Three-dimensional characterization of tissue-engineered constructs by contrast-enhanced nanofocus computed tomography

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

In order to successfully implement tissue engineered (TE) constructs as part of a clinical therapy, it is necessary to first develop and validate quality control tools that will ensure accurate and consistent TE construct release specifications. Hence advanced methods to monitor TE construct properties need to be further developed. In this study we showed proof of concept for contrast enhanced nanofocus computed tomography (CE-nanoCT) as a 'whole-construct' imaging technique with non-invasive potential that enables 3D visualization and quantification of in vitro engineered extracellular matrix (ECM) in TE constructs. In particular we performed a 3D quantitative and qualitative structural and spatial assessment of the in vitro engineered ECM, formed during static and perfusion bioreactor cell culture in 3D TE scaffolds, using two contrast agents, namely Hexabrix® and phosphotungstic acid (PTA). CE-nanoCT image data were validated by comparison to Live/Dead viability/cytotoxicity and picrosirius red staining data, and to the net dry weight of the TE constructs. When using Hexabrix® as contrast agent, ECM volume fitted linearly with net dry ECM weight independent from the flow rate used. When using PTA as contrast agent, CE-nanoCT data showed pronounced distinction between flow conditions when compared to both net dry weight and picrosirius red staining data although linearity was maintained, indicating culture-specific structural ECM differences. This was attributed to the binding specificity of this contrast agent. This novel type of information can contribute to optimize bioreactor culture conditions and potentially critical quality characteristics of TE constructs such as ECM quantity and homogeneity, facilitating the gradual transformation of 'TE constructs' in well characterized 'TE products'.