title: Revealing cytokine-induced changes in the extracellular matrix with secondary ion mass spectrometry.
abstract: Cell-secreted matrices (CSMs), where extracellular matrix (ECM) deposited by monolayer cell cultures is decellularized, have been increasingly used to produce surfaces that may be reseeded with cells. Such surfaces are useful to help us understand cell-ECM interactions in a microenvironment closer to the in vivo situation than synthetic substrates with adsorbed proteins. We describe the production of CSMs from mouse primary osteoblasts (mPObs) exposed to cytokine challenge during matrix secretion, mimicking in vivo inflammatory environments. Time-of-flight secondary ion mass spectrometry data revealed that CSMs with cytokine challenge at day 7 or 12 of culture can be chemically distinguished from one another and from untreated CSM using multivariate analysis. Comparison of the differences with reference spectra from adsorbed protein mixtures points towards cytokine challenge resulting in a decrease in collagen content. This is supported by immunocytochemical and histological staining, demonstrating a 44% loss of collagen mass and a 32% loss in collagen I coverage. CSM surfaces demonstrate greater cell adhesion than adsorbed ECM proteins. When mPObs were reseeded onto cytokine-challenged CSMs they exhibited reduced adhesion and elongated morphology compared to untreated CSMs. Such changes may direct subsequent cell fate and function, and provide insights into pathological responses at sites of inflammation.
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK; Department of Chemical Engineering, University of Washington, Seattle, WA 98195, USA; National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington, Seattle, WA 98195, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Taylor
authors.affiliation: National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington, Seattle, WA 98195, USA; Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: Buddy D
authors.initials: BD
authors.lastname: Ratner
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.
authors.forename: Lee D K
authors.initials: LD
authors.lastname: Buttery
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK. Electronic address: morgan.alexander@nottingham.ac.uk.
authors.forename: Morgan R
authors.initials: MR
authors.lastname: Alexander
mesh_terms: D000818:Animals; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D025341:Principal Component Analysis; D051381:Rats; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*
chemical_list: D024042:Collagen Type I; D016207:Cytokines
keywords: ECM (extracellular matrix); Inflammation; Osteoblast; SIMS; Surface analysis