Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs

PLoS One. 2015 Jun 15;10(6):e0130227. doi: 10.1371/journal.pone.0130227. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alloys
  • Bioreactors
  • Cells, Cultured
  • Contrast Media
  • Extracellular Matrix
  • Humans
  • Imaging, Three-Dimensional / methods*
  • Periosteum / cytology*
  • Phosphotungstic Acid
  • Staining and Labeling / methods
  • Tissue Engineering / methods*
  • Tissue Scaffolds
  • Titanium
  • Tomography, X-Ray Computed / methods*

Substances

  • Alloys
  • Contrast Media
  • Phosphotungstic Acid
  • titanium alloy (TiAl6V4)
  • Titanium

Grants and funding

MS is supported by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT/ 111457 - http://www.iwt.be/english/welcome). GK is currently financed by a postdoctoral grant of the Research Foundation - Flanders (FWO/12R4315N - http://www.fwo.be/en/). GK and LG acknowledge support by the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC grant agreement n°279100 (http://erc.europa.eu/funding-and-grants). IP was supported by the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC grant agreement REJOIND n°294191. SVV would like to acknowledge the Research Foundation - Flanders (FWO, Belgium) for financial support under the form of a post-doctoral fellowship and a Research Grant (‘Development of the ideal tissue engineering scaffold by merging state-of-the-art processing techniques’, FWO Krediet aan Navorsers). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.