Thermal and Rheological Behavior of Collagen. Chitosan blends

Collagen:chitosan blends in 1:1 ratio were prepared and characterized by Fourier transform infrared spectroscopy, thermal (DSC, TG) and rheological studies. Apparently each material maintains its behavior and addition of chitosan does not denature collagen fibers. The rheological behavior showed that adding chitosan to collagen causes a decrease of storage modulus (G’),viscous loss modulus (G”) and apparent viscosity when measured as a function of frequency. Both anionic and native collagen presented more solid-like behavior than fluid-like viscoelastic behavior. Collagen:chitosan blends exhibits a more fluid-like viscoelastic behavior. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation of ready-to-use, stockable and reconstituted collagen

Collagen is a widely used material in biomedical applications. Although processes that prepare collagen and collagen-based materials that show suitable properties after extraction exist, a ready-to-use, easily stockable, with tailored collagen concentration has not yet been developed. Using rat tail tendons, acid soluble collagen solutions were prepared by two different methods. To improve cell viability of pure collagen films, solutions with physiological pH were also prepared by mixing with NaOH solution. Specimens in the form of thin sheets were then fabricated by solvent evaporation. Next, IR spectroscopy, tensile testing techniques as well as human fibroblast cell morphology and cytotoxicity were used to validate the significant variations in the processes. The results demonstrated that, during the synthesis of collagen stock solution, lyophilization and mechanical blending had little effect on the final properties and therefore offers a method for obtaining solutions with a more homogeneous and modifiable collagen concentration and longer storage time. Neutralizing the stock solution with aqueous NaOH prior to solvent evaporation provided films that had lower mechanical properties but significantly improved biological performance.     

Antimicrobial activity of collagen material with thymol addition for potential application as wound dressing

Recently, special attention has been paid to the development of active wound dressing materials based on biopolymers. Collagen is a natural polymer, which meets the requirements of modern materials for medical applications. However, despite its unique properties, collagen has no antimicrobial activity. In this work thymol was incorporated into collagen films to meet antimicrobial properties of the material. Thymol is a naturally occurring phenolic compound recognized as an antimicrobial agent. Collagen/thymol thin films were obtained through solvent evaporation using collagen solutions containing different amounts of thymol. The structure of the obtained materials was studied using FTIR-ATR spectroscopy. The inhibition ability on the growth of several strains of microorganisms was tested. The standard ISO 22196:2007 was used to define the bactericidal properties of the material. The growth of the following bacteria on the collagen/thymol films was studied: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Enterobacter aerogenes, Candida albicans. The results showed that the growth of Staphylococcus aureus was the most inhibited compared to the other tested strains. Collagen/thymol material is more efficient against pathogens through direct contact compared to the diffusion of thymol from the material. In general, the thymol addition inhibits biofilm formation on the collagen surface.     

Comparative Study of The Yield and Physicochemical Properties of Collagen from Sea Cucumber (Holothuria scabra), Obtained through Dialysis and the Ultrafiltration Membrane

Collagen was extracted from the body wall of sea cucumber (Holothuria scabra) using the pepsin-solubilized collagen method followed by isolation using dialysis and the ultrafiltration membrane. The yield and physicochemical properties of the collagen obtained from both isolation methods, denoted as D-PSC and UF-PSC, were compared. The ultrafiltration method affords a higher yield of collagen (11.39%) than that of the dialysis (5.15%). The isolated collagens have almost the same amino acid composition, while their functional groups, referred to as amide A, B, I, II, and III bands, were in accordance with commercial collagen, as verified by Fourier Transform Infrared (FT-IR) spectroscopy. The UV-Vis absorption peaks at 240 nm and 220 nm, respectively, indicated that the collagens produced are type-I collagen. The D-PSC showed interconnecting sheet-like fibrils, while the UF-PSC exhibited a flaky structure with flat-sheets arranged very close to each other. The higher yield and comparable physicochemical properties of the collagen obtained by ultrafiltration as compared with dialysis indicate that the membrane process has high potential to be used in large-scale collagen production for food and pharmaceutical applications.     

The influence of UV light on collagen/poly(ethylene glycol) blends

The photodegradation behaviour of the collagen and poly(ethylene glycol) PEG blends has been studied by Fourier transform infrared spectroscopy (FTIR), UV-Vis spectroscopy and viscometry. Surface properties before and after UV irradiation were observed using optical microscope.Collagen and PEG were immiscible and the films obtained from the mixture were fragile with poor mechanical properties. The photochemical stability of the collagen and PEG blend was different from that of the single components. In general collagen/PEG blends are less stable under UV irradiation than pure collagen. The influence of PEG on the photochemical stability of collagen depends on its concentration in the blend. Microscope photographs showed that the surface characteristics of collagen and collagen/PEG blends in film form are not drastically altered after UV irradiation.     

The influence of UV-radiation on hyaluronic acid and its blends with addition of collagen and chitosan

In this paper, the results regarding the influence of UV-irradiation with 254?nm wavelength on the surface and mechanical properties of hyaluronic acid, hyaluronic acid/collagen and hyaluronic acid/collagen/chitosan mixtures are presented. For this study, thin films were prepared by solvent evaporation from solution of HA and mixtures made from HA/Coll and HA/Coll with 30% addition of chitosan. The surface properties of films were investigated by AFM and using contact angle measurements, allowing the calculation of surface free energy and its components. Mechanical properties of films made of biopolymeric blends before and after UV-irradiation have been investigated by mechanical testing machine.     

Behavior of collagen films in presence of structure modifiers at solid–liquid interface

Films of soluble collagen extracted from rat‐tail tendon were studied at the solid–solution interface, and the surface energy of the films was evaluated. The films transferred onto solid substrates using the Langmuir–Blodgett film (LB film) technique were characterized using Fourier transform IR attenuated total reflectance spectroscopy and atomic force microscopy. The properties of the protein in contact with different structure modifiers like basic chromium sulfate (BCS) and formaldehyde (HCHO) were analyzed for the effect of various tanning agents on the protein structure. The thermal properties of the films were studied using differential scanning calorimetry. The results show that the film of collagen treated with BCS exhibits an increase in the peak temperature and enthalpy changes compared to the pure collagen as well as the protein with HCHO. These differences are attributed to the changes in the crosslinks arising from both coordinate–covalent and covalent interactions, respectively. The atomic force micrographs showed an increase in order for the collagen film with BCS compared to the HCHO treated analogue. A similar trend is seen in the surface energy parameters of the protein films on solid surfaces on reacting with BCS and HCHO, suggesting a molecular level ordering process in collagen assemblies. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 3859–3865, 2004     

Photochemical stability of collagen/poly(ethylene oxide) blends

The photochemical stability of the blends of collagen and poly(ethylene oxide) PEO has been studied by Fourier transform infrared spectroscopy (FTIR), UV–vis spectroscopy and viscosimetry. Surface properties before and after UV irradiation were observed using an optical microscope.Collagen and PEO were immiscible in diluted solution and only small interactions between the two components in the solid state were observed. New materials based on the blending of collagen and PEO that we obtained have a different photochemical stability than those of single components. In general, collagen/PEO blends are less stable under UV irradiation than pure collagen. The influence of PEO on the photochemical stability of collagen depends on the concentration of this polymer in the blend. Microscopic photographs show that the surface characteristics of thin films of collagen/PEO blends are not drastically altered after UV irradiation.     

The review of versatile application of collagen

This review reports recent advances in the versatile application of collagen. Collagen materials have attracted great attention because they exhibit properties required in cosmetic preparations, in the biomedical field, and in the tanning industry leading to leather production. Herein, the structure and application of collagen are discussed in general, and detailed examples are also drawn from scientific literature and practical work. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and Characterization of Fluorescent Silica Nanoparticles Functionalized with Collagens of Variable Lengths

Collagen, the most abundant protein in human body, has been widely used as an excellent natural material for diverse biomedical applications due to its superior properties such as ample biological interaction sites, minimal immunogenicity and high biocompatibility. Collagens of different lengths are produced by recombinant technology and utilized to functionalize fluorescent silica nanoparticles (FNPs). The collagen‐functionalized FNPs display mono‐disperse distribution, but their sizes are dependent on the length of collagen. These modified FNPs all show nice fluorescence profile as well as low cytotoxicity, suggesting promising applications in bioimaging. We have demonstrated that various types of collagen, conveniently produced by recombinant technology, can be used to modify silica nanoparticles with nice characteristics such as mono‐dispersion, non‐interference in fluorescence and low toxicity. It may endow fluorescent silica nanoparticles with broad biological applications.     

Effect of Collagen Types,Bacterial Strains and Storage Duration on the Quality of Probiotic Fermented Sheep’s Milk

Collagen has become popular in dietary supplements, beverages and sports nutrition products. Therefore, the aim of this study was to evaluate the possibility of using various doses of collagen and collagen hydrolysate to produce probiotic sheep’s milk fermented with Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus. The effects of storage time, type and dose of collagen, and different probiotic bacteria on the physicochemical, organoleptic and microbiological properties of fermented sheep’s milk at 1 and 21 days of refrigerated storage were investigated. The addition of collagen to sheep’s milk increased the pH value after fermentation and reduced the lactic acid contents of fermented milk compared to control samples. After fermentation, the number of probiotic bacteria cells was higher than 8 log cfu g⁻¹. In sheep’s milk fermented by L. acidophilus and L. casei, good survival of bacteria during storage was observed, and there was no effect of collagen dose on the growth and survival of both strains. The addition of collagen, both in the form of hydrolysate and bovine collagen, resulted in darkening of the color of the milk and increased the sweet taste intensity of the fermented sheep’s milk. However, the addition of hydrolysate was effective in reducing syneresis in each milk sample compared to its control counterpart.     

Synthesis of Fibrin-Type I Collagen Biomaterials via an Acidic Gel

Fibrin-Type I collagen composite gels have been widely studied as biomaterials, in which both networks are usually formed simultaneously at a neutral pH. Here, we describe a new protocol in which mixed concentrated solutions of collagen and fibrinogen were first incubated at acidic pH to induce fibrinogen gel formation, followed by a pH change to neutral inducing collagen fiber formation. Thrombin was then added to form fibrin-collagen networks. Using this protocol, mixed gels containing 20 mg.mL⁻¹ fibrin and up to 10 mg.mL⁻¹ collagen could be prepared. Macroscopic observations evidenced that increasing the content of collagen increases the turbidity of the gels and decreases their shrinkage during the fibrinogen-to-fibrin conversion. The presence of collagen had a minor influence on the rheological properties of the gels. Electron microscopy allowed for observation of collagen fibers within the fibrin network. 2D cultures of C2C12 myoblasts on mixed gels revealed that the presence of collagen favors proliferation and local alignment of the cells. However, it interferes with cell differentiation and myotube formation, suggesting that further control of in-gel collagen self-assembly is required to elaborate fully functional biomaterials.     

Mineralization of Phosphorylated Fish Skin Collagen/Mangosteen Scaffolds as Potential Materials for Bone Tissue Regeneration

In this study, a potential hard tissue substitute was mimicked using collagen/mangosteen porous scaffolds. Collagen was extracted from Tilapia fish skin and mangosteen from the waste peel of the respective fruit. Sodium trimetaphosphate was used for the phosphorylation of these scaffolds to improve the nucleation sites for the mineralization process. Phosphate groups were incorporated in the collagen structure as confirmed by their attenuated total reflection Fourier transform infrared (ATR-FTIR) bands. The phosphorylation and mangosteen addition increased the thermal stability of the collagen triple helix structure, as demonstrated by differential scanning calorimetry (DSC) and thermogravimetry (TGA) characterizations. Mineralization was successfully achieved, and the presence of calcium phosphate was visualized by scanning electron microscopy (SEM). Nevertheless, the porous structure was maintained, which is an essential characteristic for the desired application. The deposited mineral was amorphous calcium phosphate, as confirmed by energy dispersive X-ray spectroscopy (EDX) results.     

Controlling coupling reaction of EDC and NHS for preparation of collagen gels using ethanol/water co-solvents

To control the crosslinking rate of the collagen gel, ethanol/water co-solvent was adopted for the reaction solvent for the collagen microfibril crosslinking. Collagen gel was prepared by using EDC and NHS as coupling agents. Ethanol did not denaturate the helical structure of the collagen and prevented the hydrolysis of EDC, but showed the protonation of carboxylate anions. In order to control the intra- and interhelical crosslink of the collagen triple helix, variations of the mole ratio of carboxyl group/EDC/NHS, and of the ethanol mole concentration were investigated. Increase in the EDC ratio against the carboxyl group increased the crosslinking rate. Furthermore, an increase in the ethanol mole concentration resulted in an increase of the crosslinking rate until ethanol mole concentration was 0.12, but showed gradual decrease as the ethanol mole concentration was further increased. This is because the adsorption of solvent by the collagen gel, protonation of carboxylate anion, and hydrolysis of EDC is at its most optimum condition for the coupling reaction when the ethanol mole concentration is 0.12. The re-crosslinking of the collagen gel showed an increase in the crosslinking rate, but did not show further increase when the coupling reaction was executed for the third time. This implied that the highest possible crosslinking rate for the intra- and interhelical is approximately 60% when EDC/NHS is used.     

Preparation and modification of collagen-based porous scaffold for tissue engineering

In the effort to generate cartilage tissues using mesenchymal stem cells, porous scaffolds with prescribed biomechanical properties were prepared. Scaffolds with interconnected pores were prepared via lyophilisation of frozen hydrogels made from collagen modified with chitosan nanofibres, hyaluronic acid, copolymers based on poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), and itaconic acid (ITA), and hydroxyapatite nanoparticles. The modified collagen compositions were cross-linked using N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) combined with N-hydroxysuccinimide (NHS) in water solution. Basic physicochemical and mechanical properties were measured and an attempt to relate these properties to the molecular and supermolecular structure of the modified collagen compositions was carried out. Scaffolds containing hydrophilic chitosan nanofibres showed the highest swelling ratio (SR = 20–25) of all the materials investigated, while collagen modified with an amphiphilic PLGA-PEG-PLGA copolymer or functionalised with ITA exhibited the lowest swelling ratio (SR = 5–8). The best resistance to hydrolytic degradation was obtained for hydroxyapatite containing scaffolds. On the other hand, the fastest degradation rate was observed for synthetic copolymer-containing scaffolds. The results showed that the addition of hydroxyapatite or hyaluronic acid to the collagen matrix increases the rigidity in comparison to the collagen-chitosan scaffold. Collagen scaffold modified with hyaluronic acid presented reduced deformation at break while the presence of hydroxypatatite enhanced the scaffold deformation under tensile loading. The tensile elastic modulus of chitosan nanofibre collagen scaffold was the lowest but closest to the articular cartilage; however, the strength and deformation to failure increased up to 200 %. Presented at the 1st Bratislava Young Polymer Scientists Workshop, Bratislava, 20–23 August 2007.     

Quantitative measurement of collagen methylation by capillary electrophoresis

Collagen methylation has been exploited in various applications involving living cells. We have observed correlation between the collagen methylation with the rate of cell proliferation in three-dimensional (3-D) microenvironment. To quantify the degree of collagen methylation, we have developed a capillary zone electrophoresis method. Using a polyvinyl alcohol-coated fused-silica capillary and UV detection at 200 nm, we have optimized pH and separated the native collagen into three major bands in phosphate buffer (50 mM, pH 2.5) with 0.05% hydroxypropylmethylcellulose. Under these conditions, the methylated collagens were separated into four major bands, which changed with different methylation reaction conditions. We propose an index to quantify the degree of collagen methylation that also correlates with their effects on cell proliferation.     

Dynameric Collagen Self-Healing Membranes with High Mechanical Strength for Effective Cell Growth Applications

The fabrication of biocompatible adaptive materials with high stiffness and self-healing properties for medical applications is a challenging endeavor. Collagen is a major extracellular matrix component acting as a substrate for cell adhesion and migration. Dynamers are constitutional polymers whose monomeric components are linked through reversible bonds, able to modify their constitution through reversible exchange of their components. In the current work, we demonstrate that the rational combination of collagen and dynameric networks connected with reversible covalent imine bonds is a very important and previously unreported strategy to provide biocompatible membranes with self-healing ability and excellent mechanical strength. The key challenge in the construction of such membranes is the required adaptive interaction between collagen chains and the dynamic cross-linkers, preventing the formation of defects. For example, by varying structure and molecular lengths of the dynamers, the tensile strength of the dynameric membranes reach over 80 MPa, more than 400 % higher than that observed for the reference collagen membrane, and the highest value for break strain found, was 19 %. The self-healing properties were observed when reconnecting two membrane pieces or even from crushed status of the membranes. Moreover, both MTT assay and confocal laser scanning microscopy method demonstrated the good biocompatibility of the collagen membranes, leaving more than 90 % viability for NIH 3T3 cells after 24 h co-culture.     

胶原蛋白-葡甘聚糖-软骨素共混膜的结构表征和物理性能研究

用溶液共混法制备了胶原蛋白-葡甘聚糖-软骨素共混膜。并进行了FPIR，XRD，SFM分析及透光率抗张强度、断裂伸长率、吸水率、透汽性、吸附性和渗透性测试。结果表明：共混膜中胶原蛋白、葡甘聚糖及软骨素之间存在着强烈的相互作用和良好的相容性，三者共混明显改善了几种材料及二元共混膜的性能。该共混膜可望用作生物医用材料。     

Thermal and mechanical properties of UV irradiated collagen/chitosan thin films

The thermal and mechanical properties of collagen/chitosan blends before and after UV irradiation have been investigated using thermal analysis and mechanical (Instron) techniques. Comparisons were made with the thermal and mechanical properties of both collagen and chitosan films. Air-dried collagen, chitosan and collagen/chitosan films were exposed to UV irradiation (wavelength 254 nm) for different time intervals. Thermal properties of collagen/chitosan blends depend on the composition of the blend and are not significantly altered by UV irradiation.Mechanical properties such as ultimate tensile strength and ultimate percentage of elongation were much better for collagen films than for collagen/chitosan films. The results have shown that the mechanical properties of the blends were greatly affected by the duration of UV irradiation. Ultimate tensile strength and ultimate percentage elongation decreased after UV irradiation of the blend. Increasing UV irradiation leads to an increase in Young's modulus of the collagen/chitosan blend.     

Niflumic acid-collagen delivery systems used as anti-inflammatory drugs and analgesics in dentistry

Collagen sponges are known to be safe and well-characterized supports for drug delivery systems. The aim of this study was to prepare, characterize and test drug delivery systems that contain collagen as support and niflumic acid as a drug. Type-I collagen and niflumic acid gels were cross-linked with different concentrations of glutaraldehyde and then freeze-dried in order to obtain collagen matrices (spongious form). The physical-chemical properties were assessed by infrared spectroscopy (FTIR) and morphological properties were evaluated by water absorption. Niflumic acid release from cross-linked collagen spongious forms was also investigated and the kinetic mechanism was discussed.