto surfaces coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the influence of decorin and biglycan on the effect of TGF-beta1 on osteoblasts. Fibril-bound biglycan and decorin influence primary osteoblast behaviour by themselves. The presence of substrate-bound biglycan or decorin influences the effect of TGF-beta1. These results may be important when designing collagen-based coatings or scaffolds for tissue engineering, including those loaded with growth factors.     

Designed triple-helical peptides as tools for collagen biochemistry and matrix engineering

Collagens, characterized by a unique triple-helical structure, are the predominant component of extracellular matrices (ECMs) existing in all multicellular animals. Collagens not only maintain structural integrity of tissues and organs, but also regulate a number of biological events, including cell attachment, migration and differentiation, tissue regeneration and animal development. The specific functions of collagens are generally triggered by specific interactions of collagen-binding molecules (membrane receptors, soluble factors and other ECM components) with certain structures displayed on the collagen triple helices. Thus, synthetic triple-helical peptides that mimic the structure of native collagens have been used to investigate the individual collagen-protein interactions, as well as collagen structure and stability. The first part of this article illustrates the design of various collagen-mimetic peptides and their recent applications in matrix biology. Collagen is also acknowledged as one of the most promising biomaterials in regenerative medicine and tissue engineering. However, the use of animal-derived collagens in human could put the recipients at risks of pathogen transmission or allergic reactions. Hence, the production of safe artificial collagen surrogates is currently of considerable interest. The latter part of this article reviews recent attempts to develop artificial collagens as novel biomaterials.     

正常椎间盘胶原的研究

腰椎间盘突出症是引起腰腿痛常见的原因。胶原作为椎间盘结构的主要成分,构成椎间盘的纤维框架,其类型与分布直接决定着椎间盘结构的强度和功能的稳定。本文利用溴化氰消化椎间盘胶原产生多肽,借助于梯度层析。SDS-PAGE及光密度定量扫描等对正常人椎间盘胶原进行了研究。结果表明:正常人椎间盘含Ⅰ型及Ⅱ型两种胶原,它们的分布呈明显而特征性的移行性变化:纤维环外层边缘以Ⅰ型胶原为主(83％),由外向内Ⅰ型胶原逐渐移行为Ⅱ型胶原,靠近髓核处以Ⅱ型胶原为主(72％);髓核中心含有Ⅱ型胶原。此为椎间盘的一个结构特性,以满足椎间盘的特殊功能的需要。     

Physical and chemical modifications of collagen gels: impact on diffusion

The extracellular matrix (ECM) represents a major barrier for delivery of therapeutic drugs, and the transport is determined by the ECM composition, structure, and distribution. Because of the high interstitial fluid pressure in tumors, diffusion becomes the main transport mechanism through ECM. The purpose of this work was to study the impact of the structure of the collagen network on diffusion, by studying to what extent the orientation and chemical modification of the collagen network influenced diffusion. Collagen gels with a concentration of 0.2-2.0% that is comparable with the amount of collagen in the tumor ECM were used as a model system for ECM. Collagen gels were aligned in a low-strength magnetic field and geometrical confinement, and chemically modified by adding decorin or hyaluronan. Diffusion of dextran 2-MDa molecules in the collagen gels was measured using fluorescence recovery after photobleaching. Alignment of the collagen fibers in our gels was found to have no impact on the diffusion coefficient. Adding decorin reduced the diameter of the collagen fibers, but no effect on diffusion was observed. Hyaluronan also reduced the fiber diameter, and high concentration of hyaluronan (2.5 mg/ml) increased the diffusion coefficient. The results indicate that the structure of the collagen network is not a major factor in determining the diffusion through the ECM. Rather, increasing the concentration of collagen was found to reduce the diffusion coefficient. Concentration of the collagen network is more important than the structure in determining the diffusion coefficient.     

Photo-cross-linking of type I collagen gels in the presence of smooth muscle cells: mechanical properties,cell viability,and function

The effectiveness of photomediated cross-linking of type I collagen gels in the presence of rat aortic smooth muscle cells (RASMC) as a method to enhance gel mechanical properties while retaining native collagen triple helical structure and maintaining high cell viability was investigated. Collagen was chemically modified to incorporate an acrylate moiety. Collagen methacrylamide was cast into gels in the presence of a photoinitiator along with RASMC. The gels were cross-linked using visible light irradiation. Neither acrylate modification nor the cross-linking reaction altered collagen triple helical content. The cross-linking reaction, however, moved the denaturation temperature beyond the physiologic range. A twelve-fold increase in shear modulus was observed after cross-linking. Cell viability in the range of 70% (n = 4, p > 0.05) was observed in the photo-cross-linked gels. Moreover the cells were able to contract the cross-linked gel in a manner commensurate with that observed for natural type I collagen. Methacrylate-mediated photo-cross-linking is a facile route to improve mechanical properties of collagen gels in the presence of cells while maintaining high cell viability. This enhances the potential for type I collagen gels to be used as scaffolds for tissue engineering.     

牛Ⅲ型前胶原的提取及抗血清的制备

 在中性pH和蛋白酶抑制剂的存在下,从新生小牛皮肤中提取Ⅲ型前胶原。利用反复盐析法将Ⅰ型胶原和前胶原与Ⅲ型胶原和前胶原分开,再经DEAE纤维素层析分离出Ⅲ型前胶原。 用提取的Ⅲ型前胶原免疫家兔,获得抗血清,用酶联免疫法测定效价达1:10O,000。经与国外标准品鉴定该提取物为Ⅲ型前胶原。     

The occurrence of two types of collagen proalpha-chain in the abalone Haliotis discus muscle.

Acid-soluble collagens were prepared from connective tissues in the abalone Haliotis discus foot and adductor muscles with limited proteolysis using pepsin. Collagen preparation solubilized with 1% pepsin contained two types of alpha-chains which were different in their N-terminal amino acid sequences. Accordingly, two types of full-length cDNAs coding for collagen proalpha-chains were isolated from the foot muscle of the same animal and these proteins were named Hdcols (Haliotis discus collagens) 1alpha and 2alpha. The two N-terminal amino acid sequences of the abalone pepsin-solubilized collagen preparation corresponded to either of the two sequences deduced from the cDNA clones. In addition, several tryptic peptides prepared from the pepsin-solubilized collagen and fractionated by HPLC showed N-terminal amino acid sequences identical to those deduced from the two cDNA clones. Hdcols 1alpha and 2alpha consisted of 1378 and 1439 amino acids, respectively, showing the primary structure typical to those of fibril-forming collagens. The N-terminal propeptides of the two collagen proalpha-chains contained cysteine-rich globular domains. It is of note that Hdcol 1alpha completely lacked a short Gly-X-Y triplet repeat sequence in its propeptide. An unusual structure such as this has never before been reported for any fibril-forming collagen. The main triple-helical domains for both chains consisted of 1014 amino acids, where a supposed glycine residue in the triplet at the 598th position from the N-terminus was replaced by alanine in Hdcol 1alpha and by serine in Hdcol 2alpha. Both proalpha-chains of abalone collagens contained six cysteine residues in the carboxyl-terminal propeptide, lacking two cysteine residues usually found in vertebrate collagens. Northern blot analysis demonstrated that the mRNA levels of Hdcols 1alpha and 2alpha in various tissues including muscles were similar to each other.     

胶原蛋白结构基础上的设计与合成

胶原蛋白在人体组织中的含量十分丰富,它是具有绕轴周期性螺旋(即三重螺旋)结构的纤维状蛋白,它为细胞粘附和大分子的沉积等提供支架结构。随着基因工程技术和生物材料科学的不断发展,以及对如何获得具有特殊功能胶原蛋白研究的不断深入,使其成为第三代生物材料中倍受瞩目的成员之一。基于胶原蛋白结构的分子设计以及合成途径值得探讨。     

Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications

Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at -20 degrees C and lyophilized to obtain a sponge, which could be stored at -80 degrees C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2-3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.     

Regulation of silk material structure by temperature-controlled water vapor annealing

We present a simple and effective method to obtain refined control of the molecular structure of silk biomaterials through physical temperature-controlled water vapor annealing (TCWVA). The silk materials can be prepared with control of crystallinity, from a low content using conditions at 4 °C (α helix dominated silk I structure), to highest content of ～60% crystallinity at 100 °C (β-sheet dominated silk II structure). This new physical approach covers the range of structures previously reported to govern crystallization during the fabrication of silk materials, yet offers a simpler, green chemistry, approach with tight control of reproducibility. The transition kinetics, thermal, mechanical, and biodegradation properties of the silk films prepared at different temperatures were investigated and compared by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), uniaxial tensile studies, and enzymatic degradation studies. The results revealed that this new physical processing method accurately controls structure, in turn providing control of mechanical properties, thermal stability, enzyme degradation rate, and human mesenchymal stem cell interactions. The mechanistic basis for the control is through the temperature-controlled regulation of water vapor to control crystallization. Control of silk structure via TCWVA represents a significant improvement in the fabrication of silk-based biomaterials, where control of structure-property relationships is key to regulating material properties. This new approach to control crystallization also provides an entirely new green approach, avoiding common methods that use organic solvents (methanol, ethanol) or organic acids. The method described here for silk proteins would also be universal for many other structural proteins (and likely other biopolymers), where water controls chain interactions related to material properties.     

Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.