Isolation and Characterisation of Major and Minor Collagens from Hyaline Cartilage of Hoki (Macruronus novaezelandiae).

The composition and properties of collagen in teleost (bony fish) cartilage have never been studied. In this study, we aimed to identify and characterise all collagen species in the nasal cartilage of hoki (Macruronus novaezelandiae). Four native collagen species were extracted using two techniques, and isolated with differential salt precipitation. We were able to assign the identity of three of these collagen species on the basis of solubility, SDS-PAGE and amino acid analyses. We found that hoki cartilage contains the major collagen, type II, and the minor collagens, type IX and type XI, which are homologous to those found in mammal and chicken cartilage. Using these extraction protocols, we also isolated a full-length type IX collagen from cartilage for the first time. In addition, we detected a 90 kDa, highly glycosylated collagen that has not been identified in any other species. For each isolate, structural and biochemical characterisations were performed using circular dichroism and Fourier transform infrared spectroscopy analyses, and the thermal denaturation properties were determined. Our results showed that the properties of hoki cartilage-derived collagens are similar to those of collagens in mammalian cartilage, indicating that teleost cartilage could provide biological ingredients for the development of biomaterials to treat cartilage-related illnesses.

Intra-fibrillar citric acid crosslinking of marine collagen electrospun nanofibres.

The physical strength of the collagen fibre network in the extracellular matrix is due to the covalent crosslinks between the molecules within the fibres (intra-fibrillar crosslinks). Citric acid was investigated as an agent to introduce crosslinks within marine collagen electrospun fibres. We used collagen films to understand the ideal conditions for citric acid crosslinking. This information was used to develop an optimised method for intra-fibrillar crosslinking in electrospun marine collagen fibres, which increased the stability of these fibres in aqueous environments. The optimised method included a spinning solution containing collagen and citric acid at pH3.5 at high concentrations (260:1 citric acid:collagen molar ratio) coupled with high temperature annealing (165°C), which resulted in the highest intra-fibrillar crosslinking density in electrospun fibres.

Guide to electrospinning denatured whole chain collagen from hoki fish using benign solvents.

Tissue engineering requires the design and manufacture of biomimetic scaffolds. Collagen-derived nanofibrous scaffolds have been intensively studied because collagen, in the form of fibrils, is one of the main components of the extra cellular matrix (ECM). Several collagen materials have been used in electrospinning studies including mammalian extracted Type I collagen and gelatin formulations. Denatured whole chain collagen (DWCC) can be prepared by heat denaturing acid-soluble collagen extracted from cold-water fish skin. This product provides a consistent source of collagen with a controlled molecular weight profile and intact alpha chains including telopeptides. In this work, we studied DWCC-water-acid systems in order to determine the effect of solution composition on nanofibre morphology. Whereas measurement of the classical physical properties of concentrated solutions failed to predict and only partially explained the electrospinning behavior of collagen derived polymers, hydrodynamic properties provided insight. All the samples are presented in ternary diagrams to map the electrospinnability of the systems. These "electrospinning maps" provide an informative resource to electrospinning collagen-derived product for biomedical or commercial applications and a practical alternative to complicated models developed for synthetic polymers.

Thermal transition properties of hoki (Macruronus novaezelandiae) and ling (Genypterus blacodes) skin collagens: implications for processing.

Hoki (Macruronus novaezelandiae) and ling (Genypterus blacodes) are cold-water fish caught in New Zealand waters. Their skins are a major component of the post-processing waste stream. Valuable products could be developed from the skins, as they are primarily composed of collagen, which has many commercial applications. We prepared acid soluble collagens (ASC) from hoki and ling skins, and analyzed their thermal denaturation properties using a Rapid Visco™ Analyzer. At slower heating rates the denaturation temperature (TD) of hoki and ling collagens decreased. This result is consistent with the model of irreversible rate kinetics for the denaturation of collagen. We determined the effects of solvents that disrupt hydrogen bonding on ASC stability. Increasing concentrations of urea from 0.1 M to 1.0 M and acetic acid from 0.1 M to 0.5 M decreased TD. This resulted from the effects of these reagents on the hydrogen bonds that stabilize the collagen triple helix.

High-throughput quantification of hydroxyproline for determination of collagen.

An accurate and high-throughput assay for collagen is essential for collagen research and development of collagen products. Hydroxyproline is routinely assayed to provide a measurement for collagen quantification. The time required for sample preparation using acid hydrolysis and neutralization prior to assay is what limits the current method for determining hydroxyproline. This work describes the conditions of alkali hydrolysis that, when combined with the colorimetric assay defined by Woessner, provide a high-throughput, accurate method for the measurement of hydroxyproline.