Student Hofstra University Brooklyn, New York, United States
Introduction:: Throughout the United States, coronary artery disease is responsible for every 1 in 4 deaths, annually. Coronary artery disease causes partial or complete blockage in the walls of arteries that are responsible for providing blood to the heart. As a result, current research focuses on creating natural, synthetic or hybrid small diameter vascular grafts. Small diameter (< 6mm) vascular grafts have not been successfully constructed in previous research; therefore, an alternative to autologous vessels, an approach to construct small diameter vascular grafts was conducted. In recent research, decellularized plants have been implemented to be used for 3D scaffolds and patches due to its similar mechanical properties to tissue. In this study, decellularized leaves were used to construct small diameter grafts for future applications.
Materials and Methods:: Leatherleaf was decellularized with 2% SDS for 3 days, followed by 6h in clearing solution (10% bleach and 0.1% Triton X-100 in deionized water) and 1h in 70% ethanol, in an orbital shaker at 37C. To assess decellularization, DNA testing was conducted for non-decellularized and decellularized leaves. To assess cell adhesion, decellularized leaves were cut into 0.5 by 0.5cm squares, coated with fibronectin and seeded with rat endothelial cells at a density of 625,000 cells/mL. Decellularized leaves were wrapped around a 2mm acrylic rod once. Gelatin and glutaraldehyde were then added to the unrolled portion of the leaf and quickly rolled until the end. The rolled leaf was held for 30s-1min to allow for cross-linking and incubated at 37C for 4h. The 3D grafts were coated with fibronectin in the lumen and rat endothelial cells were seeded into the lumen at a density of 1.5 million cells/mL. Both ends of the graft were capped with luer lock barbs for 3h to allow cell adhesion followed by being gravity fed for 5 days. Burst pressure and tensile testing were conducted to observe the grafts’ mechanical properties. To assess cell adhesion, DAPI imaging, SEM and histology were performed.
Results, Conclusions, and Discussions:: Several reagents were tested to determine the optimal pathway for decellularizing leatherleaf. DNA testing revealed 234.3±78.9 and 2.3±2.6 ng/mg tissue for non-decellularized and SDS-decellularized leatherleaf, respectively. Over 97% of DNA was removed from leatherleaf using SDS. The elastic modulus of 2D sheets of non-decellularized and SDS decellularized leatherleaf was 3.8±0.2 and 4.0±1.4MPa, respectively. Maximum modulus of SDS 3D grafts and gelatin was 1.3±0.1MPa. Maximum tensile stress at failure and failure strain were 5.5±1.1 MPa and 4.1±0.7, respectively. The burst pressure of 3D grafts was assessed to be 17.0±7.9 psi. Therefore, SDS was chosen for subsequent experiment when compared to other tested reagents. To further ensure cell removal and EC cell adhesion, DAPI imaging and scanning electron microscopy (SEM) was conducted. DAPI staining revealed cell removal and EC cell adhesion of 1296 cells/ mm2 after 5 days for 2D sheets and 1174 cells/ mm2 for 3D grafts. SEM was completed for 2D sheets of SDS-decellularized leatherleaf with and without seeded ECs. SEM revealed a monolayer of ECs on the surface of the decellularized leaves (Figure 1B). To further examine the adhered ECs, H&E staining was tested on seeded 2D sheets, 3D grafts and a control with no cells on both 2D and 3D. Histology revealed successful cell adhesion when observing the cross section of the lumen with the cells. Additionally, it revealed that the cellular material was removed while leaving the vascular structure of the leaf. We found that decellularized Leatherleaf with SDS has suitable mechanical properties when compared to native vessels. The most successful way to bind these scaffolds was through the use of cross-linked gelatin. In future studies, the scaffolds will be pre-conditioned to assess cell adhesion and strength.
