Design of experiments to evaluate the staining properties of polyoxometalates for contrast-enhanced microCT of aorta tissue
(2021) BSTE 2021 – 8th Belgian Symposium on Tissue Engineering — Location: Louvain-la-Neuve (12.November.2021)
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- Authors
Balcaen, Tim 
Hoffmann, Delia
- Abstract
- Vascular diseases are one of the leading causes of death in developed countries. To better treat these diseases, tissue engineered (TE) vascular grafts could provide a solution. However, for an improved design of these grafts, a better understanding of the relationship between the microstructure of vascular tissues and their mechanical behavior is required, as this relationship is key to obtain matching properties between the TE construct and the native tissue. The detailed microstructural characterization can be achieved by microfocus X-ray computed tomography (microCT), and in particular contrast-enhanced microCT or CECT. This novel virtual 3D histological technique combines the benefits of microCT imaging with the high discriminative power of conventional 2D histology, by using contrast-enhancing staining agents (CESAs). These CESAs enrich a specific region of the tissue under evaluation with X-ray attenuating atoms, which subsequently allows soft tissue visualization. Recently, two new CESAs have shown promise in this field: Hf-WD 1:2 POM (i.e. two Mono-WD POMs linked together by a Hafnium atom) and Mono-WD POM. However, there is still a great lack of fundamental knowledge about their staining properties i.e. the nature of chemical interactions, specificity and diffusional properties. In this study, we characterized these two CESAs in terms of their contrast enhancement and diffusion properties within the media of porcine aorta tissue. However, it is known that the Hf-WD 1:2 POM can dissociate into the Mono-WD POM and the Hf-WD 1:1 POM, which complicates observations. As a result, we added the Hf-WD 2:2 POM in a preliminary experiment, which is known to dissociate mainly into two Hf-WD 1:1 POMs. Based on this preliminary experiment, we could correlate both diffusion fronts generated by the Hf-WD 1:2 POM with the presence of Mono-WD POM and Hf-WD 1:1 POM. We used Design of Experiments to carry out these experiments efficiently (DoE, JMP software). The DoE method reduces the number of samples required and is also capable of studying complex interactions between parameters. The input parameters used in this study were: 1) the mass percentage of CESA in the staining solution, 2) the volume of the staining solution and 3) the staining time. The results revealed that both CESAs behave differently in terms of the final contrast they offer and their diffusional speed into the tissue. After analysis of the DoE, we found out that the main parameters influencing both observations are the staining time and the mass percentage of CESA. A higher initial mass percentage of CESA resulted in a higher grey value of the tissue and reduced the staining time necessary to completely stain the tissue. In addition, it was observed that for both CESAs, final grey values obtained at the intima side of the tissue, were lower than for the adventitia side. Histological analysis and comparison with high-resolution CT images revealed that the observed grey value could be correlated with the packing of the elastin fibers in the media. Near the adventitia side these fibers are more straight and hence better stacked, whereas near the intima side these fibers have a wavy appearance which inhibits efficient stacking. Moreover, the Hf-containing POMs seemed to have a higher affinity for these fibers, resulting in higher grey values. In conclusion, the Hf-containing POMs result in higher grey values and a slower diffusion, which could be linked to the interaction with the elastin fibers in the media. In future research, interactions with elastin fibers will be confirmed by performing (bio)chemical experiments. Enhancing knowledge on the staining properties of these CESAs will be beneficial for the study of the microstructure of aorta tissue and TE vascular grafts.
- Affiliations
KU Leuven Department of Chemistry, Molecular Design & Synthesis
Citations
Balcaen, T., MARCHAL, M., El Aazmani, W., Hoffmann, D., Kerckhofs, G., & Kerckhofs, G. (2021). Design of experiments to evaluate the staining properties of polyoxometalates for contrast-enhanced microCT of aorta tissue. BSTE 2021 – 8th Belgian Symposium on Tissue Engineering, Louvain-la-Neuve. https://hdl.handle.net/2078.5/269322