This article is available online at http://dmd.aspetjournals.org ABSTRACT:Cultures of primary hepatocytes and hepatoma cell line HepG2 are frequently used in in vitro models for human biotransformation studies. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food promutagens benzo[a]pyrene, dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA damage could be detected reliably with the comet assay in primary human hepatocytes, which were maintained in sandwich culture. All three promutagens caused DNA damage in primary cells, but in HepG2 no genotoxic effects of DMN and PhIP could be detected. We supposed that the lack of specific enzymes accounts for their inability to process these promutagens. Therefore, we quantified the expression of a broad range of genes coding for drug-metabolizing enzymes with real-time reverse transcription-polymerase chain reaction. The genes code for cytochromes P450 and, in addition, for a series of important phase II enzymes. The expression level of these genes in human hepatocytes was similar to those previously reported for human liver samples. On the other hand, expression levels in HepG2 differed significantly from that in human. Activity and expression, especially of phase I enzymes, were demonstrated to be extremely low in HepG2 cells. Up-regulation of specific genes by test substances was similar in both cell types. In conclusion, human hepatocytes are the preferred model for biotransformation in human liver, whereas HepG2 cells may be useful to study regulation of drug-metabolizing enzymes.

SummaryBackground-Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up.

Background-Tissue engineering using in vitro-cultivated autologous vascular wall cells is a new approach to biological heart valve replacement. In the present study, we analyzed a new concept to process allogenic acellular matrix scaffolds of pulmonary heart valves after in vitro seeding with the use of autologous cells in a sheep model. Methods and Results-Allogenic heart valve conduits were acellularized by a 48-hour trypsin/EDTA incubation to extract endothelial cells and myofibroblasts. The acellularization procedure resulted in an almost complete removal of cells.After that procedure, a static reseeding of the upper surface of the valve was performed sequentially with autologous myofibroblasts for 6 days and endothelial cells for 2 days, resulting in a patchy cellular restitution on the valve surface.The in vivo function was tested in a sheep model of orthotopic pulmonary valve conduit transplantation. Three of 4 unseeded control valves and 5 of 6 tissue-engineered valves showed normal function up to 3 months. Unseeded allogenic acellular control valves showed partial degeneration (2 of 4 valves) and no interstitial valve tissue reconstitution. Tissue-engineered valves showed complete histological restitution of valve tissue and confluent endothelial surface coverage in all cases. Immunohistological analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor), myofibroblasts (␣-actin), and matrix synthesis (procollagen I). There were histological signs of inflammatory reactions to subvalvar muscle leading to calcifications, but these were not found in valve and pulmonary artery tissue. Conclusions-The in vitro tissue-engineering approach using acellular matrix conduits leads to the in vivo reconstitution of viable heart valve tissue.

Damage to and degeneration of articular cartilage is a major health issue in industrialized nations. Articular cartilage has a particularly limited capacity for auto regeneration. At present, there is no established therapy for a sufficiently reliable and durable replacement of damaged articular cartilage. In this, as well as in other areas of regenerative medicine, tissue engineering methods are considered to be a promising therapeutic component. Nevertheless, there remain obstacles to the establishment of tissue-engineered cartilage as a part of the routine therapy for cartilage defects. One necessary aspect of potential tissue engineering-based therapies for cartilage damage that requires both elucidation and progress toward practical solutions is the reliable, cost effective cultivation of suitable tissue. Bioreactors and associated methods and equipment are the tools with which it is hoped that such a supply of tissue-engineered cartilage can be provided. The fact that in vivo adaptive physical stimulation influences chondrocyte function by affecting mechanotransduction leads to the development of specifically designed bioreactor devices that transmit forces like shear, hydrostatic pressure, compression, and combinations thereof to articular and artificial cartilage in vitro. This review summarizes the basic knowledge of chondrocyte biology and cartilage dynamics together with the exploration of the various biophysical principles of cause and effect that have been integrated into bioreactor systems for the cultivation and stimulation of chondrocytes.

Dermal skin-derived fibroblasts from rodent and human have been found to exhibit mesenchymal surface antigen immunophenotype and differentiation potential along the three main mesenchymal-derived tissues: bone, cartilage and fat. Human dermal skin-derived mesenchymal stem cells constitute a promising cell source in clinical applications. Therefore, we isolated fibroblastic mesenchymal stem-cell-like cells from human dermis derived from juvenile foreskins, which share a mesenchymal stem cell phenotype and multi-lineage differentiation potential. We could show similar expression patterns for CD14()), CD29(+), CD31()), CD34()), CD44(+), CD45()), CD71(+), CD73 ⁄ SH3-SH4(+), CD90 ⁄ Thy-1(+), CD105 ⁄ SH2(+), CD133()) and CD166 ⁄ ALCAM(+) in wellestablished adipose tissue derived-stem cells and fibroblastic mesenchymal stem-cell-like cells by flow cytometry. Immunostainings showed that fibroblastic mesenchymal stem-celllike cells expressed vimentin, fibronectin and collagen; they were less positive for a-smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. When cultured under appropriate inducible conditions, both cell types could differentiate along the adipogenic and osteogenic lineages. Additionally, fibroblastic mesenchymal stem-cell-like cells demonstrated a high proliferation potential. These findings are of particular importance, because skin or adipose tissues are easily accessible for autologous cell transplantations in regenerative medicine. In summary, these data indicate that dermal fibroblasts with multilineage differentiation potential are present in human dermis and they might play a key role in cutaneous wound healing.