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

Capella-Monsonís, Héctor; Coentro, João Q.; Graceffa, Valeria; Wu, Zhuning; Zeugolis, Dimitrios I.

doi:10.1038/nprot.2017.117

Cited by 91 publications

(72 citation statements)

References 94 publications

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“…As collagen accounts for up to 25% of total human protein, it can be considered the main component of ECM in general. 1,24 The ndings of this study are in close agreement with this fact as collagen was identied as a basic component in the in vitro generated (click)ECM in the SDS-PAGE banding pattern regardless of the azide modication.…”

Section: Qualitative Assessment Of Clickecm Compositionsupporting

confidence: 84%

“…Total collagen content via quantication and conversion of hydroxyproline (HP). For this analysis, the protocol published by Capella-Monsonis et al 24 was slightly modied. For quantifying the hydroxyproline (HP) content, lyophilised (click) ECM samples (approximately 7 mg) were transferred into Vshaped borosilicate glass vials with polytetrauoroethylene (PTFE) caps, diluted with 500 mL concentrated hydrochloric acid (HCl) and incubated overnight at 110 C. Aer allowing the samples to cool down to RT, samples were carefully ltered through a PTFE syringe lter (F 0.2 mm) in order to remove the insoluble humin fraction.…”

Section: Methodsmentioning

confidence: 99%

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Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods

et al. 2020

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Section: Qualitative Assessment Of Clickecm Compositionsupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods

et al. 2020

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“…Various assays are available to assess the purity, concentration, and crosslinking density of collagen‐based materials . Collagen extracted from different tissue sources and cell layers ( Figure ) can be characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), which separates proteins according to their molecular weight, charge, size and shape .…”

Section: Exogenous Collagen Crosslinkingmentioning

confidence: 99%

The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development

et al. 2018

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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.

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“…Several fractions of collagen were extracted from the cultures representing the sequential steps in the biosynthetic process 43 . Cell supernatants were assayed for the soluble form of secreted collagen upon concentration with Sircol Soluble Collagen Assay (Biocolor, Carrickfergus, United Kingdom) following manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Enhancement of collagen deposition and cross-linking by coupling lysyl oxidase with bone morphogenetic protein-1 and its application in tissue engineering

Rosell-García

Rodrı́guez-Pascual

2018

Sci Rep

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Cultured cell-derived extracellular matrices (ECM)-based biomaterials exploit the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, standard cell culture conditions are far from ideal given the fact that the diluted microenvironment does not favor the production of ECM components, a circumstance particularly relevant for collagen. An incomplete conversion of procollagen by C-proteinase/bone morphogenetic protein 1 (BMP1) has been proposed to severely limit in vitro collagen deposition. BMP1 also catalyzes the proteolytic activation of the precursor of the collagen cross-linking enzyme, lysyl oxidase (LOX) to yield the active form, suggesting a deficit in cross-linking activity under standard conditions. We hypothesized that the implementation of fibroblast cultures with LOX and BMP1 may be an effective way to increase collagen deposition. To test it, we have generated stable cell lines overexpressing LOX and BMP1 and studied the effect of supernatants enriched in LOX and BMP1 on collagen synthesis and deposition from fibroblasts. Herein, we demonstrate that the supplementation with LOX and BMP1 strongly increased the deposition of collagen onto the insoluble matrix at the expense of the soluble fraction in the extracellular medium. Using decellularization protocols, we also show that fibroblast-derived matrices regulate adipogenic and osteogenic differentiation of human mesenchymal stem cells (MSC), and this effect was modulated by LOX/BMP1. Collectively, these data demonstrate that we have developed a convenient protocol to enhance the capacity of in vitro cell cultures to deposit collagen in the ECM, representing this approach a promising technology for application in tissue engineering.

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

Cited by 91 publications

References 94 publications

Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods

Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods

The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development

Enhancement of collagen deposition and cross-linking by coupling lysyl oxidase with bone morphogenetic protein-1 and its application in tissue engineering

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