title: Quantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge.
abstract: Hydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.
authors.affiliation: Department of Functional Materials in Medicine and Dentistry, University of Würzburg, Pleicherwall 2, 97070 Würzburg, Germany.
authors.forename: Meike V
authors.initials: MV
authors.lastname: Beer
authors.affiliation: Department of Functional Materials in Medicine and Dentistry, University of Würzburg, Pleicherwall 2, 97070 Würzburg, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Hahn
authors.affiliation: DWI Leibniz Institute for Interactive Materials Research and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstr. 50, 52056 Aachen, Germany.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Diederichs
authors.affiliation: DWI Leibniz Institute for Interactive Materials Research and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstr. 50, 52056 Aachen, Germany.
authors.forename: Marlies
authors.initials: M
authors.lastname: Fabry
authors.affiliation: DWI Leibniz Institute for Interactive Materials Research and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstr. 50, 52056 Aachen, Germany.
authors.forename: Smriti
authors.initials: S
authors.lastname: Singh
authors.affiliation: National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, England.
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Spencer
authors.affiliation: Clinic of Surgery, Knappschafts-Hospital GmbH, University Hospital of the Ruhr-University Bochum, 44892 Bochum, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Salber
authors.affiliation: DWI Leibniz Institute for Interactive Materials Research and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstr. 50, 52056 Aachen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Möller
authors.affiliation: National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, England.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Shard
authors.affiliation: Department of Functional Materials in Medicine and Dentistry, University of Würzburg, Pleicherwall 2, 97070 Würzburg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Groll
mesh_terms: D002448:Cell Adhesion; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000502:physiology*; D006801:Humans; D008024:Ligands; D008756:Methylgalactosides / Q000737:chemistry*; D056951:Photoelectron Spectroscopy; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D008024:Ligands; D008756:Methylgalactosides; C109801:hydrogel film