(J-362) Characterizing decellularized murine pulmonary circuits using X-ray μCT

Introduction:: In this study, high-resolution 3D X-ray micro-computed tomography (μCT) was employed to analyze the structure of decellularized murine lung vasculature, and subsequently compared to native lung vasculature. To identify the pulmonary vasculature in each lung, acquired images were processed using filtering, segmentation, and skeletonization. Morphological characteristics of the pulmonary vasculature networks in both native and decellularized mouse lungs were assessed by measuring vessel length, diameter, tortuosity, and connectivity. Blood vessels were then categorized into generations according to their connections (Eric A. Chadwick 2021). The connectivity of native and decellularized lung vasculature networks were evaluated to examine the impact of decellularization on the structural features of mouse pulmonary vasculature circuits.


Materials and Methods:: Lungs from adult C57B1/6 mice (12 weeks old) were procured as per established protocols (Mohammadali Ahmadipour 2021). Briefly, mice were euthanized using CO₂ overdose, followed by extraction of heart and lung blocks from the thoracic cavity. Subsequently, the lungs were decellularized using an established detergent-based protocol to remove all cells (Mohammadali Ahmadipour 2021). A liquid contrast agent (Microfil MV-117, Flow Tech Inc.) was administered to the lungs via the pulmonary artery to improve the contrast of vasculature circuits for X-ray μCT imaging. For image acquisition, 3D images of the pulmonary circuit were obtained for each contrast-enhanced mouse lung via X-ray μCT. Reconstructed images underwent Gaussian blur refinement with a sigma (σ) value of 5 voxels as well as vesselness filtration to diminish intensity variance in vessels and void spaces. This filtration aided in avoiding segmentation errors such as non-physical gaps or vessel inconsistencies.

Image segmentation was conducted via two stages: thresholding using Otsu's technique to binarize filtered images and isolating the largest connected voxel cluster. This approach ensured a continuous path throughout the vasculature. Meanwhile, the discarded clusters were carefully examined to confirm that the contrast agent has not been separated during injection, and to ensure that no vascular structures were excluded. Vasculature volumes were subsequently skeletonized, and the Vessel Network Extraction algorithm was employed to assess the morphology of each pulmonary circuit based on vessel length, diameter, tortuosity, and connectivity (Eric A. Chadwick 2021).


Results, Conclusions, and Discussions:: A significant decrease in vascular volume was observed when comparing native and decellularized lung vascular networks, indicating an incomplete penetration of the contrast agent. The integrity of the native and decellularized lung circuits was assessed to identify the reason for this volume loss. The contrast agent permeated both arterial and venous portions of the lung circuit; however, in comparison to the native lung, the decellularized vasculature had fewer vessels per generation as well as limited connections between arterial and venous segments. Obstructions in the blood vessels and leakage through the porous extracellular matrix (ECM) of vascular walls may be responsible for the reduced number of connecting vessels and vessels per generation in decellularized lungs. Such clotting obstructions and contrast agent leakage in the decellularized lung's pulmonary circuit were visualized using X-ray μCT reconstructed images. Furthermore, leakage may be associated with an increased number of generations, reduced vessel length, and heightened vessel tortuosity in the decellularized vasculature.

In summary, current decellularization techniques may yield lung scaffolds with inadequate connectivity of the pulmonary circuit, thus making them unsuitable for fluid flow due to subtle damage to the vasculature structure. To create functional scaffolds for recellularization, it is crucial to maintain the structural integrity of the vasculature during decellularization and remove blockages so that cells have a clear pathway for the recellularization process.


Acknowledgements (Optional): :

References (Optional): : Eric A. Chadwick, Takaya Suzuki, Michael G. George, David A. Romero, Cristina Amon, Thomas K. Waddell, Golnaz Karoubi, and Aimy Bazylak. 2021. "Vessel network extraction and analysis of mouse pulmonary vasculature via X-ray micro-computed tomographic imaging." PLoS Computational Biology.

Mohammadali Ahmadipour, Pascal Duchesneau, Daisuke Taniguchi, Thomas K Waddell, Golnaz Karoubi. 2021. "Negative Pressure Cell Delivery Augments Recellularization of Decellularized Lungs." Tissue Eng Part C Methods.