An experimental toolbox for characterization of mammalian collagen type I in biological specimens


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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Figure 1: Assessment of collagen solubility.
Figure 2: Collagen purity assessment via SDS-PAGE.
Figure 3: Collagen content quantification in cell culture and tissue samples.
Figure 4: Endothermic transition assessment of wet and dry collagen samples.
Figure 5: Quantification of free amines in collagen sponges and hydrogels through TNBSA and ninhydrin assays.
Figure 6: Collagen quantification after in vitro enzymatic degradation using MMP-8 (50 U/ml).


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This work was supported by the Health Research Board, Health Research Awards Programme (grant agreement no. HRA_POR/2011/84); a Science Foundation Ireland, Career Development Award (grant agreement no. 15/CDA/3629); the Science Foundation Ireland/European Regional Development Fund (grant agreement no. 13/RC/2073); and the H2020, Marie Skłodowska-Curie Actions, Innovative Training Networks 2015 Tendon Therapy Train Project (grant agreement no. 676338). This work was also part of the Teagasc Walsh Fellowship (grant agreement no. 2014045) and the ReValueProtein Research Project (grant agreement no. 11/F/043) and was supported by the Department of Agriculture, Food and the Marine (DAFM) under the National Development Plan 2007–2013, funded by the Irish Government.

Author information

H.C.-M., J.Q.C., V.G. and Z.W. contributed equally to this work and are listed in alphabetical order. H.C.-M., J.Q.C., V.G. and Z.W. designed and conducted the experiments and analyzed the data. D.I.Z. designed and supervised the study. All authors wrote the paper.

Correspondence to Dimitrios I Zeugolis.

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Integrated supplementary information

Supplementary Figure 1 SDS-PAGE and densitometric analysis of cell-synthesized collagen.

SDS-PAGE (a) and complementary densitometric analysis (b) of Symatese bovine skin collagen type I (1.6 μg/well) standard (A); acid-extracted collagen from WS1 fibroblast cell-layers (B, C); and pepsin-extracted collagen from WS1 fibroblast cell-layers (D, E). WS1 cells were cultured for 2 days in DMEM supplemented with 0.5 % FBS, 100 μg/ml of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate with (B, D) or without (C, E) 100 μg/ml of carrageenan (CR). The collagen was extracted from the cell layer as has been described previously78–83. N=3 for all samples.

Supplementary Figure 2 Color changes for standards and samples in the quantification of collagen content.

Hydroxyproline assay (a), Pierce™ BCA Protein assay (b) and Sircol™ Collagen assay (c).

Supplementary Figure 3 Color changes for standards and samples in the quantification of free amines.

TNBSA assay (a) and ninhydrin assay (b).

Supplementary Figure 4 Qualitative/semi-quantitative analysis of enzymatic (MMP-8 at 50 U/ml) degradation of collagen hydrogels and sponges using SDS-PAGE and densitometric analysis.

Non-crosslinked and GTA crosslinked collagen hydrogels and sponges were subjected to MMP-8 degradation for 1 h, 2 h, 4 h and 8 h. Subsequently, the supernatants were collected and loaded on gels [hydrogels (a) and sponges (c)]. Semi-quantitative analysis was conducted through densitometry [hydrogels (b) and sponges (d)]. For the non-crosslinked samples, the degradation is evidenced, whilst the GTA cross-linked samples withheld enzymatic degradation for the period of time assessed.

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Capella-Monsonís, H., Coentro, J., Graceffa, V. et al. An experimental toolbox for characterization of mammalian collagen type I in biological specimens. Nat Protoc 13, 507–529 (2018).

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