title: Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs.
abstract: To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial 'design of experiments' approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.
authors.affiliation: Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium; Department of Materials Engineering, KU Leuven, Heverlee, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Sonnaert
authors.affiliation: Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium; Department of Development and Regeneration, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium; Biomechanics Research Unit, Université de Liege, Liège, Belgium.
authors.forename: Greet
authors.initials: G
authors.lastname: Kerckhofs
authors.affiliation: Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium; Department of Development and Regeneration, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Papantoniou
authors.affiliation: Polymer Chemistry and Biomaterials Group, University of Ghent, Ghent, Belgium.
authors.forename: Sandra
authors.initials: S
authors.lastname: Van Vlierberghe
authors.affiliation: Polymer Chemistry and Biomaterials Group, University of Ghent, Ghent, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Boterberg
authors.affiliation: Polymer Chemistry and Biomaterials Group, University of Ghent, Ghent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Dubruel
authors.affiliation: Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium; Department of Development and Regeneration, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium.
authors.forename: Frank P
authors.initials: FP
authors.lastname: Luyten
authors.affiliation: Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium; Department of Materials Engineering, KU Leuven, Heverlee, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Schrooten
authors.affiliation: Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium; Biomechanics Research Unit, Université de Liege, Liège, Belgium; Department of Mechanical Engineering, Biomechanics Section, KU Leuven, Heverlee, Belgium.
authors.forename: Liesbet
authors.initials: L
authors.lastname: Geris
mesh_terms: D000497:Alloys; D019149:Bioreactors; D002478:Cells, Cultured; D003287:Contrast Media; D005109:Extracellular Matrix; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D010521:Periosteum / Q000166:cytology*; D010772:Phosphotungstic Acid; D013194:Staining and Labeling / Q000379:methods; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds; D014025:Titanium; D014057:Tomography, X-Ray Computed / Q000379:methods*
chemical_list: D000497:Alloys; D003287:Contrast Media; D010772:Phosphotungstic Acid; C031462:titanium alloy (TiAl6V4); D014025:Titanium