**5. Liver bioengineering**

Tissue engineering is one of the most promising fields in regenerative medicine. As described in 1993 by Robert Langer and Joseph Vacanti it is the conjugation of biomaterials (synthetic or naturally derived) with cells, in order to generate tissue constructs that can be implanted into patients to substitute a lost function, maintain or gain new functions(43). The

maintainance of the functional hepatocytes for longer period of time in a bioreactor, complexity of the design and high cost. These challenges have delayed the entry of BAL systems in the clinic. Nonetheless, plenty of optimized designs of liver support devices are under development and undergoing clinical trials which is a sign of optimism in this area of

Hepatocyte transplantation is certainly in the forefront of new therapeutic strategies. The first successful hepatocyte transplantation into a patient was carried out in June 1992 to a French Canadian woman with familial hypercholesterolaemia. After *ex vivo* transduction with a retrovirus encoding for the human LDL receptor, the patient's hepatocytes were infused through the inferior mesenteric vein into the liver. LDL and HDL levels improved throughout the next 18 months and transgene expression was detected in a liver biopsy(22). Following this first success, other patients followed through. However, not all the patients treated had a clear benefit from the procedure(23). Since then, several other metabolic diseases have been treated with hepatocyte transplantation with different degrees of success(24-28). It has also been used as a support treatment to acute(29-31) and chronic liver diseases(30-33) in bridging severely ill patients to orthotopic liver transplantation (OLT). Low efficacy and lack of long-term therapeutic effect have been common in all these procedures. These failures could be explained by the relatively small number of hepatocytes that engraft in the recipient liver due to quality, quantity and possibly immunosuppresion protocols(34). However, transplantation of a number of hepatocytes corresponding to 1-5% of the total liver mass has been able to show a positive impact in transplanted patients, even

Due to the shortage of available human hepatocytes for transplantation, other cell sources have been used. Specifically, bone marrow derived mesenchymal stem cells(35), hematopoietic stem cells(36, 37) and fetal liver progenitor/stem cells(38) have shown to improve, to a certain extent, the condition of cirrhotic patients. The latter cell type holds an enormous potential for cell/regenerative medicine therapies due to their high expansion

Recent data suggests that human embrionic (hES) and induced pluripotent (iPS) stem cells hold great promise to regenerative applications in every medical field. Specifically for the liver, several studies have established the required pathways to differentiate a hES or iPS into a hepatic fate by using defined soluble growth factor signals that mimic embryonic development(40, 41). These cells, once transplanted into rodent livers were able to engraft and express several normal hepatic functions(42). However, more extensive characterization, as well as further safety evaluation, are needed to determine wether these

Tissue engineering is one of the most promising fields in regenerative medicine. As described in 1993 by Robert Langer and Joseph Vacanti it is the conjugation of biomaterials (synthetic or naturally derived) with cells, in order to generate tissue constructs that can be implanted into patients to substitute a lost function, maintain or gain new functions(43). The

capabilities and differentiation into hepatocytes and biliary epithelium(39).

cells will fully function as primary adult hepatocytes.

critical care and management.

if for a limited period of time(34).

**5. Liver bioengineering** 

**4. Cell therapies** 

current paradigm is suitable for the engineering of thin constructs like the bladder, skin or blood vessels. Although, in the specific case of the liver, the 3D architecture and dense cellular mass requires novel tissue engineering approaches and the development of vascularized biomaterials, in order to support thick tissue masses and be readily transplantable. Additionally to the vascular support for large tissue masses, hepatocyte function maintenance represents the ultimate aim in any organ engineering or regenerative medicine strategy for liver disease.

Hepatocytes are known to be attachment-dependent cells and lose rather quickly their specific functions without optimal media- and ECM- composition and cell-cell contacts. Also, function and differentiation of liver cells are influenced by the 3D organ architecture(44).

In the last two decades innumerous strategies for the culture of adult hepatocytes in combination with several types of 3D, highly porous polymeric matrices have been attempted(45-49). However, in the absence of vasculature, restriction in cell growth and function is common due to the limitations in nutrient and oxygen diffusion. Some of these problems are being now partially overcome with the development of bioreactors that provide continuous perfusion of culture media and gases allowing a 3D culture configuration and hepatocyte function maintenance(50-52).

The tissue engineering concept has several advantages over the injection of cell suspensions into solid organs. The matrices provide sufficient volume for the transplantation of an adequate cell mass up to whole-organ equivalents45. Transplantation efficiency could readily be improved by optimizing the microarchitecture and composition of the matrices as well as by attaching growth factors and extracellular matrix molecules to the polymeric scaffold, helping to recreate the hepatic microenvironment(44). The use of naturally derived matrices has also proved to be very helpful in hepatocyte culture(47). These matrices, besides preserving some of the microarchitecture features of the tissues that they are derived from, also retain bioactive signals (e.g., cell-adhesion peptides and growth factors) required for the retention of tissue-specific gene expression(53, 54). Additionally, cell transplantation into polymeric matrices is, in contrast to cell injection into tissues and organs, a reversible procedure since the cell-matrix-constructs may be removed if necessary.

Finally, heterotopic hepatocyte transplantation in matrices has already been demonstrated in long-term studies(55, 56). Nonetheless, initial engraftment rates are suboptimal. One of the reasons for this is the absolute requirement of the transplanted hepatocytes for hepatotrophic factors that the liver constantly receives through its portal circulation(57). Thus, the development of a tissue engineered liver construct capable of being orthotopically transplanted is essential.

Apart from cellular therapies, other early developments of experimental approaches are not showing results that will indicate clinical translation in the next few years. However, two experimental approaches that show higher level of maturity may have the potential for succesful clinical translation. The first experimental approach is the "cell sheet" technology developed by Okano *et al*. in Japan(58). Its simple configuration and fabrication allows for the stacking of up to four hepatocyte cell sheets that can readily engraft and provide a defined metabolic relief to the recipient(59). This technology has already been applied successfully to one patient with heart failure. Other technology that shows great promise is

Liver Regeneration and Bioengineering – The Emergence of Whole Organ Scaffolds 249

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tissue and organ decellularization. Our lab and others have been able to generate several decellularized scaffolds for tissue engineering applications like tissue engineering of urethra(60), heart valves(61), blood vessel(62). More recentely, Ott *et al*. developed a novel method of perfusion decellularization that is able to generate whole organ scaffolds. The use of this method allowed the decellularization of a whole heart that was later repopulated with neonatal rat cardiomyocytes. This bioengineered heart was able to contract up to 2% of the normal contractile function(63). This approach may have a tremendous potential for the field of organ bioengineering. We have recently used a similar perfusion decellularization technique to liver, pancreas, intestine and kidney generating decellularized organ scaffolds for organ bioengineering(64, 65). These bioscaffolds preserve their tissue microarchitecture and an intact vascular network that can be readily used as a route for recellularization by perfusion of culture medium with different cell populations. In an analogous fashion, Uygun *et al*. decellularized rat livers and repopulated them with rat primary hepatocytes, showing promising hepatic function and the ability of heterotopicaly transplant these bioengineered livers into animals for up to eight hours(66). Baptista *et al.* were able to take this a step further by using human primary liver progenitor/stem and endothelial cells to bioengineer a vascularized liver. These bioengineered livers displayed some of the functions of a native human liver (albumin and urea secretion, drug metabolism enzyme expression, etc), exhibiting also an endothelialized vascular network that prevented platelet adhesion and aggregation, critical for blood vessel patency after transplantation(65). Nonetheless, it is difficult to predict the outcome and the real translational value of this technology in the present days, but the potential is certainly vast. Translation of it into the bioengineering of human size livers might help mitigate the endless hurdle of organ shortage for transplantation.

#### **6. References**


tissue and organ decellularization. Our lab and others have been able to generate several decellularized scaffolds for tissue engineering applications like tissue engineering of urethra(60), heart valves(61), blood vessel(62). More recentely, Ott *et al*. developed a novel method of perfusion decellularization that is able to generate whole organ scaffolds. The use of this method allowed the decellularization of a whole heart that was later repopulated with neonatal rat cardiomyocytes. This bioengineered heart was able to contract up to 2% of the normal contractile function(63). This approach may have a tremendous potential for the field of organ bioengineering. We have recently used a similar perfusion decellularization technique to liver, pancreas, intestine and kidney generating decellularized organ scaffolds for organ bioengineering(64, 65). These bioscaffolds preserve their tissue microarchitecture and an intact vascular network that can be readily used as a route for recellularization by perfusion of culture medium with different cell populations. In an analogous fashion, Uygun *et al*. decellularized rat livers and repopulated them with rat primary hepatocytes, showing promising hepatic function and the ability of heterotopicaly transplant these bioengineered livers into animals for up to eight hours(66). Baptista *et al.* were able to take this a step further by using human primary liver progenitor/stem and endothelial cells to bioengineer a vascularized liver. These bioengineered livers displayed some of the functions of a native human liver (albumin and urea secretion, drug metabolism enzyme expression, etc), exhibiting also an endothelialized vascular network that prevented platelet adhesion and aggregation, critical for blood vessel patency after transplantation(65). Nonetheless, it is difficult to predict the outcome and the real translational value of this technology in the present days, but the potential is certainly vast. Translation of it into the bioengineering of human size livers

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