Use Of Collagen Research Articles

Synthesis of Alginate/Collagen Bioink for Bioprinting Respiratory Tissue Models.

Synthesis of bioinks for bioprinting of respiratory tissue requires considerations related to immunogenicity, mechanical properties, printability, and cellular compatibility. Biomaterials can be tailored to provide the appropriate combination of these properties through the synergy of materials with individual pros and cons. Sodium alginate, a water-soluble polymer derived from seaweed, is a cheap yet printable biomaterial with good structural properties; however, it lacks physiological relevance and cell binding sites. Collagen, a common component in the extra cellular matrix of many tissues, is expensive and lacks printability; however, it is highly biocompatible and exhibits sites for cellular binding. This paper presents our study on the synthesis of bioinks from alginate and collagen for use in bioprinting respiratory tissue models. Bioinks were synthesized from 40 mg/mL (4%) alginate and 3 mg/mL (0.3%) collagen in varying ratios (1:0, 4:1, 3:1, 2:1, and 1:1); then examined in terms of rheological properties, printability, compressive, and tensile properties and cellular compatibility. The results illustrate that the ratio of alginate to collagen has a profound impact on bioink performance and that, among the examined ratios, the 3:1 ratio is the most appropriate for use in bioprinting respiratory tissue scaffolds.

Novel polyurethane foams with titanium powder and collagen for medical uses

In this study, we aimed to develop a novel soy-based polyurethane (PU) foam material reinforced with titanium powder and collagen for use in bone tissue engineering. In this context, soy polyol-based PU foams having different (5, 10, 20 wt%) concentrations of recycled titanium powders and hydrolyzed collagen (1 wt%) were prepared and characterized by scanning electron microscopy, water contact angle measurement, and compression tests. Biocompatibility was determined by cytotoxicity analysis. It was observed that PU foams with different titanium and collagen concentrations can have optimized mechanical strength, biocompatibility, chemical composition, and morphology that resemble porous bone texture-like structure. The compressive strength of PU foam increased by 72.6% with the inclusion of 20 wt% titanium powder and slightly decreased by 9.4% with 1 wt% collagen. Cytotoxicity results revealed that PU foam with the synergistic combination of titanium powder and collagen had higher biocompatibility compared to control sample. To our knowledge, this novel biocomposite material, developed the first time in literature by incorporation of medical grade titanium powder and hydrolyzed collagen into soy-based PU matrix, has many advantages such as tunable mechanical strength, biocompatibility, low cost, and easy processability.

Synthesis and structure of the coordination complex of copper ion with collagen

The aim of this work is to synthesize, determine the composition and microstructure of complexes of copper (II) sulfate with collagen for use as a dye for textile material. Coordination complexes of copper (II) sulfate with collagen synthesized at different ratios of the initial substances by the interaction of solutions of different concentrations of copper sulfate and collagen of raw skin. With an increase in the salt content, the yield of the reaction product increases. With an excess of collagen, some of it does not bind into a complex and precipitates. With an excess of salt, almost the weight of the collagen binds into a complex and a rhombic single crystal formed. SEM and X-ray phase analysis of the starting materials and complexes carried out. The correspondence of the parameters of the crystal structure in the X-ray spectra of the complexes found for a wide ratio of the starting materials.

Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture

BackgroundHuman dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs.MethodsWe selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells).ResultsOur results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1α-dependent production and uniform distribution of collagen type I and its interactions with integrins α2β1 and α11β1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization.ConclusionThe use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.

Preparation of collagen/cellulose nanocrystals composite films and their potential applications in corneal repair.

As the main component of the natural cornea, collagen (COL) has been widely applied to the construction of corneal repair materials. However, the applications of collagen are limited due to its poor mechanical properties. Cellulose nanocrystals (CNCs) possess excellent mechanical properties, optical transparency and good biocompatibility. Therefore, in this study, we attempted to introduce cellulose nanocrystals into collagen-based films to obtain corneal repair materials with a high strength. CNCs were incorporated at 1, 3, 5, 7 and 10 wt%. The physical properties of these composite films were characterized, and in vitro cell-based analyses were also performed. The COL/CNC films possessed better mechanic properties, and the introduction of CNCs did not affect the water content and light transmittance. The COL/CNC films demonstrated good biocompatibility toward rabbit corneal epithelial cells and keratocytes in vitro. Moreover, the collagen films with appropriate ration of CNCs effectively induced the migration of corneal epithelial cells and inhibited the myofibroblast differentiation of keratocytes. A collagen film with 7 wt% CNCs displayed the best combination of physical properties and biological performance in vitro among all the films. This study describes a nonchemical cross-linking method to enhance the mechanical properties of collagen for use in corneal repair materials and highlights potential application in corneal tissue engineering.

Bioresorption of Porous 3D Matrices Based on Collagen in Liver and Muscular Tissue

Highly porous cylinder-shaped 3D matrices with diameters of 1.3 and 3 mm were obtained by lyophilization of collagen solution. A study in vivo of the mechanism and rate of resorption of the resulting material showed that complete resorption of the matrix occurred 6 weeks after their implantation into liver tissue and 3 weeks after implantation into muscle tissue of animals. Surrounding tissues were not altered or damaged. Histological analysis revealed that, simultaneously with the resorption of matrix collagen, connective tissue and blood vessels were formed. This allows us to recommend the developed porous material based on collagen for use as matrices for tissue engineering and cellular transplantation.

Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants

Short collagen-like peptides (CLPs) are being proposed as alternatives to full-length collagen for use in tissue engineering, on their own as soft hydrogels, or conjugated to synthetic polymer for mechanical strength. However, despite intended clinical use, little is known about their safety and efficacy, mechanism of action or degree of similarity to the full-length counterparts they mimic. Here, we show the functional equivalence of a CLP conjugated to polyethylene glycol (CLP-PEG) to full-length recombinant human collagen in vitro and in promoting stable regeneration of corneal tissue and nerves in a pre-clinical mini-pig model. We also show that these peptide analogs exerted their pro-regeneration effects through stimulating extracellular vesicle production by host cells. Our results support future use of CLP-PEG implants for corneal regeneration, suggesting the feasibility of these or similar peptide analogs in clinical application in the eye and other tissues. Statement of significanceAlthough biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable analogs that are functionally equivalent, and can be readily scaled-up are therefore very desirable for pre-clinical to clinical translation. Here, we demonstrate, using cornea regeneration as our test bed, that collagen-like-peptides conjugated to multifunctional polyethylene glycol (CLP-PEG) when grafted into mini-pigs as corneal implants were functionally equivalent to recombinant human collagen-based implants that were successfully tested in patients. We also show for the first time that these materials affected regeneration through stimulation of extracellular vesicle production by endogenous host cells that have migrated into the CLP-PEG scaffolds.

Enhancing the biological functions of non-animal collagens

Enhancing the biological functions of non-animal collagens

Biomedical applications of collagens.

Collagen-based biomedical materials have developed into important, clinically effective materials used in a range of devices that have gained wide acceptance. These devices come with collagen in various formats, including those based on stabilized natural tissues, those that are based on extracted and purified collagens, and designed composite, biosynthetic materials. Further knowledge on the structure and function of collagens has led to on-going developments and improvements. Among these developments has been the production of recombinant collagen materials that are well defined and are disease free. Most recently, a group of bacterial, non-animal collagens has emerged that may provide an excellent, novel source of collagen for use in biomaterials and other applications. These newer collagens are discussed in detail. They can be modified to direct their function, and they can be fabricated into various formats, including films and sponges, while solutions can also be adapted for use in surface coating technologies.

Northern pike (Esox lucius) collagen: Extraction, characterization and potential application

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the scales of northern pike (Esox lucius) were extracted and characterized. It was the first time that this species was used as sources of collagen. FT-IR and amino acid analysis results revealed the presence of collagen. Glycine accounts for one-third of its amino acid residues and specific for collagen amino acid – hydroxyproline – is present in isolated protein. The content of imino acid: proline and hydroxyproline in ASC and PSC was similar (12.5% Pro and 6.5% Hyp). Both ASC and PSC were type I collagen. The denaturation temperature of ASC and PSC were 28.5 and 27°C, respectively. Thin collagen films were obtained by casting of collagen solution onto glass plates. The surface properties of ASC and PSC films were different – the surface of ASC collagen film was more polar and less rough than PSC and we can observe the formation of collagen fibrils after solvent evaporation. ASC films showed much higher tensile properties than PSC. The obtained results suggest that northern pike scales have potential as an alternative source of collagen for use in various fields.

Chicken Collagen from Law Market Value By-Products as an Alternate Source

There has been much interest in investigating possible means of making collagen from underutilized chicken by-products and it will lead to an alternate source of collagen for use in various industries such as pharmaceuticals, cosmetics, biomedical materials, and the food industry. The objective of this research was to find methods to extract collagen from chicken skins and bones to compare the corresponding yield differences and analyze their properties. Collagen extracted by acetic acid, citric acid, alkali, one-step acetic acid and pepsin, and two-step acetic acid and pepsin extraction procedures was compared. Complete randomized design, Student’st-test, and Tukey’s test were used to analyze the samples(P<0.05). The recovered dry weights for the skin extractions were 6.1, 6.2, 5, 38.7, and 40.4% and those of bone extractions were 4.4, 4.1, 4.1, 19.1, and 20.6%, respectively. Protein, fat, and inorganic material contents of collagen preparations for skin were 62.7%, 1.5%, and 0.7% and for bone were 30.4%, 1.4%, and 0.7%, respectively. This study indicates that chicken by-products have high potential use as an alternate source of collagen.Corrigendum to “Chicken Collagen from Law Market Value By-Products as an Alternate Source”

Characterisation of acid-soluble and pepsin-solubilised collagen from jellyfish (Cyanea nozakii Kishinouye)

Annual outbreaks of the Jellyfish (Cyanea nozakii Kishinouye) in the waters of the Yellow Sea and the East China Sea are regarded as a nuisance. Thus, utilizing this jellyfish species is of great significance to reduce harm to fisheries and marine environments. The yield of the acid-soluble collagens (ASCs) from the C. nozakii umbrella was 13.0% (dry weight) and that of the pepsin-solubilised collagens (PSCs) was 5.5% (dry weight). The SDS–PAGE patterns of the ASCs and PSCs differed from that of type I collagen, which indicate the presence of (α1)3. The denaturation temperature (Td) of the collagens was approximately 23.8°C. Fourier transform infrared spectroscopy proved that the ASCs and PSCs retained their helical structures and the As, Pb, and Hg content of the collagens, detected by ICP-MS, were considerably lower than the national standards. The results suggest that collagens isolated from C. nozakii can potentially be used as an alternative source of collagen for use in various applications.

Marine Collagen Scaffolds for Nasal Cartilage Repair: Prevention of Nasal Septal Perforations in a New Orthotopic Rat Model Using Tissue Engineering Techniques

Autologous grafts are frequently needed for nasal septum reconstruction. Because they are only available in limited amounts, there is a need for new cartilage replacement strategies. Tissue engineering based on the use of autologous chondrocytes and resorbable matrices might be a suitable option. So far, an optimal material for nasal septum reconstruction has not been identified. The aim of our study was to provide the first evaluation of marine collagen for use in nasal cartilage repair. First, we studied the suitability of marine collagen as a cartilage replacement matrix in the context of in vitro three dimensional cultures by analyzing cell migration, cytotoxicity, and extracellular matrix formation using human and rat nasal septal chondrocytes. Second, we worked toward developing a suitable orthotopic animal model for nasal septum repair, while simultaneously evaluating the biocompatibility of marine collagen. Seeded and unseeded scaffolds were transplanted into nasal septum defects in an orthotopic rat model for 1, 4, and 12 weeks. Explanted scaffolds were histologically and immunohistochemically evaluated. Scaffolds did not induce any cytotoxic reactions in vitro. Chondrocytes were able to adhere to marine collagen and produce cartilaginous matrix proteins, such as collagen type II. Treating septal cartilage defects in vivo with seeded and unseeded scaffolds led to a significant reduction in the number of nasal septum perforations compared to no replacement. In summary, we demonstrated that marine collagen matrices provide excellent properties for cartilage tissue engineering. Marine collagen scaffolds are able to prevent septal perforations in an autologous, orthotopic rat model. This newly described experimental surgical procedure is a suitable way to evaluate new scaffold materials for their applicability in the context of nasal cartilage repair.

Comparative analysis of collagens extracted from different animal sources for application of cartilage tissue engineering

Mammalian collagens have been used as a base material for collagen matrices in tissue engineering applications. However, collagens of aquatic animals and human sources can potentially be utilized as a safe and viable substitute, because collagen products of bovine origin have been shown to be contaminated with some diseases. In the present study, we prepared and investigated collagen materials from several sources (bovine skin, porcine skin, amniotic membrane and starfish) as matrix biomaterials. Detailed investigations on their physicochemical and biological properties, such as amino acid composition, thermal transition temperature, molar mass, IR spectra, and cell response, suggested strong relations between their amino acid composition and intermolecular structure, thermal property, and cell response. Selectively, an amniotic membrane collagen scaffold was evaluated for cartilage tissue engineering in three types of three-dimensional 3D culture (sponge, gel and micro bead forms) and compared with a bovine matrix. Results showed that amniotic membrane collagen has a potential as an alternative source of collagen for use in tissue engineering.

Human collagen isolated from adipose tissue

Collagen, the most abundant protein in vertebrates, is a useful biomaterial in pharmaceutical and medical industries. So far, most collagen has been extracted from animals and cadavers. Herein, we suggest human adipose tissue, which is routinely abandoned after liposuction, as a plentiful source of human collagen. In this study, human collagen was obtained from adipose tissue through two successive major steps: (i) extraction of the extracellular matrix (ECM) by pulverization, centrifugation, alkaline, and alcohol treatment; (ii) isolation of collagen from ECM by pepsin treatment in dilute acetic acid. The purified human adipose-derived collagen was characterized by Fourier transform infrared spectroscopy, polyacrylamide gel electrophoresis, amino acid analysis, and circular dichroism spectroscopy. The extracted collagen showed a typical triple helix structure, good thermal stability due to abundant imino acids, and high solubility at acidic pH. The collagen greatly facilitated the adhesion and proliferation of human adipose-derived stem cells and normal human dermal fibroblasts on polystyrene plates. These results suggest that human adipose tissue obtained by liposuction can provide human collagen for use in cosmetics, pharmaceutics, and medicine.

Isolation, Characterization and Biological Evaluation of Jellyfish Collagen for Use in Biomedical Applications

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2–10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications.

Measurement of activity of collagenolytic MMP and inhibitors of MMPs using radiolabeled collagen substrate

This protocol describes how to purify and radiolabel collagen for use as a substrate to assay collagenolytic members of the matrix metalloproteinases (MMPs). This assay measures enzymes that specifically cleave native triple helical collagen. After incubation of the MMP enzyme with the collagen substrate at 37 degrees C, undigested collagen is removed by centrifugation. Radiolabeled cleaved fragments remain in the supernatant, which is then counted in a scintillation counter; a linear increase in the release of radiolabeled collagen fragments occurs with enzyme level and time. Methods are included for the activation of the proenzyme forms of these MMPs and the assay can also be adapted to measure inhibitors of the collagenolytic MMPs. This assay can be completed in 18 h.

Properties of collagen from skin, scale and bone of carp ( Cyprinus carpio)

Carp ( Cyprinus carpio) is one of the main species of freshwater fish produced in China. Acid-soluble collagens (ASC) were prepared from carp skin, scale and bone. The yields of skin ASC, scale ASC and bone ASC are 41.3%, 1.35% and 1.06% (on the dry weight basis), respectively. SDS–PAGE pattern showed that ASCs of carp skin, scale and bone were all type I collagen, which were composed of two α 1 and one α 2 chains. The molecular weight of α 2 chain is 116 KDa. The amino acid composition and peptide maps of ASCs were similar to each other, but they were totally different from those of cod skin ASC. Denaturation temperatures ( T d) of ASCs were around 28 °C. Fourier transform infrared spectroscopy proved that ASCs were integrate and native. The results suggest that carp skin, scale and bone collagens have potential as an alternative source of collagen for use in various fields.

Characterization of collagen from Japanese sea bass caudal fin as waste material

In an investigation into making more effective use of underutilized resources, Japanese sea bass caudal fin collagen was prepared and partially characterized. It was separated in two fractions, acid-solubilized collagen (ASC) and acid-insoluble collagen (AIC). The yields of ASC and AIC were about 5.2% and 36.4% respectively, on a dry weight basis. AIC consisted of only one α chain and has a chain composition of (α1)3 homotrimer. The patterns of peptide fragments of ASC and AIC were different from that of porcine skin Type I collagen. The denaturation temperatures of these collagens were about 28 °C, about 9 °C lower than that of porcine skin Type I collagen. Japanese sea bass caudal fin has a potential as an alternative source of mammalian collagen for use in various fields such as foods, medicines, and cosmetics.

The safety of reusing injectable collagen: a multicenter microbiological study.

We have previously reported pilot data regarding the safety of saving partially used syringes of a glutaraldehyde cross-linked collagen for use in subsequent treatment sessions with the same individual. That single institution study involved 56 partially used syringes cultured for aerobic bacteria. Only one weakly positive culture was detected among these 56 samples, which prompted us to carry out this expanded study involving multiple centers and different injection techniques. Samples were collected from four centers. Following periurethral injection in an office setting, 166 partially used syringes of glutaraldehyde cross-linked collagen were refrigerated for between 1 and 104 weeks (average 58). Material from all 166 syringes was then cultured qualitatively and quantitatively for both aerobic and anaerobic organisms. Collagen from one syringe grew >100,000 colonies of Escherichia coli. All other cultures were negative. In the pilot study, one culture of 56 syringes was weakly positive for coagulase-negative staphylococcus. When the results from both studies were considered together, only two of 222 partially used syringes (0.9%) were contaminated. The background risk of local infection associated with periurethral collagen injection is approximately 0.29%. Using the statistical equation 'number needed to harm', we found that a clinician would have to reuse 111 syringes at a saving of $34,965 before he or she would cause a single local injection by so doing. Therefore, we feel that it may be cost-effective and safe to reinject material from a partially used syringe of glutaraldehyde cross-linked collagen during a subsequent treatment session on an individual.