Collagen is the oldest and most abundant extracellular matrix protein that has found many applications in food, cosmetic, pharmaceutical, and biomedical industries. First, an overview of the family of collagens and their respective structures, conformation, and biosynthesis is provided. The advances and shortfalls of various collagen preparations (e.g., mammalian/marine extracted collagen, cell‐produced collagens, recombinant collagens, and collagen‐like peptides) and crosslinking technologies (e.g., chemical, physical, and biological) are then critically discussed. Subsequently, an array of structural, thermal, mechanical, biochemical, and biological assays is examined, which are developed to analyze and characterize collagenous structures. Lastly, a comprehensive review is provided on how advances in engineering, chemistry, and biology have enabled the development of bioactive, 3D structures (e.g., tissue grafts, biomaterials, cell‐assembled tissue equivalents) that closely imitate native supramolecular assemblies and have the capacity to deliver in a localized and sustained manner viable cell populations and/or bioactive/therapeutic molecules. Clearly, collagens have a long history in both evolution and biotechnology and continue to offer both challenges and exciting opportunities in regenerative medicine as nature's biomaterial of choice.

Collagen type I is the most abundant extracellular matrix protein, and collagen type I supramolecular assemblies (e.g., tissue grafts, biomaterials and cell-assembled systems) are used extensively in tissue engineering and regenerative medicine. Many studies, for convenience or economic reasons, do not accurately determine collagen type I purity, concentration, solubility and extent of cross-linking in biological specimens, frequently resulting in erroneous conclusions. In this protocol, we describe solubility; normal, reduced and delayed (interrupted) SDS-PAGE; hydroxyproline; Sircol collagen and Pierce BCA protein; denaturation temperature; ninhydrin/trinitrobenzene sulfonic acid; and collagenase assays and assess them in a diverse range of biological samples (e.g., tissue samples; purified solutions or lyophilized materials; 3D scaffolds, such as sponges and hydrogels; and cell media and layers). Collectively, the described protocols provide a comprehensive, yet fast and readily implemented, toolbox for collagen type I characterization in any biological specimen.

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Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.

In the medical device sector, bloom index and residual endotoxins should be controlled, as they are crucial regulators of the device’s physicochemical and biological properties. It is also imperative to identify a suitable crosslinking method to increase mechanical integrity, without jeopardising cellular functions of gelatin-based devices. Herein, gelatin preparations with variable bloom index and endotoxin levels were used to fabricate non-crosslinked and polyethylene glycol succinimidyl glutarate crosslinked gelatin scaffolds, the physicochemical and biological properties of which were subsequently assessed. Gelatin preparations with low bloom index resulted in hydrogels with significantly (p < 0.05) lower compression stress, elastic modulus and resistance to enzymatic degradation, and significantly higher (p < 0.05) free amine content than gelatin preparations with high bloom index. Gelatin preparations with high endotoxin levels resulted in films that induced significantly (p < 0.05) higher macrophage clusters than gelatin preparations with low endotoxin level. Our data suggest that the bloom index modulates the physicochemical properties, and the endotoxin content regulates the biological response of gelatin biomaterials. Although polyethylene glycol succinimidyl glutarate crosslinking significantly (p < 0.05) increased compression stress, elastic modulus and resistance to enzymatic degradation, and significantly (p < 0.05) decreased free amine content, at the concentration used, it did not provide sufficient structural integrity to support cell culture. Therefore, the quest for the optimal gelatin crosslinker continues.

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