Calf Skin Collagen Research Articles

Preparation and Characterization of Novel Multifunctional Wound Dressing by Near-Field Direct-Writing Electrospinning and Its Application.

Near-field direct-writing electrospinning technology can be used to produce ordered micro/nanofiber membrane dressings. The application of this technology can simply realize the control of dressing porosity, compound different functional substances, and adjust their distribution, thus improving the defects of common dressings such as insufficient breathability, poor moisture retention performance, and single function. Herein, a novel multifunctional wound dressing was prepared to utilize near-field direct-writing electrospinning technology, in which calf skin collagen type I (CSC-I) and polycaprolactone (PCL) were used as the composite matrix, Hexafluoroisopropanol (HFIP) as the solvent, and erythromycin (ERY) as an anti-infective drug component. The results show that the micro/nanofiber membranes prepared by near-field direct-writing electrospinning technology can all present a complete mesh structure, excellent thermal stability, and good moisturizing properties. Moreover, the composite fiber membrane loaded with ERY not only had obvious antibacterial properties against E. coli and S. thermophilus but also a better slow-release function of drugs (it is rare to have both in traditional wound dressings). Therefore, this experimental design can provide relevant theories and an experimental foundation for preparing a new type of medical dressing with drug loading and has good guiding significance for the application and promotion of near-field direct-writing electrospinning in medical dressings.

Physicochemical and biological properties of collagens obtained from tuna tendon by using the ultrasound-assisted extraction

This study aimed to maximize the utilization of tuna tendons, which are by-products of the tuna canning process, to obtain collagen through ultrasound-assisted extraction. Ultrasound was used to assist the extraction of vinegar-, acid-, and pepsin-soluble collagen from tuna tendons, denoted as VUTC, AUTC, and PUTC, respectively. The yield ranged from 10% to 15%, and the collagen solubility was 0.5–0.7 mg protein/mg collagen. The color differed among the collagen samples. Tuna tendon collagens comprised γ-, β-, α1-, and α2-chains, in accordance with type I calf skin collagen. Regarding the amino acid composition, there were high amounts of glycine, proline, glutamic acid, alanine, hydroxyproline, and aspartic acid. Salt concentration and pH effects on the solubility of collagen were evaluated. The samples were less soluble at higher salt concentrations and had good solubility at low pH. Fourier transform infrared spectroscopy showed the presence of a triple helix. Fractional viscosity and differential scanning calorimetry (DSC) analysis indicated a degradation temperature of 30–36 °C and 167–171 °C, respectively. Finally, tuna tendon collagen had antioxidant and immune-enhancing activities and did not exert cytotoxicity.

Türkiye Kıyılarında Dağılım Gösteren İstilacı Denizkestanesinden (Diadema setosum L., 1778) Kolajen Ekstraksiyonu ve Karakterizasyonu

In the present study, collagen was obtained from tissues of the invasive sea urchin (Diadema setosum) rapidly spread in the Mediterranean Sea. As a result, the yield of collagen isolated from sea urchin was determined to be 23.78±1.33% (dry weight). As a result of SDS-Page analysis, it was determined that it contains (α1)2α2, (Molecular Weight (MA) 124, 114 kDa) and ß chains (MA 245 kDa) similar to calfskin collagen. In the amino acid analysis of collagen, it was determined that the imino acid (proline+hydroxyproline) content was 196.1 residues/1000 residues. The functional bands of amide A (3301 cm-1), B (2924 cm-1), I (1643 cm-1), II (1550 cm-1), and III (1242 cm-1) functional bands were visualized in the FTIR spectrum. These results were like other collagen sources. Invasive sea urchin was used as a source of collagen for the first time in the present study. An alternative source of collagen to mammalian collagen, which is used commercially in many industries such as biomedicine, food and cosmetics, was isolated for the first time from D. setosum. It was proposed that marine collagen can be used as an alternative source of collagen and a functional component in areas including food, cosmetics, and pharmaceutical industries.

Deepening in the understanding of the role of collagen subunits on the differential molecular arrangement of P. glauca and M. merluccius marine collagens

Decades of extensive efforts on marine collagen extraction and characterization allowed to recognize the unique and excellent characteristics of marine collagen offering advantages over that obtained from terrestrial sources. However, not all marine collagens have the same biochemical characteristics; understanding those at molecular and supramolecular level, is crucial for optimal design of applications. One relevant aspect of collagen characterization is the analysis of its different subunits (α-chains) and their intermolecular cross-links (β- and γ-components), which ultimately determine the specific functions of a particular collagen. Collagens from a teleost and an elasmobranch species were analyzed to understand the influence of their subunit composition and intermolecular crosslinking pattern on their different physicochemical behaviour. For comparative purposes a commercial mammal collagen was included in the study. Although electrophoretic profiles showed the typical composition of type I collagen for hake, blue shark and calf collagen, molar ratios of their α-chains were different indicating a different degree of dimerization of their α2-chains with implications in the presence of a different crosslinking degree pattern. Electrophoresis, amino acid composition, hydrophobicity (RP-HPLC) and molecular weight analysis (GPC-HPLC) results, besides a peptide mapping and an antioxidant activity study of the resultant peptides, would help to understand the role of different subunit collagen composition and different crosslinking pattern in the conformation of a differential quaternary supramolecular structure within different species and its biofunctional implications. The experiments developed would allow to progress in the valorization potential of fish discards and byproducts to explore commercial uses of collagens from marine origin.

Nonlinear time-dependent mechanical behavior of mammalian collagen fibrils.

The viscoelastic mechanical behavior of collagenous tissues has been studied extensively at the macroscale, yet a thorough quantitative understanding of the time-dependent mechanics of the basic building blocks of tissues, the collagen fibrils, is still missing. In order to address this knowledge gap, stress relaxation and creep tests at various stress (5-35 MPa) and strain (5-20%) levels were performed with individual collagen fibrils (average diameter of fully hydrated fibrils: 253±21 nm) in phosphate buffered saline (PBS). The experimental results showed that the time-dependent mechanical behavior of fully hydrated individual collagen fibrils reconstituted from Type I calf skin collagen, is described by strain-dependent stress relaxation and stress-dependent creep functions in both the heel-toe and the linear regimes of deformation in monotonic stress-strain curves. The adaptive quasilinear viscoelastic (QLV) model, originally developed to capture the nonlinear viscoelastic response of collagenous tissues, provided a very good description of the nonlinear stress relaxation and creep behavior of the collagen fibrils. On the other hand, the nonlinear superposition (NSP) model fitted well the creep but not the stress relaxation data. The time constants and rates extracted from the adaptive QLV and the NSP models, respectively, pointed to a faster rate for stress relaxation than creep. This nonlinear viscoelastic behavior of individual collagen fibrils agrees with prior studies of macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. STATEMENT OF SIGNIFICANCE: Pure stress relaxation and creep experiments were conducted for the first time with fully hydrated individual collagen fibrils. It is shown that collagen nanofibrils have a nonlinear time-dependent behavior which agrees with prior studies on macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. This new insight into the non-linear viscoelastic behavior of the building blocks of mammalian collagenous tissues may serve as the foundation for improved macroscale tissue models that capture the mechanical behavior across length scales.

Biochemical and Microstructural Properties of Lizardfish (Saurida tumbil) Scale Collagen Extracted with Various Organic Acids.

The purpose of this research was to extract collagen from the scales of lizardfish (Saurida tumbil) using various acids. Acetic acid-extracted collagen (AScC) produced a higher yield (1.8 mg/g) than lactic acid-extracted collagen (LScC) and citric acid-extracted collagen (CScC) although not significantly different (p > 0.05). All extracted collagens were categorized as type I collagens with the presence of alpha chains (α1 and α2) based on the SDS-PAGE profiles. The triple-helical structure of the collagen was maintained in the AScC, LScC, and CScC as confirmed by the FTIR spectra. The UV-vis and X-ray diffraction spectra observed in all collagens were in agreement with previous work on fish scale and calfskin (commercial) collagens. The thermal stability of AScC (Tmax = 31.61 °C) was greater than LScC (Tmax = 30.86 °C) and CScC (Tmax = 30.88 °C). The microstructure of acid-extracted collagens was characterized as complex, fibrous, and multilayered, with irregular sheet-like structures. All samples were highly soluble in acidic pH (1.0–4.0) and in low concentrations of NaCl (0–20 g/L). In conclusion, the lizardfish scale collagen, particularly AScC, may be used as an alternative to terrestrial animal collagen.

Preparation and Characterization of Thermally Stable Collagens from the Scales of Lizardfish (Synodus macrops).

Marine collagen is gaining vast interest because of its high biocompatibility and lack of religious and social restrictions compared with collagen from terrestrial sources. In this study, lizardfish (Synodus macrops) scales were used to isolate acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). Both ASC and PSC were identified as type I collagen with intact triple-helix structures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopy. The ASC and PSC had high amino acids of 237 residues/1000 residues and 236 residues/1000 residues, respectively. Thus, the maximum transition temperature (Tmax) of ASC (43.2 °C) was higher than that of PSC (42.5 °C). Interestingly, the Tmax of both ASC and PSC was higher than that of rat tail collagen (39.4 °C) and calf skin collagen (35.0 °C), the terrestrial collagen. Solubility tests showed that both ASC and PSC exhibited high solubility in the acidic pH ranges. ASC was less susceptible to the “salting out” effect compared with PSC. Both collagen types were nontoxic to HaCaT and MC3T3-E1 cells, and ASC was associated with a higher cell viability than PSC. These results indicated that ASC from lizardfish scales could be an alternative to terrestrial sources of collagen, with potential for biomedical applications.

Investigation of the solubility and dispersion degree of calf skin collagen in ionic liquids

The dissolution of collagen in ionic liquids (ILs) was highly dependent on the polarity of ILs, which was influenced by their sorts and concentrations. Herein, the solubility and dispersion degree of collagen in two sorts of ILs, namely 1-ethyl-methylimidazolium tetrafluoroborate ([EMIM][BF4]) with low polarity and 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]) with high polarity in a concentration range from 10% to 70% at 10 °C were investigated. When 150 mg of collagen was added to 30 mg of ILs, the minimum soluble collagen concentration was 0.02 mg/mL in 70% [EMIM][BF4] with lowest polarity and the maximum was 3.57 mg/mL in 70% [EMIM][Ac] with highest polarity, which indicates that soluble collagen and insoluble collagen fibers were both present. For insoluble collagens, differential scanning calorimetry showed that the thermal-stability was weakened when increasing the ILs concentration and polarity, and the fiber arrangement was looser with a more uniform lyophilized structure, observed by atomic force microscopy and scanning electron microscopy. For soluble collagens, electrophoresis patterns and Fourier transform infrared spectroscopy showed that no polypeptide chain degradation occurred during dissolution, but the thermal denaturation temperature decreased by 0.26 °C~ 7.63 °C with the increase of ILs concentrations, measured by ultra-sensitive differential scanning calorimetry. Moreover, the aggregation of collagen molecules was reduced when ILs polarity was increased as determined by fluorescence measurements and dynamic light scattering, which resulted in an increased loose fiber arrangement observed by atomic force microscopy. If the structural integrity of collagen needs to be retained, then the ILs sorts and concentrations should be considered.Graphical abstract

Comparison of Physicochemical Characteristics and Fibril Formation Ability of Collagens Extracted from the Skin of Farmed River Puffer (Takifugu obscurus) and Tiger Puffer (Takifugu rubripes).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5–29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.

Comparative Studies on the Physico-chemical Characteristics of Bio-materials with Collagen from Calf and Fish Skins from Black Sea

Collagen hydrolysers are three-dimensional polymeric materials with limited cross-linking and high hydrophilicity, having multiple medical applications. The most used collagen is the one extracted from bovine skin, which is now the industrial source of collagen. Due to the outbreak of some threatening diseases such as BSE, transmissible spongiform encephalopathy, foot-and-mouth disease, researchers have sought a safer alternative to collagen. This was the marine resource, which offered multiple opportunities to capitalize on clean sea-water raw material. This paper presents a comparative study of the physico-chemical properties of collagen hydrogels derived from collagen obtained from calf and skin from the Black Sea. Physico-chemical and spectrophotometric analyzes were performed to determine the structure. Studies have been conducted to analyze rheological behavior, antioxidant activity and antimicrobial activity. The total antioxidant capacity (ACL) is higher for collagen mixtures with 40% ethyl alcohol and shows higher values for fish collagen compared to calf collagen. Antimicrobial analysis shows that all collagen hydrogels show antimicrobial activity, both gram-positive (Staphylococcus aureus ATCC 6538P) and gram-negative (Escherichia coli ATCC 10536), which increases with increasing collagen concentrations.

Electrodialysis Extraction of Pufferfish Skin (Takifugu flavidus): A Promising Source of Collagen.

Collagen is widely used in drugs, biomaterials, foods, and cosmetics. By-products of the fishing industry are rich sources of collagen, which can be used as an alternative to collagen traditionally harvested from land mammals. However, commercial applications of fish-based collagen are limited by the low efficiency, low productivity, and low sustainability of the extraction process. This study applied a new technique (electrodialysis) for the extraction of Takifugu flavidus skin collagen. We found electrodialysis to have better economic and environmental outcomes than traditional dialysis as it significantly reduced the purification time and wastewater (~95%) while maintaining high extraction yield (67.3 ± 1.3 g/100 g dry weight, p < 0.05). SDS-PAGE, amino acid composition analysis, and spectrophotometric characterization indicated that electrodialysis treatment retained the physicochemical properties of T. flavidus collagen. Heavy metals and tetrodotoxin analyses indicated the safety of T. flavidus collagen. Notably, the collagen had similar thermal stability to calf skin collagen, with the maximum transition temperature and denaturation temperature of 41.8 ± 0.35 and 28.4 ± 2.5 °C, respectively. All evidence suggests that electrodialysis is a promising technique for extracting collagen in the fishing industry and that T. flavidus skin collagen could serve as an alternative source of collagen to meet the increasing demand from consumers.

The Interaction(s) between Calf-Skin Hyaluronic Acid (Hyaluronan) and Dermal Type I Calf-Skin Collagen under 254 nm UV Radiation: Ability of Hyaluronan to Alter Qualitative and Quantitative Dimerization of Collagen Tyrosine Residues

The extracellular matrix (ECM) is the non-cellular component present within all tissues and organs, providing not only essential physical scaffolding for the cellular constituents and initiating crucial biochemical and biomechanical cues, required for tissue morphogenesis, differentiation and homeostasis. Roughly divided into two groups, these are 1) the main fibrous ECM proteins: collagens, elastins, fibronectins and laminins. 2) Classification of proteoglycans (PGs) is based on their location and binding. Although many different molecular interactions are possible, they depend on the cells’ condition (i.e. “Normal”, Aged, Wounded/Fibrotic, and cancerous). There is little or no data that addresses the influence of the surrounding ECM on dityrosine formation. As a simpler model, we have replaced total PG with hyaluronan (HA) and have used purified calf-skin collagen tyrosine, which forms dityrosine (A2) under 254 nm UV in buffered solution and (near) physiological temperatures. Our results reveal a complicated temperature dependence involving factors relating to collagen HA structure, and collagen’s photochemical activation parameters.

Isolation and characterization of acid-soluble bluefin tuna (Thunnus orientalis) skin collagen

In this study, we isolated and characterized the acid-soluble skin collagen of Pacific bluefin tuna (PBT, Thunnus orientalis). The PBT skin collagen was composed of two α chains (α1 and α2) and one β chain. The denaturation temperature of PBT collagen was low although it was rich in proline and hydroxyproline. The primary structure of PBT skin collagen was almost identical to that of calf and salmon skin collagen; however, it differed with respect to the epitope recognition of the antibody against salmon type I collagen. These results suggest that the primary structure of skin collagen was highly conserved among animal species, although partial sequences that included the epitope structure differed among collagens.

Isolation and characterisation of acid- and pepsin-soluble collagen from the skin of Cervus korean TEMMINCK var. mantchuricus Swinhoe

Acid-soluble collagen and pepsin-soluble collagen were extracted from the skin of deer, Cervus korean TEMMINCK var. mantchuricus Swinhoe. The two types of collagen were then characterised using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, amino acid composition analysis, peptide hydrolysis patterns, thermal denaturation temperature, differential scanning calorimetry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance imaging. The yield of pepsin-soluble collagen (9.62%) was greater than that of acid-soluble collagen (2.24%), but both types of collagen showed similar electrophoretic patterns with each other and with calf skin collagen. The peptide hydrolysis pattern results suggested that calf skin collagen and pepsin-soluble collagen from deer skin may be similar in terms of their primary structure. The thermal denaturation temperature of acid-soluble collagen and pepsin-soluble collagen were 36.67°C and 36.44°C, respectively, and their melting temperatures were 110°C and 120°C, respectively, which suggest high thermal stability. Fourier transform infrared showed a triple helical structure and nuclear magnetic resonance confirmed the presence of ‘hydration’ water. These results provide a basis for large-scale production and further application as alternatives to other mammalian collagens.

Extraction and characterization of collagen from Antarctic and Sub-Antarctic squid and its potential application in hybrid scaffolds for tissue engineering

Collagen is the most abundant protein found in mammals and it exhibits a low immunogenicity, high biocompatibility and biodegradability when compared with others natural polymers. For this reason, it has been explored for the development of biologically instructive biomaterials with applications for tissue substitution and regeneration. Marine origin collagen has been pursued as an alternative to the more common bovine and porcine origins. This study focused on squid (Teuthoidea: Cephalopoda), particularly the Antarctic squid Kondakovia longimana and the Sub-Antarctic squid Illex argentinus as potential collagen sources. In this study, collagen has been isolated from the skins of the squids using acid-based and pepsin-based protocols, with the higher yield being obtained from I. argentinus in the presence of pepsin. The produced collagen has been characterized in terms of physicochemical properties, evidencing an amino acid profile similar to the one of calf collagen, but exhibiting a less preserved structure, with hydrolyzed portions and a lower melting temperature. Pepsin-soluble collagen isolated from I. argentinus was selected for further evaluation of biomedical potential, exploring its incorporation on poly-ε-caprolactone (PCL) 3D printed scaffolds for the development of hybrid scaffolds for tissue engineering, exhibiting hierarchical features.

Characterisation of acid-soluble and pepsin-solubilised collagen from frog (Rana nigromaculata) skin

As the first vertebrates connecting water and land, frogs must have evolved certain specialisations. To find the difference among the collagens from amphibian, fish and mammal animals, acid-soluble collagen (ASC) and pepsin-solubilised collagen (PSC) from frog skin were isolated and characterised. The yield of PSC (19.59%, dry weight) was higher than that of ASC (1.83%, dry weight). The hydroxyproline-to-protein ratio of frog skin was 8.37%, which was higher than that of carp skin ASC (7.83%) but significantly lower than that of calf skin collagen (10.16%), which was in accordance with the living environment of frog. The denaturation temperature of frog skin collagens was approximately 31.5°C. The SDS-PAGE electrophoresis revealed that ASC and PSC were type I collagens. Fourier transform infrared spectroscopy proved that the ASC and PSC retained their helical structures. The results indicated the properties of frog skin collagen were close to those of skin collagen from freshwater fish. The frog skin collagens can potentially be used in biomaterial and other fields.

Effect of temperature on photochemical and thermal changes in calf skin collagen solutions at physiological pH

Effect of temperature on photochemical and thermal changes in calf skin collagen solutions at physiological pH

Mechanical Deformation of Cultured Cells with Hydrogels.

Polyacrylamide hydrogels can be used to culture cells in a range of stiffness that can closer mimic physiological environments. Changes in environmental stiffness have been documented in conditions such as fibrosis, cancer, and aging. In this chapter, we describe a method in which we pour gels directly into multiwell plates using a plastic support that covalently binds to the polymerizing hydrogel. The hydrogel is then crosslinked to calfskin collagen using a crosslinker. The result is a thick hydrogel, scalable to any size plate, which covers the entire surface of the well with no edge effects. The gels can be routinely assembled and are easily reproducible. These scaffolds are used as in vitro models to study fibroblast reaction to variation in environmental stiffness.

Effect of surfactants on surface activity and rheological properties of type I collagen at air/water interface

We describe the effect of three synthetic surfactants (anionic – sodium dodecyl sulfate (SDS), cationic – cetyltrimethylammonium bromide (CTAB) and nonionic – Triton X-100 (TX-100)) on surface properties of the type I calf skin collagen at the air/water interface in acidic solutions (pH 1.8). The protein concentration was fixed at 5×10−6molL−1 and the surfactant concentration was varied in the range 5×10−6molL−1–1×10−4molL−1, producing the protein/surfactant mixtures with molar ratios of 1:1, 1:2, 1:5, 1:10 and 1:20. An Axisymmetric Drop Shape Analysis (ADSA) method was used to determine the dynamic surface tension and surface dilatational moduli of the mixed adsorption layers. Two spectroscopic techniques: UV–vis spectroscopy and fluorimetry allowed us to determine the effect of the surfactants on the protein structure. The thermodynamic characteristic of the mixtures was studied using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC).Modification of the collagen structure by SDS at low surfactant/protein ratios has a positive effect on the mixture’s surface activity with only minor deterioration of the rheological properties of the adsorbed layers. The collagen/CTAB mixtures do not show that pronounced improvement in surface activity, while rheological properties are significantly deteriorated. The mixtures with non-ionic TX-100 do not show any synergistic effects in surface activity.

Mechanical property and biocompatibility of co-precipitated nano hydroxyapatite–gelatine composites

Uniform and well dispersed nano hydroxyapatite (HA)–gelatine composites were obtained by co-precipitation of hydroxyapatite and denatured calf skin collagen. The process allowed much higher concentration of hydroxyapatite to be produced over conventional hydrothermal process to improve the productivity. The effect of gelatine on the morphology, mechanical properties, and biocompatibility of hydroxyapatite particles was investigated. Fibroblast cell tests of the consolidated hydroxyapatite–gelatine composites showed that the composites have excellent biocompatibility.