Use of small angle x-ray scattering in investigations of leather and the cornea : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatū, New Zealand

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Collagen is the most abundant protein in the body and the major structural component of skin and the cornea, where it provides strength and is an important physical and chemical barrier against the environment. The biological function of collagen lies predominantly in its mechanical properties where its structural arrangement greatly influences the tissue characteristics. Understanding collagen structure, its properties and how these are affected by processing, is essential for the manufacture of skin products with superior function and when considering collagen in abnormal corneal tissue. Leather is derived from skins of various animals, providing aesthetically pleasing products that are strong and hard wearing because of their collagen structure. Collagen is comprised of fibrils which have been studied here in leather produced from skins of ovine (sheep), bovine (cattle) and cervine (deer) origins. Small angle X-ray scattering (SAXS) was used to evaluate the collagen fibril structure and alignment in leather, processed normally and by stretch-tanning, along with tear and bend testing. The average collagen fibril direction at standard sampling points in all species was perpendicular to the backbone, with the average fibril orientation relative to the backbone being 44° in cervine, 66° in bovine and 79° in ovine. The orientation index (OI) suggests the relative alignment of the fibrils, where 1 is perfectly aligned and 0 is randomly aligned. The OI was lowest in cervine (0.24), suggesting a more mesh-like arrangement, increasing in bovine (0.38) and highest in ovine (0.44) where fibrils lay more parallel to one another. There was considerable and unpredictable variability in collagen arrangements in each species but a significant difference in tear strength with ovine leather (21 N/mm) being weakest, and cervine leather (53 N/mm) stronger than bovine leather (43 N/mm), making ovine leather not suitable for high value applications like footwear. Previous correlations between leather strength and fibril alignment suggest greater alignment led to greater strength. When fibrils were aligned artificially by stretch-tanning, the OI in ovine leather increased from 0.48 to 0.79 as did the strength from 27 to 43 N/mm, making it comparable to bovine leather strength. Measurements of the bend modulus of stretch-tanned ovine leather, which was stiffer than the non-stretch tanned leather (15 vs. 34 kPa), when conditioned under increasing relative humidity environments, during which water was incorporated into leather’s collagen structure, resulted in a 66% reduction in stiffness. Examination of clinically normal sheep corneas were used to determine effects of common preservatives on collagen structures using SAXS. Compared to the control, frozen cornea, there was a significant increase in the fibril diameter and D-spacing of collagen in corneas stored for 5 days in all the preservatives studied (5% glutaraldehyde, 10% formalin, Triton X and phosphate buffered saline). Corneas from cats with corneal opacities (Florida spots) that were studied using histology, transmission electron microscopy (TEM) and SAXS showed that there was less collagen in the stroma of the lesions. Here collagen fibrils had larger and more variable diameters (32 nm vs. the normal 27 nm), and a greater relative alignment (OI) compared to normal corneas (0.43 vs. 0.29, respectively). These changes explain the opacity of the lesions as corneal transparency depends on regular small fibril diameters which are aligned orthogonally. The above studies have demonstrated the usefulness of SAXS in characterizing collagen in natural, pathological, and mechanically and chemically altered collagen-based samples.
Collagen, Structure, Small-angle x-ray scattering, Leather, Cornea, Effect of chemicals on, Diseases