A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells.
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This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Ham's F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell-polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudo-stratified columnar epithelium, with a distinct interface between tissue-engineered cartilage and epithelium. Safranin-O staining shows that tissue-engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects. (+info)
Redifferentiation of dedifferentiated bovine chondrocytes when cultured in vitro in a PLGA-collagen hybrid mesh.
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Bovine articular chondrocytes dedifferentiated and lost their ability to express articular cartilage-specific extracellular matrices such as type II collagen and aggrecan when cultured in a culture flask during in vitro multiplication. A poly(DL-lactic-co-glycolic acid) (PLGA)-collagen hybrid mesh was prepared and used to redifferentiate the dedifferentiated cells. The two passaged dedifferentiated chondrocytes were seeded in a PLGA-collagen hybrid mesh and cultured in vitro in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The gene expression of type I collagen, type II collagen, and aggrecan was analyzed after the cells were cultured in the hybrid mesh for 2-12 weeks. The expression of the gene encoding type I collagen was downregulated, whereas those of type II collagen and aggrecan were upregulated. Histological examination by hematoxylin-eosin and safranin O/fast green staining indicates that the cells regained their original round morphology. In addition, a homogeneous distribution of articular cartilage extracellular matrices was detected around the cells. These results suggest redifferentiation of the differentiated chondrocytes in the hybrid mesh. The hybrid mesh, which facilitated the redifferentiation of the dedifferentiated multiplied chondrocytes, would be an effective scaffold for the assembly of cells to regenerate three-dimensional cartilaginous tissue. (+info)
Cell and organ printing 1: protein and cell printers.
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We have developed several devices for positioning organic molecules, molecular aggregates, cells, and single-cell organisms onto solid supports. These printers can create stable, functional protein arrays using an inexpensive technology. The cell printer allows us to create cell libraries as well as cellular assemblies that mimic their respective position in organs. The printers are derived from commercially available ink-jet printers that are modified to dispense protein or cell solutions instead of ink. We describe here the modifications to the print heads, and the printer hardware and software that enabled us to adapt the ink-jet printers for the manufacture of cell and protein arrays. The printers have the advantage of being fully automated and computer controlled, and allow for the high-throughput manufacture of protein and cell arrays. (+info)
Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels.
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We recently developed a cell printer (Wilson and Boland, 2003) that enables us to place cells in positions that mimic their respective positions in organs. However, this technology was limited to the printing of two-dimensional (2D) tissue constructs. Here we describe the use of thermosensitive gels to generate sequential layers for cell printing. The ability to drop cells on previously printed successive layers provides a real opportunity for the realization of three-dimensional (3D) organ printing. Organ printing will allow us to print complex 3D organs with computer-controlled, exact placing of different cell types, by a process that can be completed in several minutes. To demonstrate the feasibility of this novel technology, we showed that cell aggregates can be placed in the sequential layers of 3D gels close enough for fusion to occur. We estimated the optimum minimal thickness of the gel that can be reproducibly generated by dropping the liquid at room temperature onto a heated substrate. Then we generated cell aggregates with the corresponding (to the minimal thickness of the gel) size to ensure a direct contact between printed cell aggregates during sequential printing cycles. Finally, we demonstrated that these closely-placed cell aggregates could fuse in two types of thermosensitive 3D gels. Taken together, these data strongly support the feasibility of the proposed novel organ-printing technology. (+info)
Cell therapy: filling the gap between basic science and clinical trials October 15-17, 2001, Rome, Italy.
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Summarized here, and in forthcoming issues of, are the concepts that emerged at a recent international workshop on cell therapy organized by The Istituto Superiore di Sanita in Rome in collaboration with Istituto Dermatopatico dell'Immacolata, Rome; Istituto Nazionale Ricerca Cancro-Centro Biotecnologie Avanzate, Genova; and University G. D'Annunzio, Chieti. The meeting intent was to provide an overview of the most recent developments in cell therapy, the future perspectives for these clinical trials, and the regulatory issues they involve, as well as a progress report on the clinical protocols that have been approved up to now in Italy. The meeting included six scientific sessions (Immunotherapy, Epithelium, Osteoregeneration, Hematopoiesis, Future Perspectives, and Overview of the National and International Regulations) and involved lectures from Italian and foreign scientists. (+info)
Reconstituted skin from murine embryonic stem cells.
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Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments. (+info)
Post-natal endothelial progenitor cells for neovascularization in tissue regeneration.
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The isolation of endothelial progenitor cells (EPCs) derived from bone marrow (BM) was an outstanding event in the recognition of 'de novo vessel formation' in adults occurring as physiological and pathological responses. The finding that EPCs home to sites of neovascularization and differentiate into endothelial cells (ECs) in situ is consistent with 'vasculogenesis', a critical paradigm well described for embryonic neovascularization, but proposed recently in adults in which a reservoir of stem or progenitor cells contributes to vascular organogenesis. EPCs have also been considered as therapeutic agents to supply the potent origin of neovascularization under pathological conditions. This review provides an update of EPC biology as well as highlighting their potential use for therapeutic regeneration. (+info)
Discrete and complementary mechanisms of protection of beta-cells against cytokine-induced and oxidative damage achieved by bcl-2 overexpression and a cytokine selection strategy.
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We have been investigating the potential utility of engineered cell lines as surrogates for primary islet cells in treatment of type 1 diabetes. To this end, two strategies that have emerged for procuring cell lines with resistance to immune-mediated damage are 1) selection of cytokine-resistant cell lines by growth of INS-1 insulinoma cells in iteratively increasing concentrations of interleukin (IL)-1beta + gamma-interferon (IFN-gamma), and 2) stable overexpression of the anti-apoptotic gene bcl-2 in INS-1 cells. Herein, we show that bcl-2-overexpressing cells are resistant to the cytotoxic effects of reactive oxygen and nitrogen species (ROS/RNS), but are only modestly protected against high concentrations of IL-1beta + INF-gamma, whereas the converse is true in cytokine selected cells. We also found that the combination of bcl-2 expression and cytokine selection confers a broader spectrum of resistance than either procedure alone, such that the resultant cells are highly resistant to cytokines and ROS/RNS, with no impairment in glucose-stimulated insulin secretion. INS-1-derived cells with combined bcl-2 expression and cytokine selection are also more resistant to damage induced by coculture with mitogen-activated peripheral blood mononuclear cells. Surprisingly, application of the cytokine selection procedure to bcl-2-overexpressing cells does not result in impairment of nuclear factor-kappaB translocation, iNOS expression, and NO production, as clearly occurs upon application of the selection procedure to cells without bcl-2 overexpression. Further investigation of the diverse pathways involved in the development of cytokine and ROS/RNS resistance may define simplified and specific strategies for preservation of beta-cell mass. (+info)