Mineralised collagen displays an improved hydrothermal stability compared to collagen that is unmineralised. The possibility of using in-vitro partial mineralisation of collagen as a method of increasing the hydrothermal stability was investigated. Remineralisation experiments using demineralised turkey leg tendon and chemically modified bovine hide collagen showed that although it was possible to grow hydroxyapatite mineral crystallites on the collagen substrate they were only present at the substrate-solution interface and as such did not give rise to an increase in hydrothermal stability. The morphology of the mineral crystallites produced in-vitro were compared with those in the naturally mineralised tendon using Scanning Electron Microscopy (SEM), Small Angle X-ray scattering (SAXS), X-ray Diffraction (XRD) and Fourier-Transform Infrared Spectroscopy (FT-IR). Differential scanning calorimetry (DSC) studies on demineralised tendon identified a previously unknown high temperature endothermic transition to be present in the thermal scan of both mineralised and unmineralised collagen during denaturation. The position of this transition was found to be affected by hydration, presence of mineral, pH, and crosslinking similar to that of the first transition. Experiments using reagents known to selectively break various non-covalent interactions within collagen indicated that the transition was due to the breaking of covalent bonds via an endothermic chemical reaction, with the most likely candidate being the hydrolysis of peptide bonds within the polypeptide backbone. Optical microscopy of collagen after heating indicated that the fibrillar structure of the collagen was destroyed during the second transition, forming an amorphous gel. Finally, the effect of the mineral phase on the hydrothermal stability of naturally mineralised collagen was discussed in context to its location within the collagen structure. It was postulated that the presence of mineral dehydrates the collagen structure, as well as decreasing the available space within the hole region.

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Wound dressing plays an important role in wound recovery and collagen interacts with the human body in such a way that it has specific advantages compared to synthetic materials. The aim of the present study was to get an optimal crosslinking agent for collagen and so the mechanical, chemical and biochemical properties of crosslinked collagen materials were investigated. Fibroblast cells are important in the process of wound healing, so the interaction of human fibroblast cells with crosslinked collagen films were investigated as well. Collagen I was isolated from bovine achilles tendons and collagen films were formed using the isolated collagen I solution. Collagen films were crosslinked with glutaraldehyde (GA), genipin, hexamethylenediisocyanate (HMDC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) at the equal concentration of 0.02 M and these crosslinked collagen films were compared with uncrosslinked collagen films (control). The surfaces of the crosslinked films were investigated using scanning electron microscopy (SEM). There was observable fibre structure on GA- and genipin-crosslinked collagen films. The tensile strength, elongation at break and low strain modulus of the crosslinked collagen films were investigated. The results showed that GA-, genipin- and HMDC-crosslinked collagen films obtained higher tensile strength than the control. Elongation at break of all the crosslinked collagen films became lower than the control. GA- and genipin-crosslinked collagen films obtained higher low strain modulus than other crosslinked collagen films and the control. The denaturation temperatures of all crosslinked collagen films were significantly higher than the control and the denaturation temperatures of GA- and genipin-crosslinked films were much higher than those of HMDC- and EDC-crosslinked films. All the crosslinked collagen films were resistant to the digestion of collagenase. These results suggest that all the crosslinking agents are effective and GA- and genipin-crosslinked films obtained more extensive crosslinking. The interaction of crosslinked collagen films with fibroblast cells was investigated, e.g. adhesion, proliferation and migration of fibroblast cells. The results demonstrated that the control, genipin- and EDC-crosslinked collagen films were conducive to cell adhesion. Fibroblast cells on the control, genipin- and EDC-crosslinked collagen films were able to proliferate after 24 hours, with increased growth after 48 hours. The fibroblast cells on the control, genipin- and EDC-crosslinked collagen films migrated directionally. The cells on genipin-crosslinked film initiated directional migration earlier than those on control- and EDC-crosslinked films. In summary, genipin crosslinked collagen films show high denaturation temperature, higher tensile strength and good biocompatibility for fibroblast cells adhesion, proliferation and migration. Genipin should be regarded as a suitable crosslinking agent for reconstituted collagen for use in wound dressing.

Vegetable tannins used in tanning of hides and skins are limited to surface reactions by their large molecular weights. The molecular weight reduces penetration into the skin and lowers the thermal stability or tanning effect. Investigation into the utilisation of small phenolic compounds such as catechin to improve penetration with subsequent in situ enzymecatalysed polymerisation may provide a novel and alternative tanning agent. In this research, catechin was oxidised by enzymatic catalysis using laccase, with the polymerisation confirmed by FT-IR and UHPLC. Tanning experiments were undertaken to measure the effect of laccase-catalysed polymerisation of catechin in the thermal stabilisation of collagen, by monitoring the change in shrinkage temperature between the treated and untreated sample of hide powder (ΔTs). This study demonstrates that the stabilisation of collagen (ΔTs) is increased with the use of in situ enzyme-catalysed polymerisation. Depolymerisation of condensed tannins is presented as an alternative source of low molecular weight phenolics to be applied in the stabilisation of collagen. In this research, mimosa tannin from the Black Wattle tree (Acacia mearnsii) was used in the depolymerisation process. Preliminary experiments on depolymerisation were undertaken using the methods laccase-mediator system and L-cysteine in mild acidic medium, both showing unsatisfactory results. Acid-catalysed depolymerisation followed by nucleophile addition is a common analytical method for determining the degree of polymerisation of proanthocyanidins. The acid-catalysed depolymerisation method was scaled-up, with the addition of pyrogallol as a nucleophile trapping agent, and powdered depolymerised mimosa was obtained. Data show an increase in the monomeric content and decrease in percentage of condensed tannins for the acid-catalysed process, indicating the potential of the depolymerisation of mimosa tannin in obtaining environmentally friendly sources of low molecular weight phenolic compounds for use in large scale/industrial applications. The depolymerised product was applied in the stabilisation of hide powder and goat skin. Using hide powder, data show that the laccase-assisted polymerisation of the depolymerised mimosa reached higher values of ΔTs in comparison with the conventional process employing unmodified mimosa. Using goat skin, the new process achieved similar values of ΔTs as the conventional process. In order to obtain similar results for goat skin as obtained with hide powder, an optimisation may be carried out. Therefore, data obtained demonstrates the potential of the new route in vegetable tanning of leather.