Optimised contrast enhanced nanoCT for volumetric analyses of in vitro manufactured tissue-engineered bone constructs

Sonnaert, Maarten Kerckhofs, Greet [UCL] Papantoniou, Ioannis Luyten, Frank Geris, Liesbet Schrooten, Jan

Introduction and aim As the field of tissue engineering (TE) matures, there is a growing need for novel imaging techniques for the characterisation of engineered constructs (i.e. cells/tissue combined with scaffolds) in a more insightful and quantitative manner. In a proof of concept study, we showed that combining the high spatial and contrast resolution of nanofocus computed tomography (nanoCT) with two different contrast agents allowed the visualisation and quantification of the neo-tissue formed in the TE constructs. Although the contrast difference between the cell-matrix fraction of the construct and the background was increased, the image quality was still limited because of metal-related image artefacts thus hindering accurate quantification in the entire scaffold. Using a design of experiment (DoE) approach we subsequently optimised the quality of the obtained datasets allowing the determination of the optimum staining time, contrast agent concentration and matrix quantity combinations. Materials, Methods and Results TE constructs were produced using Ti6Al4V scaffolds, which were seeded with human periosteal derived cells. The constructs were subsequently cultured in a 3D perfusion bioreactor system or in a static 3D setup to allow cell proliferation and matrix deposition. Post production the TE constructs were stained with Hexabrix™ or Phosphotungstic acid (PTA) prior to scanning using a Phoenix NanoTom S (GE Measurement and Control Solutions). The proof of principle study showed that the contrast enhanced nanoCT enabled the visualisation and quantification of the neo-tissue. For the Hexabrix™ stained datasets a correlation between the volume and the weight of the neo-tissue could be determined indicating that this contrast agent enables the visualisation and quantification of the entire neo-tissue fraction in the TE constructs. The DoE study showed that the image quality of the Hexabrix™ stained constructs was significantly influenced by the concentration of the staining agent. For the most optimal concentration, the image artefacts no longer affected the quantification of the ECM content. For the PTA stained constructs, staining time and concentration as well as the cell-matrix quantity had a significant effect on the contrast. Although for the most optimal staining conditions the influence of the image artefacts on the quantification of the ECM content was reduced, it could not completely be excluded. Conclusion CE-nanoCT is a non-invasive imaging technique that allows 3D visualization and quantification of in vitro engineered neo-tissue’s in TE constructs. In this study, we have shown, using a DoE approach that, when using an optimised sample preparation and staining protocol, highly quantitative datasets could be obtained despite the fact that the construct contained a metal-based scaffold.