A new method of aortic tissue decellullarization for scaffolds development
Authors: Kozin Igor , Petrov Evgenij , Kozina Tatiana , Volova Larisa , Nefedova Irina , Timchenko Pavel , Timchenko Elena , Skidanov Roman , Zelter Pavel 
Published: September 2024

Introduction and Objective. Decellularized biological scaffolds from vascular tissue can potentially replace artificial vascular conduits, which will help solve the issues of donor vessel shortage and the recipients’ immune response. A variety of decellularization techniques have been described and used to achieve effective immunogenic agent removal from a developed vascular scaffold. Although several decellularized vascular grafts are currently on the market, clinical outcomes are still poor due to graft-associated thrombosis, infection, and aneurysm. Given the increasing number of cardiovascular procedures in the world it is necessary to improve existing grafts and look for new methods for scaffolds development.   
Methods. Two human thoracic aortas were harvested from cadaveric material and decellularized with 1% formaldehyde and hexane pure for analysis. Hematoxylin-Eosin staining and Raman spectroscopy were used to confirm complete decellularization. The samples were made electron-conductive and were investigated with electron microscopy to evaluate the structure of the aortic grafts. For an in vivo experiment two grafts from human saphenous veins were decellularized using the same protocol. Afterwards they were implanted in two Flemish Giant rabbits in the abdominal aorta position with “end-to-side” anastomoses. After surgeries the rabbits received food and water ad libitum. A grafts patency was assessed with contrast computer tomography (CT) and ultrasound method. A Doppler ultrasound was used to measure velocity characteristics of blood flow through the conduits. On days 14 and 28 grafts were harvested and embedded in paraffin. Hematoxylin-eosin staining and light microscopy were used to estimate cell repopulation of decellularized implants.
Results. Hematoxylin-eosin staining of the aortas after decellularization demonstrated a complete elimination of cell nuclei. Analysis of the Raman spectra revealed a decrease in the intensity peak specific for deoxyribonucleic acid (DNA) in the decellularized aortas. On the electron microscopic images preserved aortic extracellular matrix with elastic lamellae is observed. All animals survived after surgeries. CT and ultrasound showed good patency of the grafts without signs of stenosis and thrombosis. The mean peak systolic velocity in the first third of the conduits was 122±13 cm/sec and the blood flow rate - 100±3 ml/min. After the 14th and 28th days of implantation we observed cell recellularization of the decellularized extracellular matrix and the formation of neointima on the histological images. At the 14th day the infiltration of immune cells outside of the graft persisted and at the 25th day no prolonged inflammation was observed.      
Conclusions. A new method of aortic implants decellularization with formaldehyde and hexane application makes it possible to achieve a complete elimination of cellular and nuclear contents. The small animal model demonstrated the adequate patency of the grafts, consistent cell repopulation of the extracellular matrix and absence of the hyperinflammatory immune response in a short-term period. Further investigations are required, like quantitative determination of residual DNA within the grafts after decellularization, immunohistochemistry for phenotyping of cells repopulating decellularized material after implantation.