The effects of cross linking on collagen type 1 nanostructure and nanostructural response to uniaxial tension : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatu, New Zealand
Collagen type I, is a fibrillar protein with a complex hierarchical structure, forming the
extracellular matrices of an extensive range of organs and tissues. Applications for treated
collagen materials vary vastly from commercial uses to the medical field for bioprosthetics and
tissue grafts. Glycosaminoglycan (GAG), cross links naturally bridge fibrils, whilst
glutaraldehyde is widely used as a synthetic linking agent in medical and other industries. No
consensus has been reached regarding what contribution, if any, such cross links have on
collagen structure and mechanical responses to applied stresses. This research investigated
the role of GAG and glutaraldehyde cross links on the nanostructure and nanostructural
response of type I collagen fibrils under uniaxial strain. Bovine pericardium was decellularised,
producing native samples, or further treated with glutaraldehyde or chondroitinase ABC to
produce glutaraldehyde cross linked or GAG-depleted collagen samples respectively.
Synchrotron small angle X-ray scattering (SAXS), and atomic force and polarised light
microscopy provided quantitative and qualitative information on collagen nanostructure.
Uniaxial tensile experiments in conjunction with SAXS were performed to monitor structural
changes with applied strain. Glutaraldehyde cross links constrained fibrils into more networked
isotropic structures and demonstrated a mechanical function, recruiting 45% of fibrils into
stretching which experienced strains of up to 6.4%. Comparison of native with chondroitinase
ABC-treated samples showed GAGs do not constrain fibrils into alignment and have potential
fibril lubricating effects; 12% of fibrils in native tissue experienced strains up to 4.1%, and 36%
of fibrils experienced strains up to 4.6% in the GAG-depleted tissue. A higher degree of fibril
sliding occurs in native tissue. Interestingly, whilst adult pericardia are more cross linked and
fibrils of neonatal pericardia are more aligned, both tissues share similar propensities to form
more isotropic structures with glutaraldehyde treatment. These findings build a
comprehensive picture as to the function cross linking has in collagen structure and
mechanical response at the nano-level, where such knowledge may prove useful for the
preparation of collagen materials for specific applications.
Reproduced by permission of The Royal Society of Chemistry: Kayed, H. R., Sizeland, K. H., Kirby, N., Hawley, A., Mudie, S. T., & Haverkamp, R. G. (2015). Collagen cross
linking and fibril alignment in pericardium. RSC Advances, 5, 3611-3618. doi:10.1039/c4ra10658j ;
Kayed, H.R., Kirby, N., Hawely, A., Mudie, S.T., & Haverkamp, R.G. (2015). Collagen fibril strain, recruitment
and orientation for pericardium under tension and the effect of cross links. RSC Advances, 5, 103703-103712. doi:10.1039/C5RA21870E
Reproduced by permission of the American Institute of Physics: Wells, H. C., Sizeland, K. H., Kayed, H. R., Kirby, N., Hawley, A., Mudie, S. T., & Haverkamp, R. G. (2015).
Poisson's ratio of collagen fibrils measured by small angle X-ray scattering of strained bovine pericardium.
Journal of Applied Physics, 117, 044701. doi:10.1063/1.4906325
Reproduced by permission of American Scientific Publishers: Kayed, H.R., Sizeland, K.H., Wells, H.C., Kirby, N., Hawley, A., Mudie, S.T., & Haverkamp, R.G. (2016). Age
differences with glutaraldehyde treatment in collagen fibril orientation of bovine pericardium. Journal of
Biomaterials and Tissue Engineering, 6, 992-997. doi:10.1166/jbt.2016.1532