Effect of Heat Level and Expose Time on Denaturation of Collagen Tissues.

INTRODUCTION: The applied heat level and expose time are main issues in certain operations/applications, such as a laser assisted tissue welding, preparation of collagen-based biomaterials (films, implants). Therefore, the precise investigation of these parameters is crucial. The results can serve as a guideline to assess potential effects while maintaining the functionality of the collagen structures. METHODS: Collagen tissues from rat-tail tendon, calfskin, and bones are soaked in buffer solutions, then examined by microscope at different temperature levels. RESULTS: Increase in temperature reduced the microscopically observed collagen crimp contrast for calfskin and rat-tail tendons but not for bone tissues. The contrast level for rat tail tendon decreased down to 80% of its initial value at 37, 157, and 266 s for 70, 65, and 60 °C, respectively. The decrease in the crimp contrast was about only 25% and 2% at 55 and 50 °C after 2 h, respectively. 50% drop in contrast level was occurred for the skin samples at 16, 90, 110 and 1900 s for 70, 65, and 60 °C, respectively. The bone samples, did not show any significant differences in contrast levels. CONCLUSION: The observed denaturation behaviours are in line with Arrhenius Law. This study could be expanded on to other types of tissues at wider temperature ranges to make a guideline for biological/medical processes that radiate heat in order to assess their side effects on collagen and other proteins.

Synchrotron Fourier transform infrared microspectroscopy (sFTIRM) analysis of unfolding behavior of electrospun collagen nanofibers.

Collagen nanofibers are popular extracellular matrix (ECM) materials in regenerative medicine. Electrospinning of collagen dissolved in organic solvents is widely used for fabricating anisotropic collagen nanofibers; however, such fibers are water-soluble and require cross-linking before use as scaffolds for cell culture. Herein, in-situ crosslinking during electrospinning process is suggested by using different chemical agents, namely genipin and glutaraldehyde, and physical crosslinking method (UV light). sFTIRM; Synchrotron Fourier-Transform Infrared Microspectroscopy is a powerful tool that sheds light on the molecular structure of collagen nanofibers. Applied extraction methods caused shifts on protein band positions. Electrospinning process prevents self-assembly of collagen molecules and obtained electrospun collagen nanofibers have lower band positions. Crosslinkers have effect on the secondary structure of collagen molecules. Among different crosslinkers, genipin in-situ crosslinking process perform better in preserving the native structure of electrospun collagen nanofibers than the physical crosslinking method (UV).

Production of collagen micro- and nanofibers for potential drug-carrier systems.

Two different nano- and micro-collagen fiber production methods are introduced and discussed. First one is the electrospinning method, that is very common technique to produce nanofibers from different polymeric solutions and recently collagen solutions are employed to produce nanofibers for different biomedical applications. This technique is extremely versatile method to produce nanofibers in a relatively short time, easy to control the fiber diameter and orientation with small pore sizes and a high surface area. The second method is self-assembly of collagen micro-fibers by co-extrusion method. The collagen fibers are obtained without any cross-linker, by using mainly ionic interactions. We demonstrated that self-assembled collagen fibers have well preserved their native structure (0.90 PP-II fraction), when compared with electrospun collagen fibers (0.38 PP-II fraction). However, it was only possible to produce collagen fibers with nanodimensions by using electrospinning method.

Resemblance of electrospun collagen nanofibers to their native structure.

Electrospinning is a promising method to mimic the native structure of the extracellular matrix. Collagen is the material of choice, since it is a natural fibrous structural protein. It is an open question how much the spinning process preserves or alters the native structure of collagen. There are conflicting results in the literature, mainly due to the different solvent systems in use and due to the fact that gelatin is employed as a reference state for the completely unfolded state of collagen in calculations. Here we used circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR) to investigate the structure of regenerated collagen samples and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to illuminate the electrospun nanofibers. Collagen is mostly composed of folded and unfolded structures with different ratios, depending on the applied temperature. Therefore, CD spectra were acquired as a temperature series during thermal denaturation of native calf skin collagen type I and used as a reference basis to extract the degree of collagen folding in the regenerated electrospun samples. We discussed three different approaches to determine the folded fraction of collagen, based on CD spectra of collagen from 185 to 260 nm, since it would not be sufficient to obtain simply the fraction of folded structure θ from the ellipticity at a single wavelength of 221.5 nm. We demonstrated that collagen almost completely unfolded in fluorinated solvents and partially preserved its folded structure θ in HAc/EtOH. However, during the spinning process it refolded and the PP-II fraction increased. Nevertheless, it did not exceed 42% as deduced from the different secondary structure evaluation methods, discussed here. PP-II fractions in electrospun collagen nanofibers were almost same, being independent from the initial solvent systems which were used to solubilize the collagen for electrospinning process.

In vitro observation of dynamic ordering processes in the extracellular matrix of living, adherent cells.

Collecting information at the interface between living cells and artificial substrates is exceedingly difficult. The extracellular matrix (ECM) mediates all cell-substrate interactions, and its ordered, fibrillar constituents are organized with nanometer precision. The proceedings at this interface are highly dynamic and delicate. In order to understand factors governing biocompatibility or its counterpart antifouling, it is necessary to probe this interface without disrupting labels or fixation and with sufficient temporal resolution. Here the authors combine nonlinear optical spectroscopy (sum-frequency-generation) and microscopy (second-harmonic-generation), fluorescence microscopy, and quartz crystal microgravimetry with dissipation monitoring in a strategy to elucidate molecular ordering processes in the ECM of living cells. Artificially (fibronectin and collagen I) and naturally ordered ECM fibrils (zebrafish, Danio rerio) were subjected to nonlinear optical analysis and were found to be clearly distinguishable from the background signals of diffusive proteins in the ECM. The initial steps of fibril deposition and ordering were observed in vitro as early as 1 h after cell seeding. The ability to follow the first steps of cell-substrate interactions in spite of the low amount of material present at this interface is expected to prove useful for the assessment of biomedical and environmental interfaces.

Tuning the surface-enhanced Raman scattering effect to different molecular groups by switching the silver colloid solution pH.

Silver colloids were produced for surface-enhanced Raman scattering (SERS) experiments using hydroxylamine hydrochloride as the reduction agent. The roles of hydroxylamine hydrochloride and bulk solution pH values in the formation of functional groups on the surface of silver colloids and in determining the dimensions of silver colloids were examined using Raman, Fourier transform infrared (FT-IR) and ultraviolet-visible (UV-Vis) spectroscopy, atomic force microscopy (AFM), and zeta-size measurements. The spectrum of hydroxylamine hydrochloride reduced silver colloids was compared with the spectrum of sodium borohydride reduced colloids. The effect of colloid solution pH on SERS results was demonstrated using analyte molecules with biological significance, such as ribonucleic acid, egg albumin, L-alpha-phosphatidylcholine, and glucose. In general, it was shown that at high pH values the SERS effect was more pronounced due to the surface functional groups and colloid dimensions, and sharp, high spectral intensity values were obtained. At low pH values, protonation and rapid aggregation of colloids occurred and the surface chemistry was different. Depending on the analyte, bands were shifted, broadened, and/or the enhancement effect was reduced. Using Pseudomonas aeruginosa PAO1 and Streptococcus mutans it was also shown that by changing the solution bulk pH value, it was possible to enhance the response from different molecular groups in the bacteria and obtain different spectra from the same bacteria strain and the process was reversible. It was concluded that it is possible to produce site- or molecule-specific metal colloids and to tune the SERS effect to certain functional groups of analytes by means of the pH of colloidal suspension.

Flow behavior of regenerated wool-keratin proteins in different mediums.

Keratin is abundantly present in nature and the major component of hair, wool, feather, nail and horns. Dissolution of keratin is often required when non-textile applications are demanded. However, the low solubility of keratin in water is the major problem. It becomes unstable and precipitated when stored for a long time. Therefore, it is necessary to find a good solvent that provides high stability and easy processibility. In this research, we used formic acid and dimethylformamide (DMF) to dissolve regenerated keratin protein films. It is shown that formic acid is a good solvent for regenerated keratin proteins for the purpose of storage. Transparent and stable regenerated keratin solution is obtained in formic acid.