Understanding the molecular basis of the strength differences in skins used in leather manufacture : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand

Although skin structure and its physical properties have been extensively studied, little research has been devoted to understanding the links between them. A comprehensive study of the molecular components of four animal skins commonly used to manufacture shoes, clothing and furniture was therefore undertaken in order to attempt to identify a common indicator of skin strength. The molecular architecture of the protein components of each skin was analysed using polarising, confocal and transmission-electron microscopy (TEM), small angle X-ray scattering (SAXS) and amino-acid and cross-link analysis; glycosaminoglycans were quantified and visualised using TEM; and, for the sake of completeness, total carbohydrate and lipid content were measured using a colorimetric assay and thin layer chromatography respectively. Differences in these properties were then related to different physical characteristics of each skin. The results showed that an individual mechanical property of skin such as tensile strength is complex and related to different combinations of molecular properties. For example, deer and cow skins are the strongest of the skins examined, however they derive their strength from different combinations of molecular properties. Cow skin collagen fibrils have the largest diameter, but deer skin fibrils have the smallest. On the other hand, the fibrils in deer skin frequently change direction, and have a “wavy” or crimped appearance in contrast to the fibrils in cow skin which are aligned in two main directions approximately 60 and 90 degrees apart, differences that are also reflected in the types and amount of their collagen crosslinks. Deer skin fibrils contain a higher proportion of trivalent crosslinks while cow skin fibrils contain a higher proportion of tetravalent links. For the two weaker skins, goat skin fibrils are more crimped than those of sheep skin, but both fibrils have diameters intermediate between those of cow- and deer skins and have lower mature to immature crosslink ratio. In deer skin, glycosaminoglycans are observed by TEM to link fibrils in regular arrays and are present in higher concentrations than in cow, sheep and goat skins. This study showed the relationship between the molecular structure of skin and its mechanical functions is complex, arising from different combinations of molecular features rather than just one

Strong skin, not always thick: Comparative structural and molecular analysis of deer skin and cow hide

Content: A comprehensive analysis of the molecular and structural components of deer skin and cow hide was undertaken. These skins known to be strong, however they derive their strength from different combinations of molecular and structural properties. Firstly, the physical properties of deer skin and cow hide including the tensile strength, tear strength and denaturation temperature were measured. Secondly, the structure of the collagen fibrils and glycosaminoglycans was investigated using transmission electron microscopy (TEM), and small angle X-ray scattering (SAXS). Finally, the chemical composition of deer skin and cow hide such as amino acids, crosslinks and glycosaminoglycans were analysed. Our results showed that physical properties of deer skin and cow hide are derived from different combinations of several chemical components resulting in different architecture. It was found that the large and “wavy” collagen fibres in deer skin made up of collagen fibrils with small diameters. Additionally, deer skin fibrils appeared to be linked by regular arrays of filaments of large glycosaminoglycans that are distributed uniformly. Deer skin contained higher proportion of trivalent collagen crosslinks. In contrast, the collagen fibrils in cow hide were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent collagen crosslinks, resulting in straight collagen fibres. This study suggests that although deer skin and cow hide are both strong, they have different structural and molecular features. Take-Away: Deer skin and cow hide have different structural and molecular make up which are reflected in their physical properties particularly strength. Glycosaminoglycans are important for the organisation of collagen fibrils in deer skin and cow hide. Deer skin and cow hide contain different ratios of collagen natural crosslinks which are essential collagen stability

3D-Printed Hybrid Collagen/GelMA Hydrogels for Tissue Engineering Applications

Bioprinting is an emerging technology involved in the fabrication of three-dimensional tissue constructs for the repair and regeneration of various tissues and organs. Collagen, a natural protein found abundantly in the extracellular matrix of several tissues, can be extracted from collagen-rich tissues of animals such as sheep, cows, rats, pigs, horses, birds, and marine animals. However, due to the poor printability of collagen bioinks, biocompatible collagen scaffolds that mimic the extracellular matrix (ECM) are difficult to fabricate using bioprinting techniques. Gelatin methacrylate (GelMA), a semi-synthetic polymer with tunable physical and chemical properties, has been found to be a promising biomaterial in various bioprinting applications. The printability of collagen can be improved by combining it with semi-synthetic polymers such as GelMA to develop hybrid hydrogels. Such hybrid hydrogels printed have also been identified to have enhanced mechanical properties. Hybrid GelMA meshes have not previously been prepared with collagen from ovine sources. This study provides a novel comparison between the properties of hybrid meshes with ovine skin and bovine hide collagen. GelMA (8% w/v) was integrated with three different concentrations (0.5%, 1%, and 2%) of bovine and ovine collagen forming hybrid hydrogels inks that were printed into meshes with enhanced properties. The maximum percentage of collagen suitable for integration with GelMA, forming hybrid hydrogels with a stable degradation rate was 1%. The water-soluble nature of ovine collagen promoted faster degradation of the hybrid meshes, although the structural crosslinking was identified to be higher than bovine hybrid meshes. The 1% bovine collagen hybrid meshes stood out in terms of their stable degradation rates

Strong skin, not always thick: Comparative structural and molecular analysis of deer skin and cow hide

Content: A comprehensive analysis of the molecular and structural components of deer skin and cow hide was undertaken. These skins known to be strong, however they derive their strength from different combinations of molecular and structural properties. Firstly, the physical properties of deer skin and cow hide including the tensile strength, tear strength and denaturation temperature were measured. Secondly, the structure of the collagen fibrils and glycosaminoglycans was investigated using transmission electron microscopy (TEM), and small angle X-ray scattering (SAXS). Finally, the chemical composition of deer skin and cow hide such as amino acids, crosslinks and glycosaminoglycans were analysed. Our results showed that physical properties of deer skin and cow hide are derived from different combinations of several chemical components resulting in different architecture. It was found that the large and “wavy” collagen fibres in deer skin made up of collagen fibrils with small diameters. Additionally, deer skin fibrils appeared to be linked by regular arrays of filaments of large glycosaminoglycans that are distributed uniformly. Deer skin contained higher proportion of trivalent collagen crosslinks. In contrast, the collagen fibrils in cow hide were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent collagen crosslinks, resulting in straight collagen fibres. This study suggests that although deer skin and cow hide are both strong, they have different structural and molecular features. Take-Away: Deer skin and cow hide have different structural and molecular make up which are reflected in their physical properties particularly strength. Glycosaminoglycans are important for the organisation of collagen fibrils in deer skin and cow hide. Deer skin and cow hide contain different ratios of collagen natural crosslinks which are essential collagen stability

Strong skin, not always thick: Comparative structural and molecular analysis of deer skin and cow hide

Content: A comprehensive analysis of the molecular and structural components of deer skin and cow hide was undertaken. These skins known to be strong, however they derive their strength from different combinations of molecular and structural properties. Firstly, the physical properties of deer skin and cow hide including the tensile strength, tear strength and denaturation temperature were measured. Secondly, the structure of the collagen fibrils and glycosaminoglycans was investigated using transmission electron microscopy (TEM), and small angle X-ray scattering (SAXS). Finally, the chemical composition of deer skin and cow hide such as amino acids, crosslinks and glycosaminoglycans were analysed. Our results showed that physical properties of deer skin and cow hide are derived from different combinations of several chemical components resulting in different architecture. It was found that the large and “wavy” collagen fibres in deer skin made up of collagen fibrils with small diameters. Additionally, deer skin fibrils appeared to be linked by regular arrays of filaments of large glycosaminoglycans that are distributed uniformly. Deer skin contained higher proportion of trivalent collagen crosslinks. In contrast, the collagen fibrils in cow hide were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent collagen crosslinks, resulting in straight collagen fibres. This study suggests that although deer skin and cow hide are both strong, they have different structural and molecular features. Take-Away: Deer skin and cow hide have different structural and molecular make up which are reflected in their physical properties particularly strength. Glycosaminoglycans are important for the organisation of collagen fibrils in deer skin and cow hide. Deer skin and cow hide contain different ratios of collagen natural crosslinks which are essential collagen stability

Validity and reliability of Raman spectroscopy for carotenoid assessment in cattle skin

Carotenoids are powerful antioxidants capable of helping to protect the skin from the damaging effects of exposure to sun by reducing the free radicals in skin produced by exposure to ultraviolet radiation, and they may also have a physical protective effect in human skin. Since carotenoids are lipophilic molecules which can be ingested with the diet, they can accumulate in significant quantities in the skin. Several studies on humans have been conducted to evaluate the protective function of carotenoids against various diseases, but there is very limited published information available to understand the mechanism of carotenoid bioavailability in animals. The current study was conducted to investigate the skin carotenoid level (SCL) in two cattle skin sets – weaners with an unknown feeding regime and New Generation Beef (NGB) cattle with monitored feed at three different ages. Rapid analytical and sensitive Raman spectroscopy has been shown to be of interest as a powerful technique for the detection of carotenoids in cattle skin due to the strong resonance enhancement with 532 nm laser excitation. The spectral difference of both types of skin were measured and quantified using univariate and linear discriminant analysis. SCL was higher in NGB cattle than weaners and there is a perfect classification accuracy between weaners and NGB cattle skin using carotenoid markers as a basis. Further work carried out on carotenoid rich NGB cattle skin of 8, 12 and 24 months of age identified an increasing trend in SCL with age. The present work validated the ability of Raman spectroscopy to determine the skin carotenoid level in cattle by comparing it with established HPLC methods. There is an excellent correlation of R2 = 0.96 between the two methods that could serve as a model for future application for larger population studies

Pressure Algometry Validation and Determination of Efficacy of Articaine Hydrochloride Ring Block in Antler Removal in Red Deer (Cervus elaphus)

New Zealand deer farming centres on the production of meat and velvet antler. Velvet antler removal is a painful procedure and currently, New Zealand Animal Welfare regulations dictate surgical removal of velvet antlers under lignocaine anaesthesia. To improve our knowledge on the efficacy and duration of other local anaesthetics to mitigate pain after antler removal, it is important to accurately assess and quantify pain arising from antler removal. Therefore, the current study was designed to validate mechanical nociceptive threshold (MNT) testing using a Wagner hand-held algometer, and to apply this methodology to assess the efficacy and duration of action of articaine for antler removal in deer. Baseline force (N) required to elicit the nociceptive response was recorded in 40 yearling male red deer on three alternate days. Ten of the 40 animals were selected for antler removal after administration of 4% articaine hydrochloride as a ring block. The duration of analgesic efficacy of articaine was assessed by algometry across 5 time points. There was a significant difference in MNTs among the three days (day 3 versus day 1 (p < 0.0001), day 2 versus day 1 (p < 0.0001), and day 1 versus day 2 (p < 0.01)). Positive correlations were observed between weight, antler length and thresholds. The MNT values remained above 20N for 6 h after removal of velvet antlers under the articaine ring block. This study provides valuable information about the use of MNT in red deer. These findings lay a foundation for future studies in the topics of peri-operative and postoperative pain management in deer antler removal, and a possible alternative use for articaine