Date of Award
Doctor of Philosophy (PhD)
Biomedical and Chemical Engineering and Sciences
Chris A. Bashur
Bioengineered 3D tissue constructs have gained attention as in vitro tools for the study of cell-cell and cell-matrix interactions and are being explored for potential use as experimental models for mimicking human tissues. One of the main problems in tissue engineering is the necessity to vascularize complex engineered tissues and sacrificial printing has been recognized as a possible solution to vascularization of the bioprinted tissues. Research studies have demonstrated that exposure to microgravity in space induces adaptive alterations in vascular structure and function. Changes in the morphology and gene expression is observed when endothelial cells are exposed to microgravity and radiation environment, but this needs to be further elucidated. Continuous generation of nitric oxide (NO)is essential for the survival and function of the heart and decreased production of nitric oxide causes impairment in endothelial equilibrium. However, there is still limited understanding about the pathways involved with endothelial dysfunction and oxidative stress in microgravity and therefore, is the focus of this research. The overall goals of this project were to develop and optimize bioinks of different compositions of alginate and gelatin mixed with fibrinogen along with human umbilical vein endothelial cells (HUVECs). Alginate was used to provide structural stability and gelatin helped to provide structural porosity for cells to survive within the 3D bioprinted construct. In the first study, the functionality of a 3D bioprinter comprising of an extrusion system that was designed to eliminate the need for an external laminar flow hood was evaluated. The 3D bioprinting system aimed to facilitate the process of 3D bioprinting through its ability to control the environmental parameters within an enclosed printing chamber. This was evaluated by 3D bioprinting constructs of different sizes and various bioink compositions of alginate and gelatin. The 3D constructs were seeded with HUVECs and cell compatibility/viability was evaluated depending on the printing parameters of the bioprinter. The second study comprised of preparation of bioink that was used to bioprint a 3D grid construct of 15x15x4mm using a self-contained bioprinter incorporated with a syringe-based extrusion system (SBE). The 3D bioprinted constructs were analyzed for printability, structural integrity and cell viability by performing characterization studies. It was found that all conditions were acceptable but 10% 1:9 alginate-gelatin composition was ideal for both printability, cell compatibility and degradation. Fluorescence staining and histological studies were also conducted to detect the presence of fibrin in the constructs which would appear to help in cell attachment. In the next study, the 3D bioprinted constructs of bioink concentration 5% 2:3 alginate-gelatin composition was exposed to microgravity for 24 hours and studied for the effects of microgravity on endothelial cells. Results indicated a decrease in NO production and an increase in ROS which indicates a change in endothelial cell equilibrium. We further 3D bioprinted hollow channels to accomplish vascularization by sacrificial printing. This was achieved by using Pluronic F127 as sacrificial material to print channels of 2.5mm. HUVECs were then seeded in the channels after coating it with fibronectin to help the endothelial cells to attach. The 3D bioprinted constructs were then analyzed for cell viability, wherein the results showed that the cells had survived with less cell apoptosis at day 4 when compared to day 0. Further studies were conducted to analyze the effects of microgravity on the sacrificial printed constructs and the results showed the same effect as the grid constructs where a decrease in NO and an increase in the production of ROS was observed. Future work will involve co-culture of two different cell lines to optimize the 3D bioprinted tissue construct and analyze the effects of microgravity. In conclusion, the development of a 3D bioprinted construct and microgravity related studies could benefit society in assessing the risk factors of microgravity and transform the field of tissue engineering.
Somasekhar, Likitha, "Bioink Optimization and Effects of Microgravity on 3D Bioprinted Cell Laden Constructs" (2021). Theses and Dissertations. 588.