PRODUCED FROM OVINE FORESTOMACH
8.1 IntroductionLeather is only one of many products derived from collagen-rich tissues where an
understanding of collagen structure could be advantageous. It is therefore of interest if the methods and results of this investigation are applicable to other collagen-rich products. For this reason the collagen structure of another collagen-rich product, ovine forestomach matrix, was investigated using SAXS.
Decellularized extracellular matrix (dECM) biomaterials are used for the treatment of wounds by allowing a platform on which host cell colonization can occur. These materials are produced from a number of different tissues including; dermis, urinary bladder,
pericardium, small intestine and heart valve (Crapo et al. 2011) which are processed prior to implantation to remove cellular material. dECM materials have been found to be present for 1-2 months after implantation (Badylak 2007; Lun et al. 2010). During that time, among other functions, they provide structural support (Badylak et al. 2009). The area of
implantation can undergo significant stresses and therefore it is important that these
materials can resist strain without fracturing. Therefore, the materials response to stain is of importance.
One dECM product, known as ovine forestomach matrix (OFM ), is produced from the propria subsucosa of ovine forestomach tissue (Floden et al. 2011). This material is produced for the treatment of chronic wounds such as pressure, venous and diabetic ulcers. The main component of the material is fibrillar collagen although other components such as elastin, fibronectin, and glycosoaminoglycans are also present (Floden et al. 2011). In order to study the material both unstrained and stretched sample analyses were
performed. SAXS analysis of OFM was only performed in the flat direction. This material has an average thickness of 0.25 mm and, as such, it was impractical to perform analysis in
the cross-sectional direction. Details of the experimental methods used are provided in chapter 3.
8.2 Results
Initial analysis of the OFM material found that Bragg’s scattering consistent with that of
fibrillar collagen occurred. The collagen was found to have a preferred orientation, although the strength of the preference varied with sample. The orientation index’s
measured varied between 0.2 and 0.8 with an average value of 0.54 (with a standard deviation of 0.28) indicating that there is a large variation in structure between samples. However, the d spacing was fairly consistent at an average value of 63.8 (0.2) nm. A stress-strain curve was produced from the force-extension data collected from sample stretching and is shown in Figure 8.1. As mentioned in chapter 7, the experimental method was not optimised for the determination of stretching properties such as Young’s modulus
or breaking strength. However general information about the stress-strain characteristics of the OFM sample can be determined. Necking was not observed to occur during sample stretching and there is no indication of its occurrence from the results in Figure 8.1. Stretching of OFM showed that the relationship between stress and strain was approximately linear, indicating a constant elasticity. From the slope of the curve the modulus of elasticity was calculated to be approximately 38 N/mm2. This is comparable to
that provided in literature of 44 N/mm2 (Floden et al. 2011).
During uni-axial stretching there were changes to both the orientation index and d spacing of the sample, as seen in Figure 8.2. There was a substantial increase in the orientation index of the sample, increasing from an initial value of 0.24 to 0.51 prior to failure. Similarly the increase in d spacing was also substantial, increasing from 63.5 to 68.0 nm during stretching, which equated to an extension of 7%.
Initially, application of strain resulted in an increase in the orientation index of the sample with only slight changes in the d spacing being recorded. Increased strain resulted in significant changes occurring to the d spacing, while changes in the orientation index became less significant. The orientation index increased significantly before strain levels of 0.07, stress of 2.9 N/mm2, after which the orientation index continued to increase but at a
slower rate. The d spacing gradually increased following the application of strain, with significant increases occurring above strain levels of around 0.09 (stress of 3.8 N/mm2).
Figure 8.2: d spacing and orientation versus a) strain and b) stress for a sample of OFM. d
spacing: open circle; OI: closed circle.
8.3 Discussion
Unstrained OFM samples displayed a large variation in the measured orientation index. This indicates that there is a large variability in material, presumably due to natural variation in the source material as opposed to changes caused during processing. This is probably due to the variation in the magnitude and direction of forces experienced by the material in different areas of the stomach.
The d spacing of unstrained OFM is significantly lower than those recorded for ovine leather samples. This is not unexpected as different tissues have different d spacing values. Although both products are produced from ovine tissue, OFM is produced from
forestomach while leather is produced from skin. Despite this, the d spacing for OFM is quite similar to that recorded for bovine leather.
The OFM sampled exhibited a linear stress-strain relationship. This relationship is different to those of most of the leather samples analysed, excluding flat grain samples, and is also different from those reported in literature for OFM, where samples displayed a ‘J’ type
curve (Floden et al. 2011).
The response of OFM to strain is similar to that of flat leather samples rather than cross- sectional leather samples. This is expected, as this was the same relative dimension analysed as the flat leather samples. The initial and final orientation index values are also similar to those measured for flat grain samples. The orientation index continued to increase throughout the stretching process in a manner similar to that of the flat leather samples. Similarly, the d spacing began to increase at relatively low strain rates, around 0.05. There was a substantial increase in d spacing during stretching of the OFM material (approximately 4.5 nm), equating to an extension of 7%. This extension is much greater than that recorded for leather samples under strain, indicating that the collagen fibres in OFM can withstand greater levels of extension prior to failure. This is not surprising as large forces would naturally occur to the stomach.
8.4 Conclusion
The response of OFM to uni-axial strain is similar to that of leather. When strain was first applied, the collagen fibrils reoriented to become more aligned in the direction of the strain with very little extension of the collagen fibrils themselves. As strain increased the rate of reorientation decreased, with more strain being taken up by the extension of individual collagen fibrils.
CHAPTER 9 CONCLUSIONS
9.1 Research Overview
The purpose of this investigation was to gain a better understanding of the relationship between the fibrillar collagen structure and strength of ovine leather. Fibrillar collagen is a major component of leather and it has been suggested that the physical properties of leather may be related to its structure. Advances in SAXS detection have allowed the fibrillar collagen structure of leather to be investigated in greater detail than has been previously possible. The investigation and quantification of the fibrillar collagen structure of leather and its relationship to strength has therefore been the subject of this work. The most significant finding of this work is the discovery of a linear relationship between the orientation of collagen fibrils and tear strength of leather samples. This work shows that in stronger leather the collagen fibrils are aligned mostly parallel to the plane of the leather surface (high orientation index) while weaker leather has more collagen fibrils out of this plane (low orientation index). Most significantly, the orientation index was found to correlate with the normalised tear strength of cross-sectional samples, not only of ovine leather but also when bovine leather samples were included in the data set. This
relationship was also found to be significant at all positions measured across the skin. It is thought that the relationship between orientation index and tear strength is due to the mechanism of failure in tear tests. Failure follows the path of least resistance, with tearing occurring either by separating fibres, attached by a degree of cross-linking and
entanglement, or by causing the failure of individual fibres. When fibrils are aligned with the direction of tear (low cross-sectional OI), tearing would occur by the process of fibre separation. But if fibrils were aligned perpendicular to the direction of tearing (high cross- sectional OI) failure must occur by tearing individual fibres, a process that requires
significantly more force. In this way a greater force would be required to tear samples with a high OI in the cross-sectional direction.
While this finding is obviously significant for leather manufacturing, it could also be important to other products derived from tissue that maintain their native fibrillar collagen
structure, if this relationship extends to other tissues. For example, methods could be developed to increase the strength of ECM products without the addition of cross-linking agents.
Tear strength was found to not correlate with the average amount of collagen in the samples or the orientation of collagen fibrils in the direction of the leather surface. These findings are also important for leather processing as it means that the affect of processing on these properties, or the initial state of these properties in the raw skin, will not affect the tear strength of the finished leather.
This work showed that the structure of ovine leather is reasonably consistent across the skin of the animal. This is encouraging as it indicates that the location on the skin chosen in manufacturing for ovine leather may be less important than for bovine and some other hides.
Significant changes occur to ovine skins during leather processing. The changes that occur are not just localised to the addition and removal of specific compound but also result in changes to the arrangement and structure of collagen fibrils. Results show that significant changes to the orientation index occur, with a decrease being measured during pickling followed by an increase during the remaining processing stages. Interestingly, the d spacing also changed during processing. An understanding of the changes that occur to the fibrillar collagen structure of the skin during processing are important. By understanding the changes that occur at each stage, stages that have an important affect on the final leather structure can be identified and investigated in more detail.
Investigations into the changes that occur to the fibrillar collagen structure of leather during stretching provided information on the response of leather to strain. When leather is initially placed under strain the fibres undergo reorientation and become more aligned. At higher strain levels the rate of reorientation decreases and instead strain is transferred to individual fibres. Stress is more evenly spread to individual fibrils across cross-sections in stronger samples than in weaker samples. This indicates that weak leather samples are unable to spread stress uniformly across the leather thickness, resulting instead in concentrations of stress at certain sites, which results in samples failing at lower strain levels.
The response of a different processed collagen-rich material, OFM, to uni-axial strain was found to be similar to that of leather. Initial strain resulted in the reorientation of collagen
fibrils, to be more aligned in the direction of the strain, with very little extension of the collagen fibrils themselves. As stress increased the rate of reorientation decreased, instead greater stress levels resulted in the extension of individual collagen fibrils. This response shows that the mechanisms that occur as a response to stretching in leather also occur in other processed collagen-rich material.
Many of the aims of this research, outlined in chapter 1, have been achieved. However, some have not been fully realised, in particular the aim to understand the basis for strength in leather. The relationship between structure and strength in leather has been investigated, resulting in a greater understanding of this relationship but there are still obviously other factors affecting strength that were not identified in this work.
9.2 Directions for Future Research
While this work has increased the understanding of the relationship between fibrillar collagen structure and tear strength in ovine leather there are still many areas where more investigation is required. Given the broad nature of this investigation there were areas of research where more detailed research would be advantageous. This study also produced results that would benefit from further investigation. Suggested areas of further research include;
SAXS patterns were analysed in the region of high q to determine information about the fibrillar collagen structure of the samples. SAXS patterns also include a great deal of information about collagen structure in the low q region including information about collagen fibre diameter. It is thought that the analysis of this information would increase the understanding of the relationship between fibrillar collagen structure and tear strength in ovine leather.
Analysis showed that the tear strength of the OSP was affected by sampling direction and that the relationship between orientation index and tear strength was different in OSP cross-sections cut perpendicular to the backbone to all other positions studied. Greater investigation into the structure at this position is needed. If samples were taken at a range of angles in relation to the backbone at this position the variation of structure and strength with direction in this position could be investigated in more detail.
Despite there being no strong relationships between d spacing and amount of collagen with tear strength, there was found to be a variation in both the d spacing and amount of collagen in the corium between weak and strong ovine leather samples. Therefore a more detailed investigation into the structure of the corium may increase understanding of the relationship between fibrillar collagen structure and tear strength in ovine leather.
An investigation into how samples taken at different stages of leather processing respond to strain would provide more information on how changes in structure affect the response of collagen-rich material to strain. Variation in the response to strain at different stages of processing would assist in identifying the effect of the presence of compound such as cross-linking agent and fat liquors.
An investigation into how variations in processing steps during leather production affect fibrillar collagen structure and the response to strain would be beneficial. For example; how variation in the concentration and type of cross-linking agents and variation of fat liquoring levels affects the collagen structure and how it affect the materials response to strain.
An investigation into the changes that occur to the relative distribution of the meridional intensities of SAXS patterns produced by samples under stain is recommended. This could help to identify the mechanism responsible for the changes in d spacing that was found to occur to samples under strain. Constant intensity distribution during strain would indicate that all the bonds in the helix were being extended, while the sliding of molecules would result in changes in these relative intensities.
Some of these suggestions are expected to be taken up by the PhD project that is following on from this work.