Justyna Kozłowska

Characterization of collagen/hydroxyapatite composite sponges as a potential bone substitute

Hydroxyapatite and collagen composites (HAp/Col) have the potential in mimicking and replacing skeletal bones. Their combination should prove beneficial for bone tissue engineering due to their natural biological resemblance and properties. In this study, hydroxyapatite and collagen isolated from animal tendons were used in different proportions as composites. Sponges were prepared by freezing and lyophilization of corresponding composite solutions. The properties of composite sponges, such as microstructure, chemical and physical properties were studied. In the present investigation, a collagen sponge and a composite sponge made of composite of HAp/Col were prepared in our lab and characterized by attenuated total reflection Fourier transform infra-red spectroscopy, thermogravimetric analysis and scanning electron microscopy (SEM). Infrared spectroscopy (IR) in combination with attenuated total reflection (ATR) spectroscopy is one of the most widely used technique for surface infrared analysis. The ATR-IR analysis did not indicate shift of the band corresponding to -COO(-) for none of the used HAp/Col ratios. Thermogravimetric results suggested that collagen chains had been embedded with HAp to several complexes with different thermal stabilities. SEM was used to observe the morphology and pore size of the sponges. SEM observations showed the sponges of HAp/Col with fully interconnective macroporosity.

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.

Isolation and characterization of collagen from the skin of Brama australis

Collagen was extracted from the skin of Brama australis, the fish from warm-water sea. The yield of collagen from skin of B. australis was about 1.5% on a wet weight basis of raw material. The isolated protein was confirmed as collagen by different physico-chemical techniques such as: FTIR, SDS-PAGE, and amino acid analysis. The denaturation temperature (T(d)) of obtained collagen was found to be 24°C, what is promising as an advantage for cosmetic application. According to the electrophoretic pattern, collagen consisted of two different α-chains (α1 and α2) and was classified as type I collagen. Although T(d) of obtained collagen is higher than 20 °C it is still far from T(d) of mammalian collagen.

Photochemical behaviour of hydrolysed keratin

An investigation into the influence of UV irradiation on keratin hydrolysates was carried out using UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. It was found that the absorption of keratin hydrolysates in solution increased during irradiation of the sample, most notably between 250–280 and 320–410 nm. The increase in absorbance in the region 320–410 was because of the new photoproducts formed during UV irradiation of keratin hydrolysates. The fluorescence of keratin hydrolysates was observed at 328 nm after excitation at 270 nm. UV irradiation caused fluorescence fading at 328 nm, and after 60 min of irradiation, a new broad weak band of fluorescence, attributable to new photoproducts, emerged in the UV wavelength region with emission maximum between 400 and 500 nm. FTIR spectroscopy results showed degradation of keratin under UV irradiation. A slight increase in oxidized sulphur species was also observed. The results obtained suggest that UV irradiation can be used as modifying agent for preparation of keratin hydrolysates for cosmetic applications.

New composite materials prepared by calcium phosphate precipitation in chitosan/collagen/hyaluronic acid sponge cross-linked by EDC/NHS

Nowadays, fabrication of composite materials based on biopolymers is a rising field due to potential for bone repair and tissue engineering application. Blending of different biopolymers and incorporation of inorganic particles in the blend can lead to new materials with improved physicochemical properties and biocompatibility. In this work 3D porous structures called scaffolds based on chitosan, collagen and hyaluronic acid were obtained through the lyophilization process. Scaffolds were cross-linked by EDC/NHS. Infrared spectra for the materials were made, the percentage of swelling, scaffolds porosity and density, mechanical parameters, thermal stability were studied. Moreover, the scaffolds were used as matrixes for the calcium phosphate in situ precipitation. SEM images were taken and EDX analysis was carried out for calcium and phosphorous content determination in the scaffold. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells was examined on obtained scaffolds. The results showed that the properties of 3D composites cross-linked by EDC/NHS were altered after the addition of 1, 2 and 5% hyaluronic acid. Mechanical parameters, thermal stability and porosity of scaffolds were improved. Moreover, calcium and phosphorous were found in each kind of scaffold. SEM images showed that the precipitation was homogeneously carried in the whole volume of samples. Attachment of SaOS-2 cells to all modified materials was better compared to unmodified control and proliferation of these cells was markedly increased on scaffolds with precipitated calcium phosphate. Obtained materials can provide the support useful in tissue engineering and regenerative medicine.

Modification by UV radiation of the surface of thin films based on collagen extracted from fish scales.

Collagen was extracted from fish scales (Esox lucius) through demineralization process. Thin films by solvent evaporation from collagen extracted from fish scales were prepared. The surface of thin films made of fish scales collagen was modified by ultraviolet (UV)-irradiation with the wavelength λ = 254 nm. The amino acid composition of the Esox lucius scale collagen was analyzed before and after UV-irradiation by means of high-pressure liquid chromatography. The surface properties of films were investigated using the technique of atomic force microscopy (AFM) and by means of contact angle measurements allowing the calculation of surface free energy. Measurements of the contact angle for diiodomethane (D) and glycerol (G) on the surface of fish collagen films were made and surface free energy was calculated. The structure of collagen before and after UV-irradiation was studied using Fourier-transform infrared spectroscopy. It was found that after UV-irradiation the amount of all amino acids present in collagen molecule decreased. It was found also that the contact angle and the surface free energy were altered by UV-irradiation of collagen film. AFM showed that the surface roughness of collagen films was also altered by UV-irradiation. UV-irradiation caused the decrease of surface roughness due to photochemical processes, which occurred in the top layer of collagen film. The formation of collagen fibrils after solvent evaporation was observed using AFM. The diameter of collagen fibrils was bigger for irradiated collagen film than the diameter of collagen fibrils before UV-irradiation.

Fish Scales as a Biocomposite of Collagen and Calcium Salts

Collagen for biomedical applications is mainly isolated from animal tissues (bovine or porcine skin and bovine or equine Achilles tendons). Type I collagen has been also extracted from skin, bone, fins and scales of fresh water and marine fishes. Fish scales are composed of collagen covered with calcium salts. In the present study we report the preparation of collagen from fish scales for potential cosmetic, pharmaceutical and implant applications. In our laboratory collagen was isolated from scales of Esox lucius. It was the first time that this species were used as sources of collagen. Extraction of collagen from fish scales was done in two steps. In the first step, fish scales were demineralized using EDTA. Energy dispersive X-ray analysis of demineralized scale was carried out for quantitative estimation of inorganic content. Then, demineralized fish scales were dissolved in acetic acid. Collagen isolated from Esox Lucius may serve as an attractive and safe source of collagen for biomedical and pharmaceutical applications. Fish collagen can be processed in sheet, sponges foams, injectable viscous solution, and dispersions.

Biopolymer Blends as Potential Biomaterials and Cosmetic Materials

Blends of two polymer, namely chitosan with silk fibroin or partially hydrolysed polyacrylamide (HPAM) were prepared. The surface properties of chitosan/silk fibroin and chitosan/HPAM blended films were investigated using the technique of Atomic Force Microscopy (AFM) and by means of contact angle measurements allowing the calculation of surface free energy. Measurements of the contact angle for diiodomethane (D), and glycerol (G) on the surface of chitosan films and chitosan/silk fibroin films were made and surface free energy was calculated. It was found that chitosan/silk fibroin blend surface is enriched in high surface energy component i.e. silk fibroin. The surface roughness of chitosan, silk fibroin, HPAM, chitosan/silk fibroin and chitosan/HPAM blended films differs with the composition of the blend. Film-forming polymeric blends can be potentially used as biomaterials and cosmetic materials.

Collagen/Gelatin/Hydroxyethyl Cellulose Composites Containing Microspheres Based on Collagen and Gelatin: Design and Evaluation

The objective of this study was to develop three-dimensional collagen/gelatin/hydroxyethyl cellulose composites in combination with gelatin or collagen-gelatin loaded microspheres. Microspheres were prepared by an emulsification/crosslinking method. A 1-Ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) mixture were used as a crosslinking agent for the obtained materials. The structure of the materials was studied using scanning electron microscopy (SEM) and infrared spectroscopy. Moreover, a Calendula officinalis (pot marigold) flower extract release profile of the microsphere-loaded matrices was assessed in vitro. Additionally, porosity, density, stability, swelling and mechanical properties were tested. On the basis of SEM images, the microspheres exhibited a spherical shape and were irregularly dispersed in the polymer matrix. However, it was found that the addition of microparticles to obtained materials did not significantly change their microstructure. We observed a slight decrease in the swelling properties of matrices and an increase in values of Young’s modulus. Significantly, the addition of microspheres to the polymer matrices led to improved loading capacity of materials and release performance of Calendula officinalis flower extract. This makes the collagen/gelatin/hydroxyethyl cellulose composites containing microspheres a promising and suitable vehicle for biomedical, dermatological, or cosmetic applications.