Assessment of the Effect of a Novel Contrast Agent for Contrast-Enhanced Computed Tomography, and of the X-rays, on the in vitro Behaviour of Osteoprogenitor Cells

de Bournonville, Sébastien Conceição, Francisco Geris, Liesbet Kerckhofs, Greet [UCL]

Introduction: Tissue Engineering (TE) is an interdisciplinary field aiming to provide solutions for the regeneration of organs and tissues. It has long been recognized in the field that dynamical 3D culture processes are necessary to improve the quality of complex engineered 3D tissues like bone. However, these dynamical technologies are still facing challenges due to the inability to monitor (and control) the 3D growth of the tissue inside the constructs, during in vitro culture. Contrast-enhanced X-ray computed tomography (CE-CT) came up as a promising solution to tackle these problems as it could be used as a 4D (3D space + time) non-invasive monitoring technology of tissue growth. This technique consists in using a chemical contrast agent (CA) to stain the tissue of interest in a construct (i.e. scaffold with cells), so that they become visible on X-ray CT scans. Before coupling such a technology with bioprocesses for online monitoring, one must assess its non-destructiveness as both X-rays and the presence of a chemical contrast agent could have an effect on the in vitro cell behaviour. In this study, we investigated the effect of the exposure of 2D cell cultures to an in-house developed zirconium-substituted polyoxometalate (Zr-POM) contrast agent and to X-rays, independently, on the metabolic activity of the cells. Materials and Methods: Human periosteum derived cells (hPDCs) were cultured in 2D culture vessels. Regarding the cell exposure to the CA, the cells were cultured for one week in a 24 well-plate. One (day 4) and two (days 3 and 5) staining timepoints were investigated, comparing commonly used contrast agents (Hexabrix and phosphotungstic acid, PTA) to the Zr-POM in two concentrations. For the cell exposure to the X-rays, the hPDCs were cultured for two weeks in flat tubes, while the cells were irradiated at day 4 using different X-ray doses in terms of power. Presto blue staining was used to assess daily the metabolic activity of the cells, and a Live/Dead staining was performed at the end-point of the cultures to evaluate cell viability. Results and discussion: There was no decrease in the metabolic activity of the cells that were irradiated by the X-rays, independent of the X-ray dose (Fig.1-A). Even for an expected high dose condition of 20 minutes exposition under high voltage and current (60kV and 170µA), no difference was observed on the metabolic activity. For the CA staining experiments, we have shown that PTA killed the cells, even when applying a short staining time at a relatively low concentration (Fig.1-B). While the cells were able to fully recover after staining with a Zr-POM concentration of 1.5% (for 30 minutes and 2 hours). These results show that typical staining and scanning conditions have, independently, no significant effect on the proliferation of the hPDCs. Conclusions: This study gives a first indication on the feasibility for the concept of online monitoring of hPDCs using CE-CT, in this range of settings. In future experiments, we will optimize the staining and scanning settings together, considering also several image acquisition time points over the culture. Furthermore, the effect of these conditions will also be investigated on the differentiation potential of the cells towards the chondrogenic, adipogenic and osteogenic lineages.