Copyright is owned by the Author of the thesis.  Permission is given for 
a copy to be downloaded by an individual for the purpose of research and 
private study only.  The thesis may not be reproduced elsewhere without 
the permission of the Author. 
 



 

 

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. 

 

S.J.R. Kelly 

2018





 

i 
 

Abstract  
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.  



 

ii 
 

Acknowledgements 
First and foremost I want to thank my supervisor Professor Richard Haverkamp. It has been a great honour to 

be one of you PhD students. Thank you to my co-supervisors Dr Katie Sizeland and Dr Hannah Wells for 

showing me the ropes and for your on-going support. 

The Australian Synchrotron and the SAXS/WAXS Beamline in Melbourne, Australia have provided a significant 

portion of the results presented in the publications from this thesis. To the beamline scientists Dr Nigel Kirby, 

Dr Stephen Mudie and Dr Tim Ryan, you were fundamental in data collection and processing, for that I thank 

you. 

The work on leather in Chapters 3 to 6 was made possible through the support of Dr Richard Edmonds, Dr 

Geoff Holmes and Dr Sue Cooper at the New Zealand Leather and Shoe Association. Your advice and expertise 

in the leather industry have been indispensable for these chapters.  

Chapters 5 & 6 were supported by Dr Richard Weinkamer, Dr Luca Bertinetti and Prof Peter Fratzl at the Max 

Planck Institute for Colloids and Interfaces in Potsdam, Germany. Working alongside you has been a 

memorable highlight.  I have appreciated the camaraderie and local expertise of Mrs Ingrid Zenke in using the 

Nanostar and Mr Klaus Bienert for building a custom humidity cell. In my attempted measurements of leather 

collagen structures using Raman Spectroscopy, Dr Clemens Schmitt is thanked for his time and curiosity. 

In my later work investigating collagen structures in the cornea, presented in Chapters 7 & 8, thank you to Prof 

Patrick Kelly and Dr Pompeii Bolfa from Ross University School of Veterinary Medicine in St Kitts, West Indies 

for the collaboration, Dr Fernanda Castillo from the School of Veterinary Science at Massey University for the 

samples and Ms Jordan Taylor of the Manawatū Microscopy Centre for her assistance in electron microscopy 

sample preparation and imaging. 

For this dissertation I would like to thank my proof readers for their time, interest and helpful comments. Also 

thanks to the members of my oral defence committee for their insightful questions. 

I gratefully acknowledge the funding sources that made my PhD work possible. Thank you to the New Zealand 

Leather and Shoe Research Association for co-funding the project through the LSRX1301 Ministry of Business, 

Innovation and Employment grant and to the New Zealand Synchrotron Group for providing travel funding for 

the various synchrotron trips. 

Thank you to Mum, Sean and my friends for your sanity checks and a special thank you to my Dad for all his 

insights and support.   



 

iii 
 

Contents 
Abstract ........................................................................................................................................................ i 
Acknowledgements ..................................................................................................................................... ii 
List of Figures ............................................................................................................................................... v 
Introduction ................................................................................................................................................. 1 
Chapter 1. Collagen Structure and Function ............................................................................................... 2 
Collagen Structure ....................................................................................................................................... 2 

The Collagen Molecule (Tropocollagen) .................................................................................................. 2 
Collagen Fibrils ........................................................................................................................................ 3 
Collagen Fibres ........................................................................................................................................ 4 

Mechanical Properties ................................................................................................................................. 4 
Stress and Strain ...................................................................................................................................... 5 

Skin .............................................................................................................................................................. 8 
Mechanical Properties of Skin ................................................................................................................. 9 

Leather......................................................................................................................................................... 9 
Leather Production ................................................................................................................................ 10 
Mechanical Properties of Leather ......................................................................................................... 12 

Cornea ....................................................................................................................................................... 12 
Cornea Structure ................................................................................................................................... 12 
Properties of the Cornea ....................................................................................................................... 14 

Chapter 2. Characterizing Collagen Structures.......................................................................................... 16 
Imaging at the Nano-level ......................................................................................................................... 16 

Synchrotrons ......................................................................................................................................... 16 
SAXS and WAXS ..................................................................................................................................... 18 
Electron Microscopy .............................................................................................................................. 24 

Mechanical Testing .................................................................................................................................... 25 
Tear Strength Measurements................................................................................................................ 26 
Tensile Strength Measurements ........................................................................................................... 26 
Three Point Bend Measurements .......................................................................................................... 27 

Isothermal-gravimetric Analysis ................................................................................................................ 27 
Chapter 3. Mapping Tear Strength and Collagen Fibril Orientation in Bovine, Ovine and Cervine Skins . 29 
Abstract ..................................................................................................................................................... 29 
Introduction ............................................................................................................................................... 30 
Methods .................................................................................................................................................... 31 
Results ....................................................................................................................................................... 33 
Discussion .................................................................................................................................................. 39 
Conclusions ................................................................................................................................................ 40 
Chapter 4. Artificially modified collagen Fibril Orientation Affects Leather Tear Strength ...................... 41 
Abstract ..................................................................................................................................................... 41 
Introduction ............................................................................................................................................... 42 
Methods .................................................................................................................................................... 43 
Results ....................................................................................................................................................... 45 
Discussion .................................................................................................................................................. 50 
Conclusions ................................................................................................................................................ 52 
Chapter 5. Effect of moisture content on collagen packing and stiffness in stretch-tanned leather ....... 53 
Abstract ..................................................................................................................................................... 53 
Introduction ............................................................................................................................................... 54 
Methods .................................................................................................................................................... 55 
Results ....................................................................................................................................................... 59 
Discussion .................................................................................................................................................. 65 
Conclusions ................................................................................................................................................ 67 
Chapter 6. Data on collagen structures in leather with varying moisture contents ................................. 68 
Abstract ..................................................................................................................................................... 68 
Value of the Data ....................................................................................................................................... 69 
Data ........................................................................................................................................................... 69 
Experimental Design, Materials, and Methods ......................................................................................... 73 
 

 



 

iv 
 

Chapter 7. A small angle X-ray scattering study of changes caused by preservation on the cornea ........ 74 
Abstract ..................................................................................................................................................... 74 
Introduction ............................................................................................................................................... 75 
Methods .................................................................................................................................................... 76 
Results ....................................................................................................................................................... 77 
Discussion .................................................................................................................................................. 80 
Conclusions ................................................................................................................................................ 81 
Chapter 8. Tropical Keratopathy (Florida Spots) in Cats ........................................................................... 82 
Abstract ..................................................................................................................................................... 82 
Introduction ............................................................................................................................................... 83 
Methods .................................................................................................................................................... 83 
Results ....................................................................................................................................................... 86 
Discussion .................................................................................................................................................. 91 
Conclusion ................................................................................................................................................. 93 
Overall Conclusions ................................................................................................................................... 94 
References ................................................................................................................................................. 96 
Appendix A. List of Publications .............................................................................................................. 104 
Appendix B. Poster Presentations ........................................................................................................... 106 
Appendix C. Oral Presentations .............................................................................................................. 109 
Appendix D. Statement(s) of Contribution towards Publication ............................................................ 113 

 
  



 

v 
 

List of Figures 
Figure 1. Hydrogen bonding between sequential peripheral amino acid side chains (Xx, Yy) maintaining the 
helical structure of the alpha (α) chain................................................................................................................... 2 
Figure 2. The collagen molecule triple helix. .......................................................................................................... 3 
Figure 3. Collagen fibril assembly. .......................................................................................................................... 3 
Figure 4. Collagen fibrils bundled together into fibres through cross-linkages with proteoglycans in the 
extracellular matrix. ................................................................................................................................................ 4 
Figure 5. Stress – Strain curve for a relatively aligned collagen-based tissue. ....................................................... 6 
Figure 6. Isotropic (left) and anisotropic (right) materials. ..................................................................................... 8 
Figure 7. Three primary layers of skin .................................................................................................................... 9 
Figure 8. A cross section of ovine leather showing two distinct layers ................................................................ 10 
Figure 9.  A cross section of the eye showing the location of the cornea ............................................................ 13 
Figure 10.  (a) Stroma collagen fibrils organized in parallel arrays in lamellae seen in cross section and 
transverse section. Keratocytes are interspersed between adjacent lamellae. Image of collagen fibril arranged 
into lamellae in the human cornea (b) Collagen fibrils seen in cross section are arranged in a quasi-hexagonal 
lattice by ionic forces between fibrils, proteoglycans, and water and ions in the matrix. ................................... 14 
Figure 11.  Collagen lamellae short-range order for transparency of visible light. .............................................. 15 
Figure 12.  Corneal oedema in a human after the Descemet’s layer and the endothelial cells that control 
stromal hydration have been detached. .............................................................................................................. 15 
Figure 13. Hierarchical structure of collagen and the techniques used to view its structural features. .............. 16 
Figure 14. The basic components of a synchrotron. ............................................................................................ 17 
Figure 15. Synchrotron radiation traveling down a beamline. ............................................................................. 18 
Figure 16. Small angle X-Ray scattering (SAXS) and Wide angle X-Ray scattering (WAXS) synchrotron beamline 
configurations. ...................................................................................................................................................... 18 
Figure 17. Bragg's law describing the diffraction of X-rays through a lattice. ...................................................... 19 
Figure 18. Laboratory source small and wide angle X-ray scattering (SAXS/WAXS) beamline at the Max Planck 
Institute for Colloids and Interfaces, Germany (left) and a synchrotron source SAXS/WAXS beamline at the 
Australian Synchrotron, Australia (right). ............................................................................................................. 20 
Figure 19. Equatorial and meridional scattering from (a) small angle X-ray scattering (SAXS) and (b) wide angle 
X-ray scattering (WAXS) used to gather collagen specific information. ............................................................... 21 
Figure 20. From SAXS integrated intensity profiles (a) fibril diameter and D-spacing are determined in the SAXS 
q-range with (b) further integration of the 6th order peak over the azimuthal angle to determine the preferred 
fibril orientation (orientation index, OI). From WAXS integrated profiles the (c) intermolecular spacing can be 
determined from measurements taken at this higher WAXS q-range which provides smaller structural 
information. .......................................................................................................................................................... 22 
Figure 21. Fibril orientation producing scatter patterns from an (a) anisotropic (highly aligned) sample and an 
(b) isotropic (randomly aligned) sample ............................................................................................................... 23 
Figure 22. Electron microscopy: (a) scanning electron microscopy (SEM) collecting scattered electrons at the 
detector and (b) transmission electron microscopy (TEM) collecting transmitted electrons at the detector. .... 24 
Figure 23. Scanning electron micrscopy of (a) leather cross section showing the two distinct layers and (b) 
collagen fibers in the corium layer. Transmission electron microscopy of (c) cross-section of corneal collagen 
and (d) side view of corneal collagen showing characteristic D-spacing. ............................................................. 25 
Figure 24. Tear test ............................................................................................................................................... 26 
Figure 25. Tensile test .......................................................................................................................................... 27 
Figure 26. Three point bend test setup. ............................................................................................................... 27 
Figure 27. Isothermal-gravimetric analysis connected to a humidity generator for sorption isotherms ............ 28 
Figure 28. Grid pattern used for sampling hides showing the locations of the samples taken for tear testing and 
small angle X-ray scattering (SAXS) measurements (solid grey squares) at (a) general locations and at (b) the 
official sampling position (OSP). ........................................................................................................................... 32 
Figure 29. Ovine skin (I), Bovine hide (II) and Cervine hide (III) collagen orientation, orientation index and 
directional tear strength map. .............................................................................................................................. 35 
Figure 30. Small angle X-ray scattering (SAXS) of leather from the official sampling position (OSP) of hides 
halved from neck to tail. ....................................................................................................................................... 36 
Figure 31. Collagen orientation (O) represented by the vector direction at different small angle x-ray scattering 
(SAXS) measuring positions in samples from the official sampling position ........................................................ 37 
Figure 32. Relationship between orientation (O) and tear strength measured perpendicular (filled circles) and 
parallel (hollow circles) to the backbone for (a, b) ovine (c, d) bovine and (e, f) cervine half skins/hides .......... 38 



 

vi 
 

Figure 33. Relationship between tear strength measured perpendicular (black) and parallel (grey) with the 
orientation index (OI) along the (a) backbone and (b) belly for ovine (square), bovine (circle) and cervine 
(diamond). ............................................................................................................................................................ 39 
Figure 34. Sampling positions for stretching and tearing from the official sample position (OSP) on the Ovine 
skin. ....................................................................................................................................................................... 43 
Figure 35. Orientation of sample relative to the X-ray beam to produce edge-on measurements. .................... 44 
Figure 36. An example of the SAXS pattern (a) and its corresponding intensity profile (b) for a stretched leather 
sample. Integrated intensity plots (I(q)) at the 5th diffraction peak for non-stretched (c) and stretched (d) 
leather samples measured edge-on to X-ray beam.............................................................................................. 46 
Figure 37. Bi-axial stretching and its effect on the edge-on (a) and flat-on (b) orientation index. ...................... 47 
Figure 38. Bi-axial stretching and its effect on normalized tear strength parallel to the backbone (a) and 
perpendicular (b). ................................................................................................................................................. 49 
Figure 39. Edge-on orientation index and normalized tear strength torn parallel (a) and perpendicular (b) to 
the backbone. ....................................................................................................................................................... 49 
Figure 40. Tanning under strain (ɛ) and its effect on fibril alignment. ................................................................. 50 
Figure 41. Tear propagates more (a) readily along the direction of the collagen fibrils and is (b) resisted by 
collagen when arranged perpendicular to the movement of the jaws for the tear test. ..................................... 51 
Figure 42. Scanning electron microscopy images of the (a) control, non-stretch tanned leather and (b) stretch 
tanned leather cross-sections showing the grain (top) and corium layers with a comparable scale. .................. 59 
Figure 43. 2D Scattering patterns from (a) long sample to detector configuration (3.30 m) for Small Angle X-ray 
Scattering (SAXS) measurements over a low Q-range (0.01 – 0.15 Å-1) and (b) short sample to detector (0.56 m) 
for SAXS measurements over a high Q-range (0.10 – 1.00 Å-1) ............................................................................ 60 
Figure 44. Small Angle X-ray scattering intensity spectrums for the (a, b) control and (c, d) stretched leather 
under the long camera to detector configuration (a, c) and the short camera to detector configuration (b, d) 
after relative humidity pre-conditioning. ............................................................................................................. 61 
Figure 45. Variations in D-spacing from edge-on measurements with moisture content ................................... 62 
Figure 46. Variations in lateral intermolecular spacing from edge-on measurements with moisture contents. 62 
Figure 47. Variations in the orientation index from edge-on SAXS measurement with moisture contents. ....... 63 
Figure 48. Variations in Young’s Modulus with moisture contents from relative humidity pre-conditioning ..... 64 
Figure 49. Effect of moisture content in the (a) grain (hollow shapes) and (b) corium (filled shapes) of control, 
non-stretch tanned leather (circles) and stretch tanned leather (squares) on the lateral intermolecular spacing 
(black) and Young’s modulus (blue). .................................................................................................................... 64 
Figure 50. Isothermal gravimetric analysis at various relative humidity environments to determine leather 
moisture content .................................................................................................................................................. 70 
Figure 51. Variations in D-spacing from flat on measurements with moisture content in (a) control leather and 
(b) stretch tanned leather. ................................................................................................................................... 70 
Figure 52. Variations in lateral intermolecular spacing from flat on measurements with moisture content on (a) 
control leather and (b) stretch tanned leather..................................................................................................... 71 
Figure 53. Force deflection curves for (a) control leather and (b) stretch tanned leather .................................. 73 
Figure 54. Transparency of sheep corneas preserved in 5% glutaraldehyde (G), 10% formalin (F), Triton X (T), 
phosphate buffered saline (S) and a frozen and thawed untreated control (FF). ................................................ 77 
Figure 55. Photo images of 2D small angle X-ray scattering patterns produced by frozen and thawed control 
sheep corneas (FF) and corneas preserved in 5% glutaraldehyde (G), 10% formalin (F), Triton X (T), and 
phosphate buffered saline (S). ............................................................................................................................. 77 
Figure 56. Means and standard deviations of (a) orientation indices, (b) D-spacings and (c) fibril diameters of 
collagen in frozen and thawed control sheep corneas. ........................................................................................ 78 
Figure 57. Transmission electron microscopy images of collagen fibril cross-sections in the stroma of sheep 
corneas treated with 5% glutaraldehyde (G), 10% formalin (F), Triton X (T) and phosphate buffered saline (S).79 
Figure 58. Tropical Keratopathy, eye, cat. (1) Case 3. The right cornea contains multifocal to coalescing 
leukomatous lesions of various sizes (arrows). The centre is denser than the periphery of the lesion; (2) Case 2. 
Bilateral corneal opacities. ................................................................................................................................... 84 
Figure 59. Photo images of the radial X-ray diffraction patterns produced by affected (3a) and normal (4a) 
collagen................................................................................................................................................................. 85 
Figure 60. Typical histological changes seen in hematoxylin and eosin stained sections of the left eye............. 87 
Figure 61. Tropical keratopathy, cornea (left eye), cat. ....................................................................................... 88 
Figure 62. Cornea, cat. Transmission electron microscopy.. ................................................................................ 89 
Figure 63. Collagen fibril size distribution in normal cornea (17) and in Tropical Keratopathy lesion (18) ......... 90 
Figure 64. Collagen parameters measured by small-angle X-ray scattering (SAXS) in normal corneas and those 
with Florida spots ................................................................................................................................................. 90 



Kelly 2018 

1 
 

Introduction 

Collagen-based materials are fibre composites with a hierarchical structure. Their exceptional mechanical 

properties are believed to be due to functional structural adaptations at all levels of hierarchy. Understanding 

natural collagen structures and how they impart particular characteristics in native tissue and in leather 

manufacturing will provide insights into how desirable properties can be artificially enhanced by mechanical 

and chemical processing. 

The research aims for this dissertation were to understand how collagen arrangements influence mechanical 

properties in leather and optical properties in the cornea. The following research questions were established 

to enable these to be met: 

• What is the variation in collagen alignment and strength across the skins used for leather and does this 

change between species? Are the variations significant enough that certain species or regions within a skin 

are better suited to specific applications? Findings presented in Chapter 3. 

• Highly aligned collagen fibres have been correlated with leather of high strength. In weak leather the 

alignment is low which poses the question: is it possible to artificially align collagen fibres in skins known 

to produce weak leather and what effect does this have on the leather strength? Findings presented in 

Chapter 4. 

• Strength and stiffness are intertwined properties. During stretch-tanning we increase the leather's 

strength while also increasing the stiffness. Water is known to mitigate stiffness in collagen-based 

materials. What is the effect of water on leather stiffness? Findings presented in Chapters 5 & 6. 

• Corneal collagen has a very precise arrangement allowing for optical transparency. Examined tissue has 

often been preserved which has a notable effect on tissue transparency. How do common preservatives 

interfere with the collagen structure in the cornea? Findings presented in Chapter 7. 

• Tropical Keratopathy is a corneal disease that causes opaque lesion in the cornea. Can we characterize the 

collagen structures in both normal and opaque sections of a cornea? Are the collagen structures 

comparable and can structural changes be linked to opacity? Findings presented in Chapter 8. 

These questions have formed the research incentives behind the publications presented in this thesis. The 

approach to answering these questions utilized a combination of techniques to form structure/function 

relationships in leather and the cornea. X-ray techniques for small and wide angle X-ray scattering and 

transmission electron microscopy have provided nano-structural information, while industry defined 

mechanical testing standards for leather were used to measure mechanical performance. 



Kelly 2018 

2 

Chapter 1. Collagen Structure and Function 
Collagen is the most abundant fibrous protein in animals where it provides strength and structure to a variety 

of soft and hard tissues. The key structural elements of collagen are its component fibres which are 

anisotropic, that is, they can impart different properties to tissues depending on the manner in which they are 

arranged1, 2, 3. When all are of uniform size, they confer optical transparency to the cornea4, if aligned side-by-

side they provide exceptional strength as in tendon and leather5, 6. When randomly arranged they provide 

resistance to tear propagation and flexibility for example in skin7. In the following two chapters, the collagen 

structure of the skin, leather and the cornea are reviewed and the experimental methods used to analyse 

collagen structure are described. 

Collagen Structure 

About one third of the protein in the body consists of collagen8, 9 which is the major structural component of 

many tissues including tendons, ligaments, skin,  the cornea, cartilage, bone, dentin and blood vessels10. There 

are 28 different types of collagen, each having distinct polypeptide chain components. The most abundant is 

type I collagen which is responsible for strength and flexibility in the skin and leather6, 11-16, and the structure 

and optical transparency of the cornea4, 17, 18. The amino acid sequence and fibril and fibre structure of type I 

collagen is described below. 

The Collagen Molecule (Tropocollagen) 

Collagen is composed of polypeptides containing repeating units of three amino acids ((Gly-Xx-Yy)n), primarily 

glycine (Gly) with proline, hydroxyproline or alanine16, 19. These repeating units (n = 337 to 343) are arranged 

sequentially in a left hand spiral to form an alpha (α) chain.  Each α chain is tightly coiled (3.3 amino acids per 

twist) and held in this position by hydrogen bonds between the more peripheral amino acids20 (Figure 1). 

Glycine with its small side chain, a single hydrogen molecule, is located at the centre of the helix. 

 

Figure 1. Hydrogen bonding between sequential peripheral amino acid side chains (Xx, Yy) maintaining the helical structure 
of the alpha (α) chain. 

Hydrogen bonding between amino acids in adjacent α chains and with surrounding water molecules results in 

three α chains coiling together to form a right handed triple helix which is the basic collagen molecule (also 

known as tropocollagen). Each collagen molecule contains 1050 amino acids21 and is approximately 300 nm in 

length with a diameter of approximately 1.6 nm22, 23 (Figure 2).  



Kelly 2018 

3 

 

Figure 2. The collagen molecule triple helix. There are three α chains comprising around 340 repeating amino acid 
sequences ((Gly-Xx-Yy)n) wound together in a right handed twist. The small glycine (Gly) amino acid is located in the centre 
of the helix with larger amino acids in the Xx and Yy positions. Molecules are about 1.6 nm wide and 300 nm long. 
Hydrogen bonds between amino acid side chains (Figure 1) and hydrogen bonds with surrounding water molecules 
maintain the structure.  

Collagen Fibrils 

Collagen molecules are secreted into the extracellular matrix24 where they self-assemble25 side-by-side to form 

collagen fibrils of around 50 – 200 nm in diameter (Figure 3). Although the collagen molecules lie parallel to 

the length of the fibril they are aligned in a staggered pattern, each molecule being offset from its neighbour 

by approximately 67 nm. The collagen molecules are held together in this staggered pattern by aldol cross-

linkages of lysine, hydroxylysine or arginine at the ends of the collagen molecules26-28. The areas of the 

collagen fibril where there is complete overlap of the adjacent component collagen molecules appear as dark 

bands microscopically, while areas where there is no overlap between some molecules appear as pale bands. 

The distance between a dark band, that is the area where all collagen molecules in a fibril overlap, and a pale 

band, where all the collagen molecules do not overlap, is termed the D-spacing29, 30 (or D-period). This varies 

with the types of amino acids in the fibrils and can be used to characterise the different types of collagen. 

Typically type I collagen in the skin normally has a D-spacing of 67 ± 0.5 nm31.  

 

Figure 3. Collagen fibril assembly. Cross links between collagen molecules cause a staggered arrangement creating overlap 
and gap regions resulting in the characteristic banding pattern along the collagen fibril when viewed microscopically. The 
combination of an overlap and gap region is termed the D-spacing which is approximately 67 nm in type I fibrillar collagen.  



Kelly 2018 

4 

The D-spacing is also influenced by hydrogen bonding between amino acids in the fibrils and the surrounding 

water molecules, hence the hydration state of the collagen. In this way the D-spacing in type I collagen found 

in the skin can vary from approximately 68 nm when the tissue is fully hydrated to 64 nm when dehydrated32. 

A greater variation in D-spacing exists across all type 1 collagen tissue, such as cornea, tendon and bone33. The 

diameters of the collagen fibrils vary, depending on the number of collagen molecules stacked side-by-side, 

influencing the properties of the tissues in which they are found: corneal collagen fibril diameter is around 31 

nm for optical transparency4 while fibrils in tendons are larger at 31 to 220 nm for elasticity and strength34. 

Collagen Fibres  

Collagen fibres are approximately 10 µm in diameter (in leather35) and are made up of bundles of fibrils that 

are arranged in parallel. The ultimate mechanical and transparency properties of tissue containing collagen 

fibres are partly determined by the ways in which fibrils are arranged5, 36-39. The distances separating the fibrils 

in a collagen fibre are largely determined by the matrix that surrounds them. This matrix contains 

proteoglycans that regulate the water content and also attach to the fibrils in the gap region (Figure 4). 

Electrostatic forces between proteoglycans lead to the fibrils being maintained at optimal distances for 

transparency, as in the case of the stroma,  and mechanical strength in tendons, skin and other connective 

tissue4. 

 

Figure 4. Collagen fibrils bundled together into fibres through cross-linkages with proteoglycans in the extracellular matrix. 

Mechanical Properties  

The properties of tissues containing collagen vary according to the arrangement of their collagen fibrils and 

fibres. For example, collagen fibres are found parallel to the direction of force in tendons, which provides 

maximal physical strength and resistance to rupture. At the same time the collagen fibres can act as shock 

absorbers, dissipating applied forces to some extent as fibrils and fibres slide passed one another. In the skin, 

the collagen fibres are generally more randomly arranged providing mechanical resistance to tearing in 

multiple directions. The ability of the fibrils and fibres to slide passed one-another in all directions also gives 

flexibility16. 

The number of cross-links between adjacent fibrils determines their ability to slide passed one another. More 

cross-linking between fibrils increases the stiffness of a tissue while less cross-linking facilitates sliding and 

gives flexibility and elasticity before permanent deformation occurs40. Cross-links can be artificially created 



Kelly 2018 

5 

between collagen molecules41-44 with aldehydes being widely used in tanning to impart mechanical strength to 

leather products45 and in histology to impart rigidity to soft tissue samples46. 

The ability of collagen fibrils to slide passed one another depends on the amount of contact they have with 

one another. The more contact a fibril has with its neighbour, the higher the internal friction generated when 

forces are applied and fibres are forced to slide passed one another. A parallel arrangement, or high relative 

alignment, of fibres generates the greatest internal friction due to high fibril-to-fibril contact. This means that 

tissues with highly aligned fibrils and fibres, such as tendon, have greater longitudinal strength due to both the 

physical strength of the collagen itself and also the internal friction between adjacent fibres. In the leather 

industry, areas of skin with highly aligned collagen are selected for tanning and manufacture of leather 

products that need to be able to resist linear forces such as belts, straps and harnesses6, 47.  

Tissues with collagen fibrils that have a more random arrangement, or low relative alignment, can be more 

easily stretched in all directions and therefore are more flexible. Similarly, the low relative alignment means 

the tissue is more resistant to tearing forces in any direction. Areas of skin that contain less aligned collagen 

fibres are selected for leather products that need to be flexible and tear resistant, for example gloves and 

clothes7. The alignment of collagen in different animals used in leather production varies, with cattle generally 

having greater alignment of collagen in their skins than sheep47. 

Various methods have been developed to measure the mechanical properties of skin.  

Stress and Strain 

Application of force (stress: tensile and/or compression) to materials while continuously recording the strain 

(deformation) provides insights into the materials mechanical properties. Stress (σ) represents an applied force 

over an area, while strain (Ɛ) represents the length of elongation relative to the original length of the material 

(Equation 1).  

σ =  F
A

      ;    ε =  (L−Lo)
Lo

 

Equation 1. Stress (σ) and strain (Ɛ) where F is the applied force, A is the area over which the force is applied, L is the 
length of elongation and LO is the original length of the sample. 

A plot of stress versus strain reveals the thresholds for elastic and plastic deformation by the yield strength 

and fracture point respectively, as well as other parameters such as the Young’s modulus, (discussed below) 

and ultimate strength that is the maximum force the sample can withstand. 

Analysing changes in collagen structure under varying stress has provided important information on how 

forces are dissipated by collagen-based materials (Figure 5). Initially, mechanical stress applied to a collagen 

containing tissue results in elastic deformation where knots in fibrils (so called fibrillar crimps which are seen 

to a greater extent in tendon due to an additional undulation of molecules48) are straightened out. This 

dissipates some of the applied strain resulting in only a slow increase in stress seen at the start of the stress-

strain curve, also known as the heel region (Figure 5 (a-b)) which is characteristic of entropic elasticity. After 

the crimping is removed, additional stress causes fibres to rotate and align in the direction of the strain49. The 



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6 

greater order/alignment of fibres relative to one another results in a more tightly packed fibre arrangement 

(Figure 5 (b-c))2. This creates a greater resistance to stress due to the greater internal friction between the 

more aligned fibres and the stretching of the cross-links resulting in linear elastic behaviour50. The ‘yield 

strength’ is the maximum stress that can be applied before the collagen structure is permanently altered, that 

is, with stresses up to the yield strength the collagen remains elastic and can return to its original shape when 

the stress is removed. Beyond this elastic deformation phase the effects of applied forces are no longer 

reversible and plastic deformation occurs. Here the collagen fibrils become stretched as a result of inter- and 

intra-fibrillar sliding with permanent elongation of the helical structure shown by an increase in the D-spacing 

(Figure 5 (d)). Above the ultimate strength, the fibrils and fibres rupture and ultimately the tissue fails at the 

fracture point (Figure 5 (e)).  

 

Figure 5. Stress – Strain curve for a relatively aligned collagen-based tissue during five stages of tensile loading and their 
relation to a schematic representation of the collagen fibres over the two major stages of elastic and plastic deformation. 
Applied strain is in the vertical direction. (a) Material at rest; (b) after fibril crimps are removed, marking the end of the 
heel region, the collagen fibres begin to straighten in the direction of strain; (c) fibres continue to straighten as more fibres 
orientate along the axis of strain making the material more tightly packed; (d) fibres begin to stretch; (e) fibres fracture.  

Flexibility (Young’s Modulus)  

The Young’s modulus, also referred to as the ‘elastic’ or ‘tensile’ modulus, is an intrinsic property of a material, 

which is a measure of material flexibility along the axis of strain in Pascal’s. This mechanical property is defined 

as the relationship between stress and strain in the linear elastic region of the stress-strain curve according to 

the factor of proportionality in the linear region under bend forces (Equation 2).  

𝑑𝐹
𝑑𝑥

=
4 𝐸 𝑎3𝑏
𝐿3

 

Equation 2. Young's Modulus (E) for a beam where L is the length of the beam, F is the force applied to the middle of the 

beam, a and b are the width and height of the beam, and x is the beam deflection due to the force applied. 

The Young’s modulus of a material tells us how much the material can be expected to deform along an axis 

when opposing forces are applied. Typical Young’s modulus values range from > 1 GPa in flexible materials like 



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rubber and low-density polyethylene, to values of > 10 GPa in stiffer materials like wood and bone. The 

Young’s modulus for collagen has been studied extensively under various methods resulting in a range of 

values for the same tissue. For example, hydrated individual fibrils have a Young’s modulus of approximately 

0.9 GPa51, collagen from rat tail tendon has a range of values from 3 - 20 GPa and bovine Achilles tendon of 2 - 

7 GPa, depending on the experimental method used, cross-linkages between fibrils and tissue hydration 

state14, 52. Flexural testing using a three-point bend test, applying a force perpendicular to ovine leather 

samples reveals its anisotropic two layer structure where the Young’s modulus values vary depending on the 

sample orientation. The difference between the Young’s modulus grain side up and corium side up is 

approximately 1.5 GPa16, 53 suggesting the grain to be more resistant to compression and/or the corium being 

less resistant to tension. 

The linear portion of a stress-strain curve, when bend forces are applied, is used to determine the materials 

bend/ flexural modulus (Equation 3). Data is collected from a three-point bend test, applying forces 

perpendicular to the sample (technique discussed in Section 2). The bend modulus is a measure of the 

materials stiffness under flexural deformation where the materials surface is submitted to the greatest values 

of stress.   

Ebend =  
𝑑𝐹
𝑑𝑥

 
1

4𝑤
 

Equation 3. Bend Modulus (Ebend) F is the force applied to the middle of the beam; w is the width of the beam, and x is the 
beam deflection due to the force applied. 

 Poisson’s ratio 

When comparing a material’s ability to resist distortion under a mechanical load, rather than altering its 

volume, Poisson’s ratio offers a measure to compare performance when a material is strain elastically under 

tensile forces. Materials under tensile forces experience elongation in the direction of the applied force and 

often contraction in the orthogonal direction, hence a negative strain in the direction perpendicular to the 

elongation strain. This relationship is summarized into the Poisson’s ratio (Equation 4) which can help to 

differentiate between materials that are isotropic or anisotropic (Figure 6). In a stable isotropic material, 

where the material has the same properties regardless of measurement direction, we expect a theoretical 

upper limit of 0.5 for Poisson’s ratio with a lower limit of -1. When a material has a negative Poisson’s ratio 

they are regarded as Auxetic materials since they become thicker perpendicular to the applied force. In an 

anisotropic material, where there are direction specific properties, it is possible for the Poisson’s ratio to 

exceed 0.5. However anisotropic materials can still exhibit a Poisson’s ratio below 0.554. 

ν = −
ε lateral

ε axial
=  

Ly −  Lo,y 
Lo,y

Lx −  Lo,x 
Lo,x

 

Equation 4. Poisson’s ratio (ν) where Ɛlateral is the strain in the direction of the applied force, Ɛaxial is the strain orthogonal to 
the applied force, Ly is the length after elongation, Lo,y is the original sample length in the lateral direction, Lx is the length 
after contraction and Lo,x is the original sample length in the axial direction. 



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Figure 6. Isotropic (left) and anisotropic (right) materials (Lo,x, Lo,y) under tensile forces (F) resulting in elongation, lateral to 
the applied force direction, Ly and contraction orthogonal to the applied force in the axial direction, Lx. 

Collagen-based materials are anisotropic in that the properties are direction specific, tailored to suit their 

function in living tissue, for example, tendons require direction specific strength and elasticity for 

performance. Since collagen is anisotropic, it is possible to get a Poisson’s ratio of > 0.5, suggesting the volume 

of the collagen fibrils decreases with strain. A range of collagen-based materials have been investigated. The 

Poisson’s ratio of the individual collagen fibrils, when the material is under tension, have been measured and 

values have been determined for bovine pericardium (ν = 2.1)55, human patella cartilage (ν = 1.3)56 and tendon 

fascicles (ν = 4)57. 

 

Skin 

The skin is a major organ in the body with three main functions: protection, temperature and moisture 

regulation and sensation58. It consists of three main layers, the epidermis which is the outer most layer, the 

dermis and the subcutaneous layer (Figure 7). The epidermis is the thin tough outer layer which is composed 

mainly of keratinocytes providing waterproofing and some protection for the thick fibrous dermis layer. The 

dermis is mostly made up of type I collagen and elastin and is the main protection for the body against 

external trauma. The subcutaneous layer sits below the dermis and enables the skin and underlying muscles to 

move independently59. It also acts as a site of energy storage (fat), to insulate the body, and it is the site where 

blood vessels, nerve endings and hair follicles are found. 



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Figure 7. Three primary layers of skin: the epidermis, dermis with its collagen and elastin network and subcutaneous layer 
embedded with blood vessels, hair follicles, sweat glands and fat. 

Mechanical Properties of Skin  

The protective function of skin is largely provided by its mechanical properties of flexibility and strength. 

Flexibility is a material’s ability to reversibly deform in response to an applied force while strength is a 

material’s ability to withstand an applied force until it ruptures. In skin, flexibility and strength are mainly due 

to interweaved and interlaced collagen and elastic fibres in the dermis60, 61. The elastin fibres contribute 

primarily to the elasticity of the skin while the collagen primarily provides strength. Tear resistance is an 

important measure of skin strength and depends on collagen fibre arrangement in the dermis. It indicates the 

ability of skin to resist an introduced tear front under tensile loading62. Tear resistance is widely used in the 

leather industry as a measure of the strength of various areas of skin from different species following tanning.  

 

Leather 

Tanning is the process used to transform skins into leather that has greater strength and is suitable for 

protective and hardwearing products, such as shoes, saddles and harnesses. Tanning can also impart flexibility 

to the final leather product making it more suitable for products such as gloves. During the tanning process, 

various mechanical and chemical processing steps are used to preserve the collagen network responsible for 

strength and flexibility, while some skin components are removed. The latter include proteins, and fats and 

carbohydrates that can be acted upon by spoilage bacteria leading to putrefaction and undesirable changes to 

the collagen network. Leather consists mainly of the collagen rich dermis that remains after tanning. It is 

characterized by two structurally different layers, separated by a transition zone (Figure 8). The upper surface 

is called the grain, which consists of small (0.1 µm diameter) densely packed collagen fibres that represent the 

remains of the sub-epithelial skin tissues and give leather its surface texture and appearance. Below the grain 

is the corium which is made up of larger (100 µm diameter) collagen fibres and is largely responsible for the 

bulk strength of leather63. The fibrils that make up these two distinct layers are similar, and it is the way these 

fibrils assemble into fibres that accounts for the differences in the physical properties of these layers16. 



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Figure 8. A cross section of ovine leather showing two distinct layers, separated by a transition zone: the grain with small 
densely packed fibres and the corium with larger, more loosely packed fibres. 

Leather Production 

To create leather with desirable properties, targeted chemical processing steps are used in tanning to alter 

specific skin components (Table 1). For example, the addition of calcium hydroxide during the unhairing stage 

targets cystine bonds found exclusively in keratin, allowing just the hair to be removed64. Approximately 70% 

of the protein in skin is made up of type I collagen and it is the principal concern of the tanner to leave this 

collagen intact while removing the remaining proteins and other skin structures65, 66.  

Table 1. Bovine skin composition, the relevant tanning stage and the resulting composition of hides at the official sampling 
position (OSP). Created from Sharphouse et al (1971)67. 

Skin Component Tanning Stage Composition of Hide at the OSP 

Collagen Entire process 70% 
Keratin Unhairing 2% 
Albumins/Globulin Liming, De-liming and Bating 1% 
Glycoproteins/ Proteoglycans Fleshing, Liming, De-liming and Bating 1% 
Fats Fleshing, Liming, De-liming and Bating 2% 
Water Skin preparation and Curing 24% 

Leather Production Process 

Beamhouse operations, that is all the processes from curing to tanning, are reviewed below. 

  

Curing. Since slaughterhouses and tanneries are usually at separate sites, skins are salted to prevent 

putrefaction during storage before tanning. This curing process results in loss of water from the skins, which 

then need to be rehydrated by soaking in water. The process also helps to remove surface contaminants such 

as soil. 

Tanning.  

Step 1: Liming and Unhairing. This step is to remove the epidermis and the other structural proteins, mainly 

hair composed of keratin. During unhairing the skins are immersed in an alkaline solution, typically lime 



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(calcium hydroxide) or soda ash (sodium carbonate), which causes chemical degradation of the hair shaft. 

More specifically the chemicals break down cystine linkages, a primary structural component of keratin, which 

allows the hair to be released from the hair follicle. The liming stage also results in the removal of unwanted 

material, such as grease and fats, and the matrix between collagen fibres which leads to an influx of water into 

the skin. Penetration of water into the collagen fibres causes them to swell and burst, thereby exposing 

collagen fibrils to subsequent chemical treatments resulting in better tanning and dyeing. 

Step 2: Fleshing. Residual flesh is removed from the skin with a fleshing machine that helps to mechanically 

expel excess water from the skin and at the same time even out variations in thickness. Skins of even thickness 

are important for the later processing stages to allow for uniform chemical penetration. 

Step 3: Deliming and Bating. Deliming is performed by adding acid salts to neutralize the alkaline solutions in 

the skins. The lowered pH encourages rapid protonation of basic groups in the collagen fibres and further 

opening out of the collagen structure. The lower pH also facilitates bating which is the addition of proteases to 

remove collagen fibrils that have degraded and disintegrated. This leads to the production of a softer leather68. 

Step 4: Pickling. The skins are acidified in citric acid that stops the bating and facilitates the penetration of 

mineral tanning agents added in the next step. 

Step 5: Tanning. This involves the addition of tanning agents that often cause complex chemical reactions that 

lead to crosslinking of collagen fibrils to produce the final leather product. There are a variety of tanning 

agents available, with each producing characteristic cross-linkages. These agents stabilize the collagen and 

prevent its degradation while at the same time maintaining flexibility and introducing colour and texture. The 

major tanning methods, and in turn resulting leather types, are listed below:  

• Vegetable-tanned leather is produced with tannins extracted from vegetable matter. It is not stable in 

water and tends to discolour as well as lose its flexible, smooth texture if soaked and left to dry.  

• Chrome-tanned leather is made by adding chromium sulphate and other chromium salts which produce a 

more supple and pliable leather when compared to vegetable tanned leather. This leather is known as wet 

blue, as the chromium imparts a blue colour.  

• Aldehyde tanned leather is produced by the addition of glutaraldehyde or oxazolindine compounds and is 

referred to as wet white leather due to its pale cream appearance. Marketed as chrome-free leather, it 

can typically be found in infant shoes and car interiors. Under the aldehyde tanned leather category there 

is a further classification depending on the source of the tannin. This is as follows:  

o Formaldehyde tanned leather is  produced by a form of aldehyde tanning which is beginning to be 

phased out due to people’s sensitivity to formaldehydes and toxicity concerns.  

o Brain tanned leather undergo a labour intensive process during which animal brains are emulsified to 

extract oils used as the tannin. These leathers are known for their exceptional softness and their 

ability to be washed.  

o Chamois leather is processed in a similar way to brain tanned leather but cod liver oil is used as the 

tannin.   

Step 6: Neutralizing and Dyeing. Here a sodium bicarbonate solution is applied to raise the skin pH to neutral, 

which enables the leather dyes to more readily penetrate the leather and ensure more even colouration.  

Leather dyes are anionic and come in a variety of colours. The intensity and evenness of the final colouration 

can be varied by pre-treatment with various fixatives and multiple applications of different dyes.  



Kelly 2018 

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Step 7: Drying. Removing the remaining water in the leather facilitates the stabilization of the chemical 

properties of the leather. Drying can be achieved through toggling where skins are stretched over a vertical 

frame and allowed to either air dry in a well-ventilated room or in a blast oven for a short period of time. 

Step 8: Finishing. Once most of the water has been removed from the leather, the final stages of the process 

can begin. The leather is re-dampened and left to equilibrate at room temperature. When a residual moisture 

content of around 10-20% is reached, the leather is staked by passing it through a set of weighted rollers. 

Staking relaxes the structure by loosening any tight fibres and softens the leather. Surface coatings can then be 

applied to improve the colour or even out the texture. 

Mechanical Properties of Leather 

Various mechanical characteristics of leather have been defined in a series of ISO Standards creating a 

normalized method for comparing properties of leather between species (Table 2).  

Table 2. The mechanical characteristics of bovine and ovine leather at the official sampling position (OSP).  

Parameter Bovine Leather at the OSP Ovine Leather at the OSP Reference 

Tear strength (N/mm) 63 20 - 40 69 
Tensile Strength (N/mm2) 22 10 70 71 
Young’s Modulus (GPa) 10 2 72 16 
Orientation Index 0.49 0.42 - 0.46 47 
D-spacing (nm) 63.5 64.2 47 
Fibril diameter (nm) 59 - 62 61 - 63 69 
Fiber diameter (µm) 0.1 (grain) 100 (corium) 0.1 (grain) 100 (corium) 35 

 

Cornea 

The cornea is the outer clear layer of the eye that meets the surrounding opaque sclera at a region called the 

limbus. The cornea is around 0.5 mm thick and performs a number of functions including protection and 

focusing light onto the lens and retina for vision. These functions are intimately related to the macro- and 

microscopic structure of the cornea that are described below.  

Cornea Structure 

The cornea is made up of five parallel layers (Figure 9). The outermost is the epithelium which is coated in a 

tear film and consists of around 6 to 12 layers of epithelial cells. Beneath, and supporting the epithelium, is 

Bowman’s layer which is made up of various types of collagen (types I, III, V and VII). Bowman’s layer merges 

with the stroma which is a dense layer of type I collagen making up 90% of the thickness of the cornea. On the 

inside of the cornea, against the vitreous humor in the anterior chamber, is Descemet’s layer which is a thin 

layer of connective tissue and an endothelium, a single layer of non-dividing cells that maintains the hydration 

status of the cornea. 



Kelly 2018 

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Figure 9.  A cross section of the eye showing the location of the cornea, anterior chamber, vitreous humor and sclera. The 
cornea is composed of five parallel layers, from the outer most epithelium to the deeper Bowman’s layer, stroma, 
Descemet’s layer and endothelium. 

Epithelium 

As the outer most layer of the eye, the epithelium forms a barrier between the environment and the inner eye 

structures. It is also important for absorbing ultraviolet (UV) radiation, protecting the underlying structures 

from the harmful effect of UV rays73, 74. The epithelium is about 50 µm thick and consists of several layers of 

rapidly growing and easily regenerated epithelial cells which are anchored to Bowman’s layer. 

Bowman’s Layer 

Bowman’s layer lies between the epithelium and the underlying stroma. It constitutes a 10 µm thick layer of 

randomly oriented collagen fibrils that merge with the collagen lamellae of the stroma and anchor the 

overlying epithelial cells. 

Stroma  

This thick (about 0.2 mm) fibrous layer consists of collagen fibrils (mainly type I but also some type V) and an 

inter-fibrillar matrix consisting mainly of proteoglycans (PGs) (Figure 10a) which are produced by keratocytes 

that are interspersed between lamellae (collagen fibrils arranged in flat sheets). The keratocytes can also 

repair damaged tissue by changing into fibroblasts which replace damaged tissue with scar tissue75. The 

lamellae are made up collagen fibrils that lie parallel to one another in sheets that then interweave and 

interlace with adjoining lamellae (Figure 10). There are over 300 layers of lamellae in the stroma17 with most 

interweaving found in the anterior cornea, where the stroma inserts into the Bowman’s layer. It is thought 

that this increase in interweaving contributes to the stability and shape of the anterior cornea76, 77. In cross-

section, the collagen fibrils within the lamellae have a quasi-regular hexagonal lattice arrangement with a 

centre-to-centre interfibrillar distance of 40 to 65 nm78 (Figure 10b).   



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Figure 10.  (a) Stroma collagen fibrils organized in parallel arrays in lamellae seen in cross section and transverse section. 
Keratocytes are interspersed between adjacent lamellae. Image of collagen fibril arranged into lamellae in the human 
cornea (taken from Komai and Ushiki 199179). (b) Collagen fibrils seen in cross section are arranged in a quasi-hexagonal 
lattice by ionic forces between fibrils, proteoglycans, and water and ions in the matrix.  

The regular spacing of the fibrils depends on complex ionic forces between adjacent fibrils and also between 

the fibrils and the proteoglycans in the matrix (PGs)80, 81, 82. These forces are influenced by the hydration state 

of the stroma. Proteoglycans and ions in the matrix create an osmotic pressure which draws water into the 

stroma from tears and the anterior chamber. The hydration state, however, ultimately depends on active 

transport mechanisms in the endothelial cells which effectively remove water from the stroma83. Disruption of 

the hydration control mechanisms results in fluid accumulation in the stroma that disrupts the quasi-regular 

hexagonal lattice which is required for optical transparency83.  

Descemet’s Layer 

This thin layer, 8 to 10 µm thick, provides a barrier between the anterior chamber of the eye and the cornea. It 

consists of type IV and VIII collagen and supports the endothelium.  

Endothelium 

The endothelium is a single layer of non-dividing of cells which controls the exchange of ions and water 

between the cornea and the vitreous humor. It maintains the cornea in a slightly dehydrated state which is 

important for the optimal spacing of collagen fibrils and transparency84.  

Properties of the Cornea 

The collagen of the cornea provides support and maintains the cornea in the  shape required to focus light 

onto the retina with minimal scatter85. At the same time, it prevents changes in the shape when the extra 

ocular eye muscles exert forces on the globe during eye movement.  

Transparency 

The cornea must be transparent to allow light to pass onto the retina with minimal reflection and refraction. 

The epithelium is transparent because the refractive indices of the cytoplasm and cell organelles are within a 



Kelly 2018 

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range that does not produce scattering of light. This is due to the presence of specialized crystalline proteins 

and high levels of enzymes such as aldehyde dehydrogenase and transketolase in the cytoplasm of the 

epithelial cells86. The transparency of the stroma is mainly due to the precise arrangement of its component 

collagen fibrils 78 which comes about because of ionic forces between adjacent fibrils, and between fibrils and 

the proteoglycans and water molecules in the matrix80. 

Collagen Arrangement for Transparency  

The collagen fibrils found in the stroma of the cornea are much smaller and have more uniform diameters and 

D-spacing than those found in other areas such as the skin and tendons (approximately 31 to 34 nm and 65 nm 

respectively)17, 87. Although it has been calculated that 95% of incident light is transmitted through the cornea, 

how these light waves pass through the myriad of fibrils in the cornea is unknown4. It is known, however, that 

the size and spacing of the corneal stromal collagen fibrils is critical for transparency. It is thought that fibril 

diameters need to be constant to ensure that there is perfect destructive interference between incident light 

and the light that encounters fibrils and is reflected (back-scatter)4. Also, the distances between adjacent 

collagen fibrils need to be restricted, with the stromal fibrils having a short-range order4 (Figure 11). 

 

Figure 11.  Collagen lamellae short-range order for transparency of visible light where all fibrils have the same diameter of 
about 31 nm and no fibrils are closer than 62 nm from centre-to-centre. Incoming light is traveling from left to right with a 
wavelength of 500 nm.  

Where there is damage to the epithelium or endothelium and Descemet’s layer, water flows into the cornea 

(oedema) and disrupts the normal interfibrillar distances with a loss of transparency and resulting in opacity of 

the cornea (Figure 12).   

    

Figure 12.  Corneal oedema in a human after the Descemet’s layer and the endothelial cells that control stromal hydration 
have been detached (indicated by the white arrow) and is no longer functional. Photograph by Brice Critser from the 
Department of Ophthalmology at the University of Iowa.  



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16 

Chapter 2. Characterizing Collagen Structures 
Collagen is the most abundant protein in mammalian bodies and its mechanical properties are largely due to 

its hierarchical structure (Figure 13). The component amino acids of collagen are assembled to form triple 

helices that combine in a staggered array to form fibrils that are bundled together to form fibres. To 

thoroughly evaluate collagen’s hierarchical structure and properties, analytical techniques are required.  

 

Figure 13. Hierarchical structure of collagen and the techniques used to view its structural features and mechanical 
properties at length scales from Angstroms to millimetres.  

Below follows a description of two X-ray techniques, small and wide angle x-ray scattering (SAXS and WAXS), 

which were used to investigate collagen structure on the nano-scale in leather and the cornea. Further, 

mechanical tests are described, including tensile, tear and three-point bend tests, which were used to 

characterize the mechanical properties of collagen in leather.  

 

Imaging at the Nano-level 

The nano-structure of collagen containing materials has been widely studied using analyses by means of SAXS 

and WAXS5, 47, 55, 88-92. SAXS and WAXS are X-ray techniques which provide structural detail on the nano-scale 

based on intensities of the scattering diffraction patterns made by the X-rays when scattered by atoms in the 

sample93. In diffraction studies, the structural resolution that can be obtained depends on the wavelength of 

the incident radiation. The X-rays used in SAXS and WAXS have wavelengths between 0.01 and 0.2 nm which 

have been found to be suitable for providing structural detail on the nano-scale in a wide range of biological 

samples63, 89, 90, 92, 94.  

Synchrotrons  

Initially an unwanted by-product of particle accelerators, synchrotron radiation has become a multidisciplinary 

tool for investigating nano-scaled structures across a range of fields from medical imaging and forensic 

science, to basic and applied research in a variety of fields including protein structure and mineral composition 

and more recently, manufacturing techniques for nano-scaled lithography for micro-electronics95-98. Currently 



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there are over 50 synchrotron facilities in operation across the world. The closest to (and partially funded by) 

New Zealand is the Australian Synchrotron, located in Melbourne, which accelerates electrons in a circular 

orbit to produce extremely bright light. The term synchrotron is in fact short for synchrotron light source. It 

has six basic components namely; the electron gun, linear accelerator (linac), booster ring, storage ring, beam 

lines and workstations (Figure 14).  

 

Figure 14. The basic components of a synchrotron. 

At the Australian Synchrotron the electron gun (the cathode at the beginning of the linac) contains a barium 

compound that is heated to 1000 degrees Celsius to enable electrons to be emitted (thermionic emission). 

These electrons are directed in a stream about the thickness of a human hair towards an anode at the end of 

the linac in bunches of around 100 million electrons spaced just two nano-seconds apart, at almost 60% the 

speed of light. There is a very high voltage between the electron gun cathode and anode (90 kV) causing the 

electrons to reach speeds close to the speed of light, roughly 100 billion km/h, 99.9987% the speed of light. 

The linac feeds into the booster ring where magnetic fields direct the electron beam around the ring. Here 

radio frequency cavities microwaves are used to add energy to the electrons each time they go passed the 

cavities. After 600 milliseconds and 1.38 million laps, the electrons bunches have 30 times the energy they had 

when they left the linear accelerator, and now travel at 99.99998%  the speed of light. They are then moved 

into their final home, a long vacuum chamber operating at a similar pressure to the moon’s atmosphere, called 

the storage ring. Rather than a true circle, the storage ring is more of a multisided shape covering an area of 

approximately 100 m across. Within the storage ring are a series of magnets and insertion devices (wigglers, 

undulators and bending magnets) that cause the electron beam to bend and travel in a snaking path around 

the circumference of the ring. As the electron beam bends, radiation/synchrotron light is emitted*. The 

tightness of the bend depends on the strength of the magnetic field applied and influences the wavelength of 

the radiation emitted. A tight bend produces short wavelength radiation, such as X-rays, while a gentler curve 

                                                                 
* For energy to be exchanged between the electron beam and a light wave, an undulator is needed. The undulator consists of 

alternating dipole magnets which cause the electron beam to oscillate. They add a velocity component to the electron beam in the 
transverse direction. The transverse component of the electron velocity and the vector of the light wave must point in the same direction 
to get an energy transfer from the electron to the light wave. The light wave, traveling at the speed of light, then has to shift by half an 
optical wavelength in a half period of the electron trajectory in order not to get ahead of the large, slower traveling electron beam. This is 
important for sustaining a steady energy transfer from the electron beam to light wave along the entire undulator. A slip of this magnitude 
allows the transverse velocity and the electromagnetic field of the light wave to remain parallel. 



Kelly 2018 

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produces wider wavelength radiation such as infrared rays. In this way, specific radiation can be produced in 

the storage ring by the placement of insertion devices across the beam line to create and channel radiation 

(Figure 15). 

 

Figure 15. Synchrotron radiation traveling down a beamline after being emitted from bends in the electron path caused by 
insertion devices (undulators, bending magnets and wigglers) in the storage ring, at the opening to the beamline. 

Beamlines usually consist of two hutches (radiation shielding enclosures); the first is the optical hutch which 

contains focusing mirrors, monochromators, and vacuum tubes to modify the incoming radiation into the 

required beam. X-ray beams can have a wide range of wavelengths depending on their source. A 

monochromator enables a beam of a more specific wavelength to be obtained for sample analysis. The second 

is the experimental hutch which houses the sample stage for mounting and manipulating samples, and the 

radiation detectors to measure the radiation that has interacted with the sample and has been scattered.  

Some beamlines have multiple end stations, with more than one experimental hutch, meaning all the optics 

don’t necessarily need to be in the first hutch. For example, the focusing mirrors in the SAXS/WAXS beamline 

at the Australian Synchrotron are located in the experimental hutch.  

SAXS and WAXS 

SAXS and WAXS describe two ranges of angles over which scattered radiation from a sample can be detected 

(Figure 16). 

 

Figure 16. Small angle X-Ray scattering (SAXS) and Wide angle X-Ray scattering (WAXS) synchrotron beamline 
configurations. 2D detectors are placed to capture scattered rays from a range of angles relative to the incident X-ray (SAXS 
(0.1 - 10°) and WAXS (5 - 60°)).  



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Theory 

The 2D radiation detector99 allows the full 360° scatter pattern from a sample to be collected  with background 

subtraction to remove non-sample related data such as background air scatter, Kapton tape scatter, water 

scatter and buffer scatter - essentially any scatter unrelated to the sample and sample environment can be 

removed. SAXS or WAXS beamline setups are achieved by moving the 2D detector further or closer to the 

sample, respectively. For SAXS, X-rays scattered at low angles relative to the incident X-ray (0.1 - 10° from the 

incident X-ray) are captured, providing structural information in the 10 - 600 nm range. For WAXS, X-rays 

scattered at higher angles (5 - 60° from the incident X-ray) are captured, providing structural information in 

the range of 1 - 60 nm. Measurements across both the SAXS and WAXS range provide detailed insights into the 

sample’s shape and size through its macromolecular structure. Non-scattered X-rays travel directly through the 

sample and are blocked by the beam stop, which is placed to protect the detector. 

Scatter patterns are the result of constructive and destructive interference of the scattered X-rays. Regions of 

constructive interference are defined as Bragg’s peaks according to Bragg’s law (Equation 5) which describes 

the scatter intensity at different scatter angles, giving insights into the samples interatomic structure. At small 

scatter angles, the Guinier model100 allows an estimate of particle size through the radius of gyration 

parameter and the extrapolated intensity at zero angle scatter, a region we are unable to measure due to the 

beam-stop.  The derivation of Bragg’s law can be seen when considering two beams with identical wavelengths 

and phase approaching a crystalline solid (Figure 17). When the beams are scattered by two different atoms in 

the sample, relative to one another, one beam will traverse an extra length of 2𝑑 𝑠𝑖𝑛(𝜃), where d is the 

spacing between planes and θ is the angle between the incident X-ray and the scattering planes. Constructive 

interference occurs when the length of the scattered rays travelled path is equal to an integer multiple of the 

wavelength of the incident X-ray, causing the scattered rays to constructively overlap resulting in a greater 

detected intensity. 

n λ = 2 d sinθ 

Equation 5. Bragg's law where n is the peak order, λ is the wavelength of the incident X-ray, d is the interplanar distance 
and θ is the half angle between the scattering plane and the incident beam. 

 

Figure 17. Bragg's law describing the diffraction of X-rays through a lattice where λ the wavelength of the incident X-ray, θ 
is the half angle between the scattering plane and the incident beam, d is the interplanar distance and d sin θ is half the 
path length difference between the two waves undergoing interference.  



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From the radial distribution of intensities on a scatter pattern, we can get an overall idea of the general sample 

structure. To gather further information from the scatter patterns, radial averaging is used to give a scattering 

intensity (𝐼(𝑞)) as a function of the momentum transfer (𝑞), allowing us to determine structural features when 

combined with Bragg’s law (Equation 6). 

q =
2 π n
𝑑

 

Equation 6. Scatter vector q where 𝐪 =  𝟐𝐝 𝐬𝐢𝐧𝛉
𝛌

  according to Bragg’s law, d is the interplanar distance, n is the peak order 
and q is the corresponding scattering vector. 

From the relationship defined above, the scattering vector range (also known as q-range) from SAXS analysis is 

0.001 – 0.6 nm-1, providing structural information between 10 and 600 nm, while WAXS analysis has a 

scattering vector range of 0.01 – 0.1 nm-1 providing sample structural information between 1 and 60 nm. 

Sources 

X-ray sources can be from a laboratory or a synchrotron source (Figure 18), each providing varying 

wavelengths and flux abilities. Laboratory X-ray generators produce wavelengths in the range of 0.1 - 0.2 nm 

while synchrotron sources have a broader range of 0.03 - 0.35 nm. Flux is a scalar quantity defined as the 

surface integral of the component of a vector field perpendicular to the surface at each point. A synchrotron 

source can achieve an energies between 8 and 12keV (a total photon flux between 2 and 8 x 1012 

photons/second) from the synchrotron radiation given off in the storage ring. High fluxes allow samples with 

weak scattering abilities to be analysed under greater intensities, producing good contrast in scatter patterns. 

Laboratory X-ray sources on the other hand achieve X-ray energies in the range of 1 - 4 keV reducing the 

intensity range and therefore resolution in scatter patterns. However, greater resolution can be achieved with 

a laboratory source of X-rays if longer times are used. 

    

Figure 18. Laboratory source small and wide angle X-ray scattering (SAXS/WAXS) beamline at the Max Planck Institute for 
Colloids and Interfaces, Germany (left) and a synchrotron source SAXS/WAXS beamline at the Australian Synchrotron, 
Australia (right). 

Benefits 

SAXS/WAXS imaging is an easy method for determining protein structures since it does not require 

crystallography like other protein imaging techniques, such as macromolecular crystallography. It has the 

capability of giving structural information between 1 and 600 nm allowing sample volumes to be small with 

short experimental runs (depending on the X-ray source) and minimal sample preparation. 



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Considerations 

Measuring small scattering angles accurately can be difficult as the diameter of the main beam can be unstable 

because of divergent beams appearing from the X-ray source. This effect can be lessened if large concave 

mirrors are used to focus the main beam and in the process minimize these divergent beams. Such mirror 

adjustments are not possible with laboratory based SAXS/WAXS instruments, which instead use collimation 

devices for beam focusing. In point-collimation devices, pinholes, are used to focus the X-ray beam while 

inline-collimation devices, slits, are used for focussing X-ray beams onto samples. 

Samples that contain components with random orientations of dissolved or partially ordered molecules need 

special consideration since a loss of information can result from spatial averaging of the scatter patterns. Such 

samples are more appropriately examined using crystallography. Sample stage mounting at strategic positions 

can help to reduce the effects of spatial averaging. Introducing more sophisticated sample chambers, to 

control environments (ambient temperature or humidity) or to apply mechanical strain during measurements, 

can provide additional data on samples.  

Encasing samples in films, typically Kapton and Quartz, with known scattering characteristics help to isolate 

samples in a desired environment. However, careful consideration needs to be taken when selecting films so 

that they do not interfere with the scattered intensities produced by the sample. 

Applications for Collagen Materials 

SAXS and WAXS scatter patterns from collagen-based materials provide insights into the arrangement and 

characteristics of fibrils (Figure 19). The position and intensity of the scatter pattern rings result from structural 

order. The relative peak location provides inter-planar measurements of the fibril diameter69, D-spacing88 and 

intermolecular spacing14, 94, while the intensity distribution of rings relates to structural order such as fibril 

orientation88,70 (Figure 20). 

 

Figure 19. Equatorial and meridional scattering from (a) small angle X-ray scattering (SAXS) and (b) wide angle X-ray 
scattering (WAXS) used to gather collagen specific information from integrated intensities over the azimuthal q-range (I(q)) 
from a collagen-based sample. The white arrows indicate the peaks of interest for D-spacing (a) and intermolecular spacing 
(b) on the scatter patterns.  



Kelly 2018 

22 

 

Figure 20. From SAXS integrated intensity profiles (a) fibril diameter and D-spacing are determined in the SAXS q-range 
with (b) further integration of the 6th order peak over the azimuthal angle to determine the preferred fibril orientation 
(orientation index, OI). From WAXS integrated profiles the (c) intermolecular spacing can be determined from 
measurements taken at this higher WAXS q-range which provides smaller structural information.  

Fibril diameter 

The scattering of X-rays occurs exclusively at the interface of two media with different capacities to scatter 

radiation, for example, a solid matrix and a pore space containing air. The scatter intensity versus the 

scattering angle (I(q)) are therefore determined by the geometry of the matrix-pore interface at various length 

scales derived from the q-range. The area under the I(q) curves can be used to determine fibril diameters in a 

collagen-based sample. 

D-spacing 

Collagen’s D-spacing arises from the precise arrangement of adjacent tropocollagen molecular chains, which 

are staggered axially. This leads to distinct and highly regular 'gap' and 'overlap' regions along the 

macromolecular assembly. The D-spacing reflects an average lateral intermolecular separation within the fibril, 

and is derived from a broad equatorial peak (Figure 20 (a)), usually the 6th order peak for a I(q) plot on the 

SAXS q-range. The location of the maximum of the 6th order peak, fitted with a Gaussian curve, is used to 

calculate the collagen periodicity (D-spacing) using the relationship defined by Bragg’s Law (Equation 6). 

Fibril Orientation 

Plotting the measured intensities over the full azimuthal angle range at the 6th order peak on a SAXS q-range 

provides information on the fibrils preferred orientation (Figure 20 (b)). The peak intensity relates to the 

alignment of the collagen fibres, while the peak width relates to the delamination or misalignment of the 

collagen fibrils. Two methods can be used to numerically define the organization within a fibrous structure, 

namely orientation index (OI) and Herman’s orientation. These have been used to determine the preferred 

fibril orientation from the intensities collected on SAXS scatter patterns. OI has been used to characterize 

alignment in leather88, cornea18, pericardium103 and acellular dermal matrix104 structures while Herman’s 

orientation has been used on a range of fibrous materials including skin7, bone105 and  various scaffolds106-108. 



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Orientation Index 

The orientation index (OI) is calculated from the spread in the SAXS scatter intensity over the azimuthal angle 

of the D-spacing peak in the q-range of 0.045 − 0.055 Å from a SAXS scatter pattern from a fibrous sample. The 

OI is defined as 90°−𝑂𝐴
90°

 , where OA is the minimal azimuthal angle range, centred at 180°, which contains 50% 

of the micro fibrils63, 109. The OI range is from a perfect anisotropic alignment (OI = 1) (Figure 21 (a)) through to 

isotropy (OI = 0) (Figure 21 (b)).  

 

Figure 21. Fibril orientation producing scatter patterns from an (a) anisotropic (highly aligned) sample and an (b) isotropic 
(randomly aligned) sample where θ is the scattering angle and 2θ is the maximum angle collected by the detector. 

 2.2.1.5.3.2 Herman’s Orientation 

Herman’s orientation (f) (Equation 7) is used to describe the extent of orientation of fibres relative to an axis of 

interest110, 111 from SAXS scatter patterns where complete fibre alignment is represented by f = 1 (Figure 21 

(a)),  and randomly orientated fibres are represented by f= 0 (Figure 21 (b)). 

𝑓 =  0.5 3∑ 𝐼(Ø) 𝑠𝑖𝑛2Ø 𝑐𝑜𝑠Ø 𝑑(Ø)𝜋
0
∑ 𝐼(Ø) 𝑐𝑜𝑠Ø 𝑑(Ø)𝜋
0

− 1  

Equation 7. Herman's orientation (f) where Ø is the azimuthal angle made by the fibre axis centred at 180° and I(Ø) is the 
intensity at a specified azimuthal angle. f is equal to 1 for a perfectly oriented fibrils and is 0 when the fibril orientation is 
completely random. 

Intermolecular spacing 

With the detector placed close to the sample (Figure 16) to capture wider scattering angles (WAXS) in the q-

range of 0.1 – 1 Å-1, small structural detail such as the intermolecular spacing in a collagen-based material can 

be determined. Using a technical graphing and data analysis package with a peak fitting function (Igor Pro) the 

peak location in an integrated intensity profile, focused on the 0.35 – 0.45 Å q-range, can be determined. The 

peak fit function can determine the peak maximums relative to their position on the scattering vector scale (q). 

Placing the peak location into the relationship derived previously (Equation 6) where n = 1 we can determine 

the intermolecular spacing (d).  



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Electron Microscopy  

Theory 

Electron microscopy utilizes an electron source, commonly a tungsten filament or lanthanum hexaboride 

source, connected to high voltage (typically 100 - 300 kV) to illuminate samples. With the wavelength of an 

electron being much shorter than that of light, magnifications of up to x 106 can be achieved. Electron 

microscopy takes two forms, namely scanning electron microscopy (SEM) and transmission electron 

microscopy (TEM). For SEM, a thin gold or graphite coating is applied to the surface of a sample. The electrons 

reflected from the coating enable the surface topography of the sample to be examined (Figure 22 (a)). In 

TEM, samples need to be embedded in a resin to allow for ultrathin sectioning (50 – 100 nm thick). An ultra-

thin section of a sample lowers its density and allows electrons to interact with the full sample thickness 

before reaching the detector (Figure 22 (b)). This creates images with information on the samples internal 

structure. The image’s contrast depends primarily on differences in electron density within the sample, where 

dark regions in the final image belong to parts of the sample with a high electron density and light areas 

belong to parts with low electron density. Staining can be used to highlight specific features by increasing the 

contrast. Staining with heavy metals like osmium tetroxide and lead, which scatter electrons due to their high 

atomic weight, improves the contrast detected by the microscope. 

 

Figure 22. Electron microscopy: (a) scanning electron microscopy (SEM) collecting scattered electrons at the detector and 
(b) transmission electron microscopy (TEM) collecting transmitted electrons at the detector.  

Benefits 

TEM and SEM are complementary microscope techniques. SEM views surfaces, while TEM views the interior of 

extremely thin films. Electron microscopy provides a high resolution image of the structure and the facilities 

are readily accessible in most research institutions. 

Considerations 

TEM is used in situations where the interior structure of a sample is to be investigated while SEM is used when 

the surface of a sample is to be studied. While coating a sample with gold or graphite spray for SEM is 

relatively simple, for TEM sample preparation is more complex. First, the sample for TEM has to be stabilized, 

in the case of biological samples with fixatives such as glutaraldehyde, and stained with chemicals that 

emphasize the capacities of different materials/tissues to conduct electrons. After embedding in resin, 



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25 

sections of the sample are cut which are thin enough (around 100 µm) to enable the passage of electrons 

through the material. SEM has lower resolution than TEM, micrometres vs. nanometres. 

Applications for Collagen Materials 

Measurements of the fibril diameter, D-spacing and inter-fibrillar packing can be made with a good set of TEM 

images (Figure 23) using an image processing and analysing tool such as ImageJ18, 112. TEM images also provide 

an idea of short order arrangements and structural patterns. SEM can be used to gather information on longer 

ordered arrangements and surface textures. 

 

Figure 23. Scanning electron micrscopy of (a) leather cross section showing the two distinct layers and (b) collagen fibers in 
the corium layer. Transmission electron microscopy of (c) cross-section of corneal collagen and (d) side view of corneal 
collagen showing characteristic D-spacing. Images were captured at the Manawatū Microscopy Centre, Palmerston North, 
New Zealand. 

 

Mechanical Testing 

Mechanical testing records a material’s response to an applied force for quantitative measurements to 

compare different materials. Quantitative measurements include strength, defined as the maximum force a 

sample can withstand over a defined surface area and stiffness, defined as a materials ability to resist 

deformation forces over a defined area in the material’s elastic region. Since these measurements are useful 

for comparing properties of different materials, a range of standardized test methods have been developed to 

allow comparable measurements to be recorded. Combinations of tests have been determined for leather by 

the International Organization for Standardization (ISO) to evaluate leather properties. These are discussed in 

detail below. 



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Tear Strength Measurements 

Tear testing gives a true representation of material strength since the materials failure is the result of a tear 

propagating from a tear front (puncture), representative of its failure in practice (Figure 24 (b)). The industry 

defined test (ISO 3377-2:2016) determines the maximum tear load a sample can withstand until failure. The 

testing criterion requires two sample types to be taken from the leather within a specific location, relative to 

the tail and backbone of a whole hide, called the official sampling position (OSP) (Figure 24 (a)).  At this 

location one sample (dimensions specified in the standard) is cut parallel to the backbone and the other 

perpendicular to account for the natural collagen alignment that occurs in hides15. The strength of the hide is 

then determined by the average force required to tear both orientation specific samples, normalized for 

thickness. 

 

Figure 24. Tear test (a) sampling location and orientation relative to the backbone of a skin/hide where L is the total length 
of the hide and the OSP is the official sampling position defined by ISO 2418:2017 and (b) the tensile forces F applied to a 
tear sample with tear fronts defined by the ISO standard. 

Tensile Strength Measurements 

The industry defined tensile test for leather (ISO 3376:2011) determines the maximum elongation force a 

sample can withstand before failure. As for the tear test, two sample types (dimensions specified in the 

standard) are cut relative to the backbone at the OSP, to account for the natural collagen alignment in skins. 

This method of determining strength is less preferred, because the failure mechanism is less likely to occur in 

practice (i.e.: application of an even force to a sample cross section until failure) (Figure 25). 

 



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Figure 25. Tensile test (a) sampling location and orientation relative to the backbone of a skin/hide where L is the total 
length of the hide and the OSP is the official sampling positi