[Hepatic tissue engineering]. / Tissue Engineering der Leber.

Today liver transplantation is the only curative option for the treatment of end-stage liver diseases. A major limitation of liver transplantation is the donor organ shortage. Therefore, tissue engineering based cell transplantation is currently under investigation with the aim to replace liver tissue and function. The principle of tissue engineering is the notion of an interaction between a cell and a three-dimensional matrix. The matrix serves as a scaffold and guides a three-dimensional cell assembly. In addition, the matrix provides for a regulation of cell proliferation and function by cell-matrix interactions. In cultures of hepatocytes a regulation of cell proliferation and specific function by using three-dimensional matrices and by modifying the surface with isolated molecules of the extracellular matrix has been demonstrated. Furthermore, a beneficial effect of a flow bioreactor system on cell viability and function was observed. In addition, a system for heterotopic hepatocyte transplantation on polymeric matrices was developed in an animal model. In this transplantation model a long-term proliferation and function of transplanted hepatocytes was shown. The major limitation of matrix-based transplantation systems is the high initial cell loss, most probably due to an insufficient vascularisation. Thus, the development of vascularised matrices and the creation of bile ducts remain major problems in the technologies of hepatic tissue engineering and have to be addressed to enable further advances towards clinical applications.

Foetal hepatocyte transplantation in a vascularized AV-Loop transplantation model in the rat.

The use of foetal liver cells (FLC) in the context of hepatic tissue engineering might permit efficient in vitro expansion and cryopreservation in a cell bank. A prerequisite for successful application of bioartificial liver tissue is sufficient initial vascularization. In this study, we evaluated the transplantation of fibrin gel-immobilized FLC in a vascularized arterio-veno-venous (AV)-loop model. FLC were isolated from embryonic/foetal (ED 16) rat livers and were enriched by using magnetic cell sorting (MACS). After cryopreservation, FLC were labelled by pkh-26. Cells were transplanted in a fibrin matrix into a subcutaneous chamber containing a microsurgically created AV-loop in the femoral region of the recipient rat. The chambers were explanted after 14 days. Subcutaneous implants without an AV-loop and cell-free implants served as controls. Fluorescence microscopy of the constructs was used to identify pkh-26(+)- donor cells. Characterization was performed by RT-PCR and immunhistology (IH) for CK-18 and CD31. Transplantation of FLC using the AV-loop permitted a neo-tissue formation in the fibrin matrix. A high-density vascularization was observed in the AV-loop constructs as shown by CD31 IH. Viable foetal donor cells were detected which expressed CK-18. FLC can be successfully used for heterotopic transplantation. Fibrin matrix permits rapid blood vessel ingrowth from the AV-loop and supports engraftment of FLC. It is therefore an appropriate environment for hepatocyte transplantation in combination with microsurgical vascularization strategies. Transplantation of fibrin gel-immobilized FLC may be a promising approach for the development of highly vascularized in vivo tissue-engineering-based liver support systems.

Fetal and adult liver stem cells for liver regeneration and tissue engineering.

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.

Submucosal plexus of dilatated gut disappears after ligation in chicken embryos: preliminary results.

INTRODUCTION: Dilatation and impaired function of the gut is a condition often seen in newborns with bowel obstruction caused by intestinal atresia. In a previous experimental study in chicken embryos, we established a model to study ultrastructural changes during the development of the enteric nervous system after small bowel ligation. The aim of this study is to investigate the changes of the enteric nervous system (ENS) after gut ligation. METHODS: 56 chicken embryos were investigated. In the operation group fertilized eggs and the allantoic membrane were opened and the small bowel was ligated on embryonal day (ED) 11. The controls were sham-operated. The gut was prepared and harvested for analysis on ED 11, 12, 13, 14, 15, 16, 17 and 18. Silver staining or staining of the specimens for acetylcholinesterase (AchE) was performed. RESULTS: A marked dilatation of the bowel was observed three days after operation (ED 14). The submucosal (PSM) and myenteric plexus (PM) appeared normal at this time, however silver staining showed rarification of the neuronal axonal network between the myenteric and submucosal plexus. Later, on ED 16 an additional rarification of the submucosal plexus was also seen in the operation group using AchE staining, compared to the controls. DISCUSSION: The data suggest that distension of the gut hinders normal development of the ENS in the gut ligation model of chicken embryos. The changes were observed sequentially, starting with rarification of the axonal network between the PM and PSM. Future studies will be required to show whether the changes of the ENS are reversible.

Hepatocytic gene expression in cultured rat mesenchymal stem cells.

The origin of liver cells from distinct bone marrow stem cells, eg, hematopoietic stem cells or multipotent adult progenitor cells has been recently described using in vitro studies. Cell culture experiments revealed the key role of growth factors and the organ-specific environment for the induction of liver-specific genes. We investigated the in vitro potential of rat mesenchymal stem cells to differentiate into hepatocytic cells in cocultures with isolated rat liver cells. Rat mesenchymal stem cells (MSCs) propagated in culture, and transduced with green fluorescent protein (GFP) were cloned. Cells from selected clones were either cultured under liver-stimulating conditions, using serum free medium supplemented with HGF, EGF, SCF, and FGF-4 alone on fibronectin-coated surfaces, or cocultured with freshly isolated rat liver cells. Cocultured cells were harvested after two weeks and sorted into GFP-positive (GFP+) and GFP-negative (GFP-) cells. RT-PCR for liver specific markers CK-18 and albumin were performed on the different cell populations. After 2 weeks, the specified culture conditions led to the expression of albumin and CK-18 RNA in GFP-positive sorted MSCs from the cocultures, whereas MSCs cultured without liver cells did not express the studied genes. The results indicate, that when cocultured with liver cells MSCs from the bone marrow have the potential to differentiate toward hepatocytic cells in vitro. We conclude that MSC may possess an enhanced capacity to differentiate into functional liver cells. Additionally, environmental factors seem to be crucial for specific and directed differentiation.

Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: comparison with heterotopically transplanted liver grafts.

BACKGROUND: Hepatocyte transplantation using polymeric matrices is under investigation as an alternative therapy for metabolic liver diseases. Long-term engraftment of hepatocytes in polymers has been demonstrated. However, the metabolic activity of hepatocytes in such devices has never been assessed in direct comparison with liver grafts. METHODS: Hepatocyte and partial liver transplantation were evaluated in the scurvy-prone osteogenic disorder Shionogi rat model. Biodegradable poly glycolic acid matrices seeded with hepatocytes equivalent to 20% of the recipient's liver mass, or 20% liver grafts were heterotopically transplanted into ascorbic acid- (AsA) deficient recipients. Recipients of cell-free matrices or AsA-deficient liver grafts served as controls. Recipients were set on AsA-free diet after transplantation. Plasma AsA levels, AsA concentrations in liver and adrenal gland tissue, and body weight ratios were assessed and H&E histology was performed. RESULTS: Recipients from the control groups showed symptoms of scurvy at 1 month after cessation of AsA supply. Hepatocyte transplantation and auxiliary liver transplantation prevented symptoms of scurvy and increased plasma and tissue AsA levels and body weight ratios. AsA levels in recipients of 20% liver grafts were comparable to normal control animals. CONCLUSIONS: Hepatocytes transplanted in polymeric matrices are able to compensate for liver-based metabolic deficiencies. Hepatocyte transplantation improves plasma AsA levels in AsA-deficient recipients. However, auxiliary liver grafts are superior to hepatocyte grafts in improving metabolic parameters. Further research work is needed to increase the efficiency of liver cell transplantation with regard to a clinical application.

Priming of hepatocytes for cell culture by partial hepatectomy prior to cell isolation.

The combination of ex vivo gene transfer and a sufficient transplant model for hepatocytes may permit treatment of single enzyme-based metabolic liver diseases. Induction of replicative potential (priming) in hepatocyte cultures may enhance the efficiency of gene transfer under stable in vitro conditions. It is known that hepatocyte replication is increased in vivo after partial hepatectomy. We investigated the effect of partial hepatectomy prior to cell isolation on hepatocytes in vitro. Male Lewis rats served as donors. Hepatocytes were isolated by collagenase digestion from either intact livers or from livers 48 h after 70% hepatectomy (PH). Cells were seeded on collagen-coated culture dishes with hormone-supplemented culture media. Hepatocyte morphology, number, albumin secretion rate, and mono-ethyl-glycin-xylidid (MEGX)-biotransformation capacity were assessed on days 1, 3, and 5 in culture. PH significantly increased hepatocyte number and albumin secretion of cultured hepatocytes over the whole observation period. In contrast, MEGX-biotransformation capacity was significantly decreased. Morphology of cultured hepatocytes was not affected by PH prior to hepatocyte isolation. These results suggest a prolonged and complex response of hepatocytes to PH in vitro. Hepatocyte priming by PH is a promising approach toward stable cultures of proliferating hepatocytes and may provide a model for in vitro studies of hepatic regeneration mechanisms. Further research on hepatocyte priming toward an application in ex vivo gene transfer and hepatic tissue engineering seems justified.

Interaction of hepatocytes and pancreatic islets cotransplanted in polymeric matrices.

Heterotopic hepatocyte transplantation (HcTx) in polymeric matrices may become an alternative to liver transplantation for metabolic disorders. Hepatotrophic stimulation by means of a portocaval shunt operation is an established, but invasive, procedure used to optimize hepatocyte engraftment in matrices. We evaluated hepatocyte and pancreatic islet cotransplantation (ICT) as an alternative noninvasive approach to hepatotrophic stimulation. Lewis rats served as donors and recipients. Hepatocytes and islets were isolated using collagenase digestion and seeded into polyvinylalcohol matrices. HcTx and ICT were compared with HcTx plus portocaval shunt and HcTx without stimulation. Matrices were investigated at 1, 3, and 6 months after implantation: the test methods applied were trichrome staining, PAS, immunohistochemistry for insulin, glucagon and incorporated BrdU, and in situ hybridization for albumin RNA. Hepatocytes expressed albumin RNA and formed conglomerates without atypias in all animals. ICT and portocaval shunting increased the number of hepatocytes and BrdU uptake. Alpha cells migrated into the islet-surrounding hepatocytes, whereas beta cells remained immobile. It is concluded that ICT and portocaval shunting supported engraftment of hepatocytes in polymeric matrices equally well. ICT did not interfere with recipient glucose metabolism and did not induce hyperproliferative premalignant foci within the transplanted hepatocytes. The technique is an attractive approach to hepatotrophic stimulation of bioartificial liver equivalents.

Influence of pancreatic islets on growth and differentiation of hepatocytes in co-culture.

Improvement of cell culture conditions in hepatic tissue engineering may permit cell/tissue banking and the generation of liver tissue equivalents for transplantation. In these systems, continuous hepatotrophic stimulation is still necessary. We investigated the stimulatory effects of pancreatic islets on hepatocytes in co-culture and characterized the stimulatory mechanisms. Hepatocytes and pancreatic islets were harvested from Lewis rats. Cells were cultured on collagen dishes either with nonstimulated media (controls and co-cultures with low or high islet rate) or stimulated media (controls and co-cultures). To characterize stimulatory mechanisms, additional co-cultures with membrane separation, with antiinsulin, antiglucagon, and with both antibodies were examined. Hepatocyte numbers, albumin secretion rate by enzyme-linked immunoadsorbent assay, and monoethylglycinxylidid biotransformation values by fluorescence polarization immunoassay were assessed. A radioimmunoassay measured insulin and glucagon concentrations. In groups with nonstimulated media, cell number was higher in co-cultures with low islet rate, and albumin secretion rate was increased in co-cultures with high islet rate compared to controls. MEGX biotransformation was decreased in co-cultures. In groups with stimulated media, co-culture had no impact on cell number or albumin secretion rate. Hepatocyte numbers and albumin secretion rates were not changed in co-cultures after membrane separation. Islet effects on hepatocytes were reduced in co-cultures with antiinsulin, antiglucagon, or both antibodies. Pancreatic islets provide stimulation for hepatocytes in vitro. Islet effects were mediated by soluble factors, and are dependent on insulin and glucagon. These results permit further investigations towards three-dimensional transplantable hepatocyte-islet devices for continuous in vitro and in vivo stimulation.

Long-term differentiated function of heterotopically transplanted hepatocytes on three-dimensional polymer matrices.

Hepatocyte transplantation using porous matrices is under investigation as an alternative therapy for certain liver diseases. For this purpose, long-term function of transplanted hepatocytes is mandatory. This problem has not been sufficiently investigated yet. In this study Lewis rats were used as donors and recipients. Stimulated (group A, portocaval shunt) or unstimulated (group B) hepatocytes were transplanted into prevascularized polyvinyl-alcohol matrices. Cell-free matrices served as controls (group C). Matrices were harvested between 1 h and 1 year after implantation and analyzed by morphometry; albumin RNA in situ hybridization; and cytokeratin-, actin-, desmin-, and macrophage-specific antigen immunohistology. The hepatocyte number significantly decreased within the first week following implantation. Between 1 month and 1 year after transplantation a significant increase in hepatocyte number was noted in groups A and B. Albumin transcripts of transplanted hepatocytes were at normal levels at all times except for group B after 1 year. The immunohistology suggested engraftment of nonparenchymal liver cells. We conclude that 3-dimensional matrices provide a sufficient environment for long-term engraftment of transplanted liver cells. The hepatocytes are able, despite suboptimal initial engraftment, to repopulate the scaffold for at least half of the recipient's life span and maintain cell-specific function after sufficient stimulation.

Is there an optimal concentration of cotransplanted islets of Langerhans for stimulation of hepatocytes in three dimensional matrices?

BACKGROUND: Hepatocyte transplantation using three-dimensional matrices is under investigation as an alternative therapy for several liver diseases. For sufficient transplantation results hepatotrophic stimulation is necessary. We investigated the stimulatory effect of cotransplanted pancreatic islets in different ratios. METHODS: Lewis rats were used as donors and recipients. A portocaval shunt (group A) or sham operation (groups B-G) was performed 1 week before hepatocyte transplantation. Four polyvinyl-alcohol matrices each containing 1.25 x 10(7) hepatocytes (groups A and B) or 1.25 x 10(7) hepatocytes and 125 (C), 250 (D), 500 (E), or 750 (F) islets were implanted between small bowel mesenteric leaves. In group G, medium soaked matrices were implanted. One month after implantation, specimens were harvested and investigated using albumin-RNA in situ hybridization, and insulin, glucagon, and bromodesoxy uridine immunohistochemistry. The hepatocyte area was assessed using image analysis. RESULTS: Hepatocyte area and proliferation ratio increased depending on the number of cotransplanted islets with a peak at 40 islets per 1 million hepatocytes (group E). Cotransplantation of islets in higher concentrations did not further increase hepatocyte area or proliferation ratio. Hepatocytes in all groups expressed albumin RNA at normal transcription levels as compared to standard liver sections. Islets displayed insulin and glucagon in physiological distribution. DISCUSSION: Three-dimensional matrices provide a sufficient environment for transplanted hepatocytes and islets. The hepatotrophic effect of cotransplanted islets is comparable to portocaval shunting and has a saturation limit at 40 islets per 1 million hepatocytes. For further application of islet cotransplantation, this ratio seems to be preferable.

Heterotopic hepatocyte transplantation utilizing pancreatic islet cotransplantation for hepatotrophic stimulation: morphologic and morphometric evaluation.

Hepatocyte transplantation using three-dimensional (3D) matrices is under evaluation as an alternative therapy for liver diseases. It is known that hepatotropic stimulation optimizes hepatocyte engraftment. We investigated hepatotrophic stimulation by portocaval shunt operation (PCS) or pancreatic islet cotransplantation (ICT) over a period of 6 months. Lewis rats served as donors and recipients, respectively. One week prior to hepatocyte implantation PCS (group A) or a sham operation (groups B-D) was performed. Four polyvinyl-alcohol matrices, each containing 1.25 x 10(7) hepatocytes (groups A and C), 1.25 x 10(7) hepatocytes and 500 islets (group B), or cell-free culture medium (group D, control) were implanted between recipients' small-bowel mesenteric leaves. One, 3, and 6 months after implantation eight polymers from each group were harvested and analyzed by morphometry, PAS reaction, and immunohistochemistry for insulin, glucagon, and bromodesoxyuridine. Morphologically healthy-appearing hepatocytes were found in all cell transplantation groups at all times. Stimulation by either PCS or ICT significantly increased hepatocyte area at 1 and 6 months compared to unstimulated specimens (group C). Over time, an increase in hepatocyte area was noted in all groups. There were no significant differences in proliferation ratios between the three experimental groups. The initially reduced PAS reaction became normal after 3 months. 3D matrices provided a sufficient environment for transplanted hepatocytes and islets. Hepatocytes proliferated and maintained differentiation independent of hepatotrophic stimulation for at least 6 months when 3D matrices were utilized. ICT efficiently stimulated transplanted hepatocytes by means of hepatocyte area. These results justify further research on hepatocyte transplantation and ICT with regard to clinical application.