In situ structural studies during denaturation of natural and synthetically crosslinked collagen using synchrotron SAXS.

Collagen is an important biomacromolecule, making up the majority of the extracellular matrix in animal tissues. Naturally occurring crosslinks in collagen stabilize its intermolecular structure in vivo, whereas chemical treatments for introducing synthetic crosslinks are often carried out ex vivo to improve the physical properties or heat stability of the collagen fibres for applications in biomaterials or leather production. Effective protection of intrinsic natural crosslinks as well as allowing them to contribute to collagen stability together with synthetic crosslinks can reduce the need for chemical treatments. However, the contribution of these natural crosslinks to the heat stability of collagen fibres, especially in the presence of synthetic crosslinks, is as yet unknown. Using synchrotron small-angle X-ray scattering, the in situ role of natural and synthetic crosslinks on the stabilization of the intermolecular structure of collagen in skins was studied. The results showed that, although natural crosslinks affected the denaturation temperature of collagen, they were largely weakened when crosslinked using chromium sulfate. The development of synergistic crosslinking chemistries could help retain the intrinsic chemical and physical properties of collagen-based biological materials.

Molecular and structural insights into skin collagen reveals several factors that influence its architecture.

Although the biomechanical properties of skin and its molecular components have been extensively studied, little research has been devoted to understanding the links between them. Here, a comprehensive analysis of the molecular components of deer and cow skins was undertaken in order to understand the basis of their physical properties. These skins were chosen because they are known to be strong yet supple, exhibiting properties that have been exploited by man for centuries. Firstly, the tensile strength, tear strength and denaturation temperature of deer and cow skins were measured. Secondly, the organisation of the collagen fibrils and presence of glycosaminoglycans in each skin was investigated using polarising microscopy (PM), laser scanning confocal microscopy (LSCM), transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS). Finally, amino acid, crosslink and glycosaminoglycan analyses were carried out on both skins in the study. The results of the study showed that individual physical properties such as tensile strength of the skin are derived from different combinations of biomolecular components which are reflected in collagen architecture. The "wavy" organisation of collagen fibres in deer skin was associated with a small fibril diameter, uniform glycosaminoglycan distribution and higher proportion of trivalent crosslinks. In contrast, the collagen fibrils in cow skin were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent crosslinks, resulting in straight fibres. This study showed for the first time that the relationship between the structure of collagen in skin and its biomechanical functions is complex, arising from different architectural and molecular features including organisation of collagen fibres, diameters of collagen fibrils, distribution and amount of glycosaminoglycans and types and concentrations of crosslinks.

Rapid analysis of pyridinoline and deoxypyridinoline in biological samples by liquid chromatography with mass spectrometry and a silica hydride column.

Pyridinoline and deoxypyridinoline crosslinks are biomarkers found in urine for collagen degradation in bone turnover. For the first time, a rapid, sensitive, and ion-pairing free method is described for the analysis of pyridinoline and deoxypyridinoline using ultra-high performance liquid chromatography with Cogent Diamond Hydride column and detection by Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry. The separation was achieved using both isocratic and gradient conditions and run time <5 min under isocratic conditions of 20% acetonitrile in water containing 0.1% formic acid. Pyridoxine was used as an internal standard and relative standard deviation of the retention times of both pyridinoline and deoxypyridinoline were <1%. The limit of detection was 0.082 ± 0.023 µM for pyridinoline and 0.118 ± 0.052 µM for deoxypyridinoline. The limit of quantitation was 0.245 ± 0.070 µM for pyridinoline and 0.354 ± 0.157 µM for deoxypyridinoline. The method was validated by the detection and quantitation of both pyridinoline and deoxypyridinoline in skin and urine samples.