Protocol

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

  • Nature Protocols volume 13, pages 507529 (2018)
  • doi:10.1038/nprot.2017.117
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Abstract

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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Acknowledgements

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

Author notes

    • Héctor Capella-Monsonís
    • , João Q Coentro
    • , Valeria Graceffa
    •  & Zhuning Wu

    These authors contributed equally to this work.

Affiliations

  1. Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), National University of Ireland Galway (NUI Galway), Galway, Ireland.

    • Héctor Capella-Monsonís
    • , João Q Coentro
    • , Valeria Graceffa
    • , Zhuning Wu
    •  & Dimitrios I Zeugolis
  2. Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway (NUI Galway), Galway, Ireland.

    • Héctor Capella-Monsonís
    • , João Q Coentro
    • , Valeria Graceffa
    • , Zhuning Wu
    •  & Dimitrios I Zeugolis

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dimitrios I Zeugolis.

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