The field of liver regeneration and liver support remains one of the most complex and unsolved medical problems. According to the American Liver Foundation, 25 million Americans suffer from liver and biliary diseases. In 1993, liver disease became the seventh leading cause of death in the United States, and each year, about 30,000 people die from end-stage liver failure. There are currently few effective treatments for severe liver diseases. In contrast to other end-stage organ failures, liver transplantation is the only established successful treatment for liver failure. Again, the discrepancy between supply and demand of organs is staggering. It is estimated that approximately 5,000 liver transplants were performed in 2000. However, almost 1,700 prospective recipients died in 2001 while waiting for a liver for transplantation. At present, there are over 18,000 people waiting for a liver transplant [124].

In efforts to overcome the severe donor organ shortage, several alternative therapies have been explored. These include split liver cadaveric grafts, living donor transplants, xenografts, and selected cell transplantations [125-130]. Several approaches have also been developed for the transplantation of hepatocytes. Direct injections of cell suspensions have been carried out in a variety of locations such as the liver, spleen, or pancreas [131-133]. In addition, approaches involving encapsulations or microcarrier beads have also been assessed [134, 135]. Transplanted hepatocytes were able to maintain normal hepatocellular architecture and demonstrated functional ability for a limited time. However, it has been difficult to achieve a sufficient cell mass to replace lost function.

The difficulty of liver regeneration stems in part from the vast complexity of the tissue. The liver is the largest internal organ and consists of several cell types arranged in a highly complex architecture. It is highly vascularized and performs a large number of metabolic functions. Hepatocytes, the major liver cell type, are anchorage-dependent cells and require an insoluble extracellular matrix for survival, reorganization, proliferation, and function. In addition, they are highly metabolic cells and require close proximity to nutrient and oxygen supply. To achieve a higher cell number and structural support for hepatocytes, hepa-tocyte transplantation combined with synthetic, highly porous, biodegradable scaffolds was proposed. Initial studies demonstrated the survival of transplanted he-patocytes on porous, biodegradable polymer disks in a peripheral site and in the small intestine mesentery in rats [136, 137]. Transplanted hepatocytes expressed liver-specific functions and survived for extended periods. However, a significant decrease in cell number was noted after transplantation.

To improve cell viability, a new approach exploring prevascularization was pursued. Empty scaffolds were placed between the leaves of the mesentery or subcutaneous pockets to promote fibrovascular ingrowth prior to cell injection. This led to improved cell engraftment and survival, but a large number of cells were lost within a week [138, 139]. Further improvement in cell engraftment and growth was achieved by considering the self-regulation of liver mass, i.e., transplanted livers will grow or atrophy to reach an appropriate size for the recipient. It was conjectured that transplanted hepatocytes were actively suppressed in the recipient due to the presence of a healthy native liver. To assess this conjecture, recipient animals underwent partial hepatectomies or portacaval shunts, resulting in an increased delivery of hepatotrophic factors to the systemic circulation and reduced clearance by the native liver. It was shown that hepatotrophic stimulation led to a significant improvement in cell survival [140-143]. Using this approach, a mass of Wistar rat hepatocytes equivalent to a whole liver was transplanted in Gunn rats, which have a genetic deficiency of glucuronyl transferase activity, showing unconjugated hyperbili-rubinemia. Over a course of several weeks, a decrease in serum bilirubin levels was observed [144, 145]. The methodology of combined cell/polymer transplantation and surgical hepatotrophic stimulation has also been extended to large animals [146-148]. Another approach to overcome the insufficient engraftment has been to improve vascularization by local delivery of angiogenic factors. In one study, bFGF was incorporated into degradable scaffold, and increased angiogen-esis and hepatocyte engraftment were observed [149]. A recent approach to overcome the critical limits of nutrient and oxygen diffusion has been the development of polymer scaffolds that can be implanted directly into the bloodstream [150]. Hepatocytes were dynamically seeded onto these scaffolds and placed in a flow reactor. The engrafted hepatocytes showed excellent survival with a high rate of albumin synthesis [151, 152].

A novel approach is the creation of a scaffold with an integrated vascular network to provide immediate access to the blood supply after implantation. A versatile scaffold fabrication method termed three-dimensional printing (3DP) offering unprecedented control over the geometry and architecture including controlled porosity and ingrowth channels was used to create complex three-dimensional biodegradable scaffolds [153]. Hepatocytes attached to the scaffold and survived under dynamic culture conditions in vitro. Albumin synthesis was demonstrated, and the hepatocytes reorganized into histotypical structures in the channels of the scaffold [154, 155]. The concept of prevascularization to provide improved cell engraft-ment and mass transfer of oxygen and nutrients was recently further refined through the adaptation of silicon microfabrication. This methodology is based on semiconductor wafer process technology originally developed for integrated circuits (IC) and microelec-tromechanical systems (MEMS). Silicon microfabrication offers submicron-scale resolution over several orders of magnitude from 0.1 mm to tens of centimeters [156]. Since this range covers the relevant physiological length scales from capillaries to large vessels, a concept was developed to create a complete branching vascular circulation in two dimensions on silicon wafers and subsequently build up three-dimensional structures by stacking or rolling. In a first demonstration, hepato-cytes and endothelial cells were cultured on silicon and Pyrex wafers patterned with trenches reminiscent of a vasculature. Hepatocyte sheets were lifted off, folded into compact three-dimensional configurations, and implanted into rat omenta. This resulted in the formation of vascularized hepatic tissue [157]. Subsequent advanced have been the development of a computational model to create tissue-specific vascular networks and the transfer of this methodology to biocompatible polymers [158, 159]. Current research is in progress to transfer the process methodology to biodegradable polymers to arrive at the ultimate goal of thick, vascu-larized tissue-engineered organs (Fig. 16.6).

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