Liver disease is a leading cause of mortality in the United States resulting in over 30,000 deaths annually. Allogenic liver transplantation represents the treatment of choice for end-stage liver failure, but the shortage of viable donor livers, the need for immunosuppressive drugs, and high cost are all limiting factors. The concept of whole organ engineering of the liver has emerged as a potential solution. This approach involves perfusion decellularization of a xenogeneic liver followed by repopulating the resultant three-dimensional biologic scaffold with an autologous cell source. Current limitations preventing this approach from becoming a clinical reality include the re-establishment of a non-thrombotic microvasculature and an effective method for delivering functional parenchymal cells to their native location within the three-dimensional scaffold.
The first objective of this work was to determine the effects of commonly used decellularization agents upon the resulting scaffold and its ability to support endothelial cell attachment and growth. Results showed that when a tissue is decellularized with harsh anionic detergents, such as sodium dodecyl sulfate, it is stripped of naturally occurring bioactive components and the native fiber architecture of the extracellular matrix is significantly damaged. Conversely, when less harsh non-ionic detergents are used for decellularization, such as Triton X-100, the resulting scaffold maintains the native microstructure of the extracellular matrix resulting in improved endothelial cell attachment. Thus, the choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the resultant bioscaffold.
The second objective of the present work was to systematically investigate key variables associated with reconstructing a functional hepatic vascular network. Four factors of endothelial cell seeding (1) rate of media perfusion, (2) seeding density, (3) duration of culture, and (4) the addition of an anti-thrombotic heparin coating were investigated by means of two outcomes: endothelial coverage of the scaffold vasculature and cell viability. Within three days of culture, seeded human endothelial cells attached to the three-dimensional liver scaffold, displayed a normal flattened appearance, and formed microvasculature throughout.
The final objective of this work was to develop a preferred method of delivering hepatocytes to achieve effective and viable cell engraftment, anatomically appropriate spatial location, and functionality. Two hepatocyte seeding techniques were developed and evaluated: (1) syringe injection through the Glisson's capsule and (2) infusion through portal and hepatic veins. Metabolic activity and cell viability of the engrafted hepatocytes was evaluated by quantification of albumin and urea production, engrafted cell morphology, and expression of hepatic specific genes. Results also showed high hepatocyte viability (>80%), excellent cell morphology, albumin and urea production, and hepatocyte specific gene expression with the syringe injection technique. The findings from this work represent notable steps toward clinical translation of whole organ engineering.
|School:||University of Pittsburgh|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 77/08(E), Dissertation Abstracts International|
|Keywords:||Biology scaffolds, Biomaterials, Extracellular matrix, Liver tissue engineering, Whole organ engineering, regenerative medicine|
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