Date of Award

12-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Nagatomi, Jiro

Committee Member

Webb, Ken

Committee Member

Visconti, Richard

Committee Member

Purves, Todd

Abstract

In recent years, tissue engineering of the bladder has undergone many technological advances. Autologous urothelial cells have been used in animal models and have shown increased performance for ureteral reconstruction compared to unseeded grafts. However, since patients may lack a reliable source of native urothelial cells due to the nature of their specific bladder disease, autologous cells are not an ideal source clinically, and an alternate cell source must be explored. Adipose derived stem cells (ADSCs) are an attractive cell source for such regenerative medicine applications as they have been extensively studied for their multipotential differentiation, immunosuppressive properties, ease of harvest, and relative abundance. The present doctoral thesis investigates the effect of growth factors present in defined keratinocyte serum-free medium (KSFM), all-trans retinoic acid (ATRA), and environmental conditions on ADSCs to explore their efficacy as a potential donor source for urothelial tissue regeneration. Using serum-free conditions, hADSCs were exposed to defined KSFM and ATRA over a 21-day culture period, and effects on morphology and phenotype marker expression were examined. The results indicated that hADSCs differentiated towards urothelial-like cell lineage without co-culturing or serum-containing medium after 14 days of culture in defined KSFM, while the effect of ATRA supplementation was limited to morphology changes and not on expression of markers tested. Furthermore, a fibronectin/gelatin layer-by-layer coating technique was used to develop a multilayer tissue construct with both undifferentiated and differentiated hADSCs (cultured using defined KSFM) and UROtsa cells to investigate cell morphology, protein expression, and dextran permeability across the multilayer construct. In hADSCs, cell aggregation occurred in differentiated and coated samples over 24 hours of culture in Transwell® inserts. Fibronectin/gelatin coating technique applied to both UROtsa cells and growth membrane resulted in a multilayer with expression of CK-17, CK-20, UPIb, and ZO-1 urothelial protein markers and less aggregation compared to hADSC. Dextran permeability results after 24 hours indicated diffusion of 4 and 70 kDa dextran across cell constructs with no differences between cell types. Lastly, a tension bioreactor system capable of applying constant tension on cell-seeded tissue engineering constructs was calibrated. Mechanical conditioning may play a role in development of functional tissue in vitro to increase overall strength and compliance of tissue engineered constructs. In the present study, the bioreactor was tested with three materials (parafilm, PCUU, and T1107 hybrid hydrogel) to demonstrate its capability to apply desired loading parameters based on user settings and to record force/distance vs. time profiles. In summary, the results provide novel evidence that, through a combination of physical and chemical factors, hADSCs differentiate towards urothelial-like lineage under defined KSFM culture after 14 days. Fibronectin/gelatin cell and Transwell® membrane coating promotes in vitro formation of urothelial-like multilayer constructs using UROtsa cells. A tension bioreactor system has been calibrated to apply controlled mechanical force stimuli on soft biomaterials intended to reproduce the in vivo bladder filling for functional tissue engineering. The knowledge gained from the present study will serve as foundation for future studies focusing on multilineage and functional bladder tissue engineering using adipose-derived stem cells for the treatment of bladder disorders.

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