Katie H. Sizeland

Collagen orientation and leather strength for selected mammals.

Collagen is the main structural component of leather, skin, and some other applications such as medical scaffolds. All of these materials have a mechanical function, so the manner in which collagen provides them with their strength is of fundamental importance and was investigated here. This study shows that the tear strength of leather across seven species of mammals depends on the degree to which collagen fibrils are aligned in the plane of the tissue. Tear-resistant material has the fibrils contained within parallel planes with little crossover between the top and bottom surfaces. The fibril orientation is observed using small-angle X-ray scattering in leather, produced from skin, with tear strengths (normalized for thickness) of 20-110 N/mm. The orientation index, 0.420-0.633, is linearly related to tear strength such that greater alignment within the plane of the tissue results in stronger material. The statistical confidence and diversity of animals suggest that this is a fundamental determinant of strength in tissue. This insight is valuable in understanding the performance of leather and skin in biological and industrial applications.

Poisson's ratio of collagen fibrils measured by small angle X-ray scattering of strained bovine pericardium

Type I collagen is the main structural component of skin, tendons, and skin products, such as leather. Understanding the mechanical performance of collagen fibrils is important for understanding the mechanical performance of the tissues that they make up, while the mechanical properties of bulk tissue are well characterized, less is known about the mechanical behavior of individual collagen fibrils. In this study, bovine pericardium is subjected to strain while small angle X-ray scattering (SAXS) patterns are recorded using synchrotron radiation. The change in d-spacing, which is a measure of fibril extension, and the change in fibril diameter are determined from SAXS. The tissue is strained 0.25 (25%) with a corresponding strain in the collagen fibrils of 0.045 observed. The ratio of collagen fibril width contraction to length extension, or the Poissons ratio, is 2.1 ± 0.7 for a tissue strain from 0 to 0.25. This Poissons ratio indicates that the volume of individual collagen fibrils decreases with increasing strain, which is quite unlike most engineering materials. This high Poissons ratio of individual fibrils may contribute to high Poissons ratio observed for tissues, contributing to some of the remarkable properties of collagen-based materials.

Changes to Collagen Structure during Leather Processing

As hides and skins are processed to produce leather, chemical and physical changes take place that affect the strength and other physical properties of the material. The structural basis of these changes at the level of the collagen fibrils is not fully understood and forms the basis of this investigation. Synchrotron-based small-angle X-ray scattering (SAXS) is used to quantify fibril orientation and D-spacing through eight stages of processing from fresh green ovine skins to staked dry crust leather. Both the D-spacing and fibril orientation change with processing. The changes in thickness of the leather during processing affect the fibril orientation index (OI) and account for much of the OI differences between process stages. After thickness is accounted for, the main difference in OI is due to the hydration state of the material, with dry materials being less oriented than wet. Similarly significant differences in D-spacing are found at different process stages. These are due also to the moisture content, with dry samples having a smaller D-spacing. This understanding is useful for relating structural changes that occur during different stages of processing to the development of the final physical characteristics of leather.

Age Dependent Differences in Collagen Alignment of Glutaraldehyde Fixed Bovine Pericardium

Bovine pericardium is used for heart valve leaflet replacement where the strength and thinness are critical properties. Pericardium from neonatal animals (4–7 days old) is advantageously thinner and is considered as an alternative to that from adult animals. Here, the structures of adult and neonatal bovine pericardium tissues fixed with glutaraldehyde are characterized by synchrotron-based small angle X-ray scattering (SAXS) and compared with the mechanical properties of these materials. Significant differences are observed between adult and neonatal tissue. The glutaraldehyde fixed neonatal tissue has a higher modulus of elasticity (83.7 MPa) than adult pericardium (33.5 MPa) and a higher normalised ultimate tensile strength (32.9 MPa) than adult pericardium (19.1 MPa). Measured edge on to the tissue, the collagen in neonatal pericardium is significantly more aligned (orientation index (OI) 0.78) than that in adult pericardium (OI 0.62). There is no difference in the fibril diameter between neonatal and adult pericardium. It is shown that high alignment in the plane of the tissue provides the mechanism for the increased strength of the neonatal material. The superior strength of neonatal compared with adult tissue supports the use of neonatal bovine pericardium in heterografts.

Collagen cross linking and fibril alignment in pericardium

The influence of natural cross linking by glycosaminoglycan (GAG) on the structure of collagen in animal tissue is not well understood. Neither is the effect of synthetic cross linking on collagen structure well understood in glutaraldehyde treated collagenous tissue for medical implants and commercial leather. Bovine pericardium was treated with chondroitinase ABC to remove natural cross links or treated with glutaraldehyde to form synthetic cross links. The collagen fibril alignment was measured using synchrotron based small angle X-ray scattering (SAXS) and supported by atomic force microscopy (AFM) and histology. The alignment of the collagen fibrils is affected by the treatment. Untreated pericardium has an orientation index (OI) of 0.19 (0.06); the chondroitinase ABC treated material is similar with an OI of 0.21 (0.08); and the glutaraldehyde treated material is less aligned with an OI of 0.12 (0.05). This difference in alignment is also qualitatively observed in atomic force microscopy images. Crimp is not noticeably affected by treatment. It is proposed that glutaraldehyde cross linking functions to bind the collagen fibrils in a network of mixed orientation tending towards isotropic, whereas natural GAG cross links do not constrain the structure to quite such an extent.

Acellular dermal matrix collagen responds to strain by intermolecular spacing contraction with fibril extension and rearrangement

Acellular dermal matrix (ADM) materials are used as scaffold materials in reconstructive surgery. The internal structural response of these materials in load-bearing clinical applications is not well understood. Bovine ADM is characterized by small-angle X-ray scattering while subjected to strain. Changes in collagen fibril orientation (O), degree of orientation as an orientation index (OI) (measured both edge-on and flat-on to the ADM), extension (from d-spacing changes) and changes to intermolecular spacing are measured as a result of the strain and stress in conjunction with mechanical measurements. As is already well established in similar systems, when strained, collagen fibrils in ADM can accommodate the strain by reorienting by up to 50° (as an average of all the fibrils). This reorientation corresponds to the OI increasing from 0.3 to 0.7. Here it is shown that concurrently, the intermolecular spacing between tropocollagen decreases by 10% from 15.8 to 14.3Å, with the fibril diameter decreasing from 400 to 375Å, and the individual fibrils extending by an average of 3.1% (D-spacing from 63.9 to 65.9nm). ADM materials can withstand large strain and high stress due to the combined mechanisms of collagen reorientation, individual fibril extension, sliding and changes in the molecular packing density.

Artificially modified collagen fibril orientation affects leather tear strength: Artificially modified collagen fibril orientation affects leather tear strength

BACKGROUND Ovine leather has around half the tear strength of bovine leather and is therefore not suitable for high-value applications such as shoes. Tear strength has been correlated with the natural collagen fibril alignment (orientation index, OI). It is hypothesized that it could be possible to artificially increase the OI of the collagen fibrils and that an artificial increase in OI could increase tear strength. RESULTS Ovine skins, after pickling and bating, were strained biaxially during chrome tanning. The strain ranged from 2 to 15% of the initial sample length, either uniformly in both directions by 10% or with 3% in one direction and 15% in the other. Once tanned, the leather tear strengths were measured and the collagen fibril orientation was measured using synchrotron-based small-angle X-ray scattering. CONCLUSION The OI increased as a result of strain during tanning from 0.48 to 0.79 (P = 0.001) measured edge-on and the thickness-normalized tear strength increased from 27 to 43 N mm-1 (P < 0.001) after leather was strained 10% in two orthogonal directions. This is evidence to support a causal relationship between high OI (measured edge-on), highly influenced by thickness, and tear strength. It also provides a method to produce stronger leather.

The influence of water, lanolin, urea, proline, paraffin and fatliquor on collagen D-spacing in leather

Water interacts with collagen to alter the structure at the fibrillar scale and therefore the mechanical properties of collagen. Humectants or moisturizers also alter the mechanical properties and fibril structure. The nature of these interactions and relationship between the different additives is not well understood. Changes in collagen D-spacing in leather were measured by synchrotron based small angle X-ray scattering in samples stored at various relative humidities and treated with lanolin, fatliquor, urea, proline or paraffin. The D-spacing increased with rising humidity and with increasing lanolin or fatliquor content, but not with treatment with urea, proline or paraffin. Strength increased with the addition of lanolin. Lanolin and fatliquor were shown to act as humectants whereas the other components did not act in this way. The Hofmeister effect is shown not to be a factor in the change in D-spacing, since samples treated with either proline or urea exhibited the same behavior. Different agents used in leather treatment and skin care function by different mechanisms, with collagen water retention being important for some additives but not others.

Effect of collagen packing and moisture content on leather stiffness

Applications for skin derived collagen materials, such as leather and acellular dermal matrices, usually require both strength and flexibility. In general, both the tensile modulus (which has an impact on flexibility) and strength are known to increase with fiber alignment, in the tensile direction, for practically all collagen-based tissues. The structural basis for flexibility in leather was investigated and the moisture content was varied. Small angle X-ray scattering was used to determine collagen fibril orientation, elongation and lateral intermolecular spacing in leather conditioned by different controlled humidity environments. Flexibility was measured by a three point bending test. Leather was prepared by tanning under biaxial loading to create leather with increased fibril alignment and thus strength, but this treatment also increased the stiffness. As collagen aligns, it not only strengthens the material but it also stiffens because tensile loading is then applied along the covalent chain of the collagen molecules, rather than at an angle to it. Here it has been shown that with higher moisture content greater flexibility of the material develops as water absorption inside collagen fibrils produces a larger lateral spacing between collagen molecules. It is suggested that water provides a lubricating effect in collagen fibrils, enabling greater freedom of movement and therefore greater flexibility. When collagen molecules align in the strain direction during tanning, leather stiffens not only by the fiber alignment itself but also because collagen molecules pack closer together, reducing the ability of the molecules to move relative to each other.

Tropical Keratopathy (Florida Spots) in Cats

The authors used microscopy and synchrotron-based small-angle X-ray scattering analysis (SAXS) to describe lesions macroscopically typical of tropical keratopathy (“Florida spots”) from 6 cats on St Kitts. Microscopically, there were varying degrees of epithelial hyperplasia and thinning of the cornea (by 4% to 18%) due to loss of corneal stroma associated with dense accumulations of collagen in the superficial stroma. The collagen fibrils in lesions were wider and had more variable diameters (39.5 ± 5.0 nm, mean ± SD) than in normal corneas (25.9 ± 3.6 nm; P < .01). There were occasional vacuoles (<1 μm) in the corneal epithelial basement membrane but no evidence of inflammation, edema, stromal neovascularization, fibrosis, acid-fast organisms, or structures suggestive of a fungal organism. SAXS analysis showed collagen fibril diameters and variation in size were greater in stroma containing the lesions compared to normal corneas (48.8 ± 4.5 nm vs 35.5 ± 2.6; P < .05). The d-spacing of collagen in the stroma of lesions and normal corneas was the same, but the average orientation index of collagen in lesions was greater (0.428 ± 0.08 vs 0.285 ± 0.03; P < .05). A survey revealed Florida spots lesions were static over time and became less obvious in only 1 of 6 affected cats adopted on St Kitts and taken to areas in the US where lesions are not reported. An anterior stromal collagen disorder with various degrees of epithelial hyperplasia is the pathologic hallmark of lesions clinically identical to Florida spots in cats from St Kitts.