J. Skopinska-Wisniewska

Chemical and thermal cross-linking of collagen and elastin hydrolysates

Chemical and thermal cross-linking of collagen soluble in acetic acid and elastin hydrolysates soluble in water have been studied. Solutions of collagen and elastin hydrolysates were treated using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Moreover, diepoxypropylether (DEPE) has been used as cross-linking agent. Films made of collagen and elastin hydrolysates were also treated with temperature at 60°C and 100°C to get additional cross-links. The effect of cross-linking has been studied using FTIR spectroscopy, thermal analysis, AFM and SEM microscopy. Mechanical and surface properties of materials have been studied after cross-linking. It was found that thermal and mechanical properties of collagen and elastin materials have been altered after thermal treatment and after the reactions with EDC/NHS and/or DEPE. Surface properties of collagen materials after chemical cross-linking have been modified. Thermal and chemical cross-linking of collagen films lead to alteration of polarity of the surface.

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.

Dialysis as a method of obtaining neutral collagen gels

Collagen gels are useful materials for medicine and tissue engineering. They are generally obtained by chemical cross-linking of the protein chains. However, other kinds of interactions can also stabilize the structure. In our investigations we employed dialysis against deionised water as a method of neutralization of collagen solution. This promoted the creation of stable, flexible, transparent gel composed only of collagen and water. The FTIR-ATR spectroscopy showed that changing pH of the solution caused organization of collagen chains into triple-helical motifs similar to native protein. As a result, thermal stability of the material improved and the surface was more polar than in case of collagen film obtained from acidic solution. The freeze-drying of the gel provided the relatively stiff, porous material, which returned to its original shape after deformation. We expect that the method of obtaining neutral collagen gels can be widely applied for preparation of scaffolds for tissue engineering.

Collagen/elastin hydrogels cross-linked by squaric acid.

Hydrogels based on collagen and elastin are very valuable materials for medicine and tissue engineering. They are biocompatible; however their mechanical properties and resistance for enzymatic degradation need to be improved by cross-linking. Up to this point many reagents have been tested but more secure reactants are still sought. Squaric acid (SqAc), 3,4-dihydroxy 3-cyclobutene 1,2-dione, is a strong, cyclic acid, which reacts easily with amine groups. The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5%, 10% and 20% of SqAc and neutralized via dialysis against deionized water were tested. Cross-linked, 3-D, transparent hydrogels were created. The cross-linked materials are stiffer and more resistant to enzymatic degradation than those that are unmodified. The pore size, swelling ability and surface polarity are reduced due to 5% and 10% of SqAc addition. At the same time, the cellular response is not significantly affected by the cross-linking. Therefore, squaric acid would be regarded as a safe, effective cross-linking agent.

Collagen/Polypropylene composite mesh biocompatibility in abdominal wall reconstruction.

Background: Intraperitoneal placement of polypropylene mesh leads to extensive visceral adhesions and is contraindicated. Different coatings are used to improve polypropylene mesh properties. Collagen is a protein with unique biocompatibility and cell ingrowth enhancement potential. A novel acetic acid extracted collagen coating was developed to allow placement of polypropylene mesh in direct contact with viscera. The authors’ aim was to evaluate the long-term influence of acetic acid extracted collagen coating on surgical aspects and biomechanical properties of polypropylene mesh implanted in direct contact with viscera, including complications, adhesions with viscera, strength of incorporation, and microscopic inflammatory reaction. Methods: Forty adult Wistar rats were divided into two groups: experimental (polypropylene mesh/acetic acid extracted collagen coating) and control (polypropylene mesh only). Astandardized procedure of mesh implantation was performed. Animals were killed 3 months after surgery and analyzed for complications, mesh area covered by adhesions, type of adhesions, strength of incorporation, and intensity of inflammatory response. Results: The mean adhesion area was lower for polypropylene mesh/acetic acid extracted collagen coating (14.5 percent versus 69.9 percent, p < 0.001). Adhesion severity was decreased in the experimental group: grades 0 and 1 were more frequent (p < 0.04 and p < 0.002, respectively) and grade 3 was less frequent (p < 0.0001). An association between adhesion area and severity was found (p < 0.0001). Complications, strength of incorporation, and intensity of inflammatory response to the mesh were similar. Conclusions: Visceral adhesions to polypropylene mesh are significantly reduced because of acetic acid extracted collagen coating. The collagen coating does not increase complications or induce alterations of polypropylene mesh incorporation.

Is dialdehyde starch a valuable cross-linking agent for collagen/elastin based materials?

AbstractnCollagen and elastin are the main structural proteins in mammal bodies. They provide mechanical support, strength, and elasticity to various organs and tissues, e.g. skin, tendons, arteries, and bones. They are readily available, biodegradable, biocompatible and they stimulate cell growth. The physicochemical properties of collagen and elastin-based materials can be modified by cross-linking. Glutaraldehyde is one of the most efficient cross-linking agents. However, the unreacted molecules can be released from the material and cause cytotoxic reactions. Thus, the aim of our work was to investigate the influence of a safer, macromolecular cross-linking agent—dialdehyde starch (DAS). The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5 and 10xa0% of DAS and neutralized via dialysis against deionized water were tested. The homogenous, transparent, stiff hydrogels were obtained. The DAS addition causes the formation of intermolecular cross-linking bonds but does not affect the secondary structure of the proteins. As a result, the thermal stability, mechanical strength, and, surprisingly, swelling ability increased. At the same time, the surface properties test and in vitro study show that the materials are attractive for 3T3 cells. Moreover, the materials containing 10xa0% of DAS are more resistant to enzymatic degradation.

New Amniotic Membrane Based Biocomposite for Future Application in Reconstructive Urology.

Objective Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model. Material and Methods Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-E-caprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material. Results Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson’s trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young’s elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process. Conclusions Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.

Blood Vessel Matrix Seeded with Cells: A Better Alternative for Abdominal Wall Reconstruction—A Long-Term Study

Purpose. The aim of this study was to present abdominal wall reconstruction using a porcine vascular graft seeded with MSC (mesenchymal stem cells) on rat model. Material and Methods. Abdominal wall defect was prepared in 21 Wistar rats. Acellular porcine-vascular grafts taken from aorta and prepared with Triton X were used. 14 aortic grafts were implanted in place, of which 7 grafts were seeded with rat MSC cells (Group I), and 7 were acellular grafts (Group II). As a control, 7 standard polypropylene meshes were used for defect augmentation (Group III). The assessment method was performed by HE and CD31 staining after 6 months. The mechanical properties have been investigated by Zwick&Roell Z0.5. Results. The strongest angiogenesis and lowest inflammatory response were observed in Group I. Average capillaries density was 2.75, 0.75, and 1.53 and inflammatory effect was 0.29, 1.39, and 2.72 for Groups I, II, and III, respectively. The means of mechanical properties were 12.74 ± 1.48, 7.27 ± 1.56, and 14.4 ± 3.7u2009N/cm in Groups I and II and control, respectively. Conclusions. Cell-seeded grafts have better mechanical properties than acellular grafts but worse than polypropylene mesh. Cells improved mechanical and physiological properties of decellularized natural scaffolds.

Use of Adipose-Derived Stem Cells to Support Topical Skin Adhesive for Wound Closure: A Preliminary Report from Animal In Vivo Study

The aim of this study was to determine the local and systemic effects of adipose-derived stem cells (ADSCs) as a component of topical skin adhesive in an animal artificial wound closure model. In presented study the cosmetic effects, histological analysis, mechanical properties, and cell migration have been assessed to evaluate the usefulness of ADSCs as supporting factor for octyl blend cyanoacrylate adhesive. The total of 40 rats were used and divided into six groups. In the Study Group, ADSCs were administered by multipoint injection of the six surrounding intrawound areas with additional freely leaving procedure of the cells between the skin flaps just before applying adhesive to close the wound. Five control groups without using ADSCs, utilizing different types of standard wound closure, were created in order to check efficiency of experimental stem cell therapy. In our study, we proved that ADSCs could be used effectively also as a supportive tool in topical skin adhesive for wound closure. However we did not achieve any spectacular differences related to such aspects as better mechanical properties or special biological breakthroughs in wound healing properties. The use of stem cells, especially ADSCs for wound closure can provide an inspiring development in plastic and dermatologic surgery.

Influence of glycosaminoglycans on the properties of thin films based on chitosan/collagen blends

Thin films based on chitosan, collagen, and glycosaminoglycans isolated from fish skin were obtained by solvent evaporation. The films were characterized by different analyses, e.g. surface free energy determination, swelling tests, roughness, mechanical and thermal measurements. Moreover, the degradation studies were carried out by the film treatment with collagenase. The results showed that the properties of the films based on chitosan and collagen can be modified by the glycosaminoglycans addition. It was noticed that the addition of glycosaminoglycans enhances the surface hydrophilicity and reduces surface free energy. Surfaces of films modified by glycosaminoglycans (GAGs) show more roughness which inhibits the risk of biofilm formation. The highest films swelling was obtained after 2u202fh immersion in phosphate-buffered saline (PBS). After their immersion in PBS, the films were more elastic, which was assumed on the basis of the elongation at break values higher than in the case of films on a dry surface. The proposed films can create biocompatible coatings for biomedical applications.