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PubMed2001

Frequency- and duration-dependent effects of cyclic pressure on select bone cell functions.

The present study demonstrated unique correlations between characteristic parameters of mechanical loading and osteoblast functions. Specifically, osteoblast proliferation was dependent on the frequency and on the duration of the applied cyclic pressure stimulus: decreased cell proliferation was only observed when these cells were exposed to cyclic pressure at 1.0-Hz (but not at 0.25-Hz) frequency for 1 h (but not for 20 min) daily for 5 days. In contrast, endothelial cells were not responsive to cyclic pressure, whereas fibroblast proliferation increased under similar test conditions. Most important, cyclic pressure affected various osteoblast genes differently: exposure of osteoblasts to cyclic pressure (at 1.0-Hz frequency for 1 h daily) resulted in enhanced transcription and translation of alkaline phosphatase after 5 days; the same mechanical stimulus, however, did not affect osteopontin mRNA expression during the same time periods. These findings provide cellular and molecular level information, which is not only important in elucidating the correlation between mechanical loading and bone homeostasis, but can be useful in development of new technology in skeletal tissue engineering.

Animals↗

PubMed2006

Spatially resolved non-invasive chemical stimulation for modulation of signalling in reconstructed neuronal networks.

Functional coupling of reconstructed neuronal networks with microelectronic circuits has potential for the development of bioelectronic devices, pharmacological assays and medical engineering. Modulation of the signal processing properties of on-chip reconstructed neuronal networks is an important aspect in such applications. It may be achieved by controlling the biochemical environment, preferably with cellular resolution. In this work, we attempt to design cell-cell and cell-medium interactions in confined geometries with the aim to manipulate non-invasively the activity pattern of an individual neuron in neuronal networks for long-term modulation. Therefore, we have developed a biohybrid system in which neuronal networks are reconstructed on microstructured silicon chips and interfaced to a microfluidic system. A high degree of geometrical control over the network architecture and alignment of the network with the substrate features has been achieved by means of aligned microcontact printing. Localized non-invasive on-chip chemical stimulation of micropatterned rat cortical neurons within a network has been demonstrated with an excitatory neurotransmitter glutamate. Our system will be useful for the investigation of the influence of localized chemical gradients on network formation and long-term modulation.

Animals↗

PubMed1999

Overview of the clinical development of rituximab: first monoclonal antibody approved for the treatment of lymphoma.

Rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) is a genetically engineered monoclonal antibody for the treatment of non-Hodgkin's lymphoma. This chimeric mouse/human, immunoglobulin GI kappa anti-CD20 antibody mediates complement-dependent cell lysis and antibody-dependent cellular cytotoxicity. It also has been shown to sensitize chemoresistant human lymphoma cell lines and to induce apoptosis. It was approved by the Food and Drug Administration on November 26, 1997, for the indication of relapsed or refractory, CD-20 positive, B-cell, low-grade or follicular non-Hodgkin's lymphoma Rituximab is the first monoclonal antibody approved for the treatment of cancer and the first single agent approved specifically for therapy of a lymphoma. The recommended dose is rituximab 375 mg/m2 intravenously weekly x4 infusions. Treatment is well tolerated and outpatient therapy is feasible. Adverse events are mostly grades I and 2, occurring primarily with the first infusion. In a phase II single-agent clinical trial, the overall response rate was 50%, with a median time to progression in responders of 10.2 months. In a larger multicenter trial involving 166 patients, the overall response rate was 48% with 6% complete and 42% partial responses. Median time to progression for responders was 13.2 months and median duration of response was 11.6 months. A 40% response rate has been observed on re-treatment with rituximab. Activity also has been seen in patients with bulky disease. Combination studies have been performed with interferon, cyclophosphamide/doxorubicin/vincristine/prednisone, and radioimmunotherapy. Rituximab, the first monoclonal antibody approved for the treatment of cancer, is safe and effective in treating patients with relapsed or refractory, CD-20 positive, B-cell, low-grade or follicular non-Hodgkin's lymphoma.

Antibodies, Monoclonal↗

PubMed2005

Co-expression and immunity of Legionella pneumophila mip gene and immunoadjuvant ctxB gene.

The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplified by PCR respectively. The amplified cDNA was ligated to the pcDNA3.1(+) vector. The recombinant plasmids pcDNA3.1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR, and further confirmed by sequencing analysis. NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB according to the Lipofection method. Transient and stable products of the co-expression of the mip gene and ctxB gene were detected by immunofluorescence and Western blotting. The results showed that NIH3T3 cells were successfully transfected, and that the transiently and stably co-expressed products can be detected in the transfected cells. To detect the humoral and cellular immune response in immunized mice induced by the co-mmunization of the mip and ctxB genes, female BALB/c mice were immunized intramuscularly with pcDNA3.1-mip and pcDNA3.1-ctxB. The results showed that the specific antibody titer and the cytotoxic T-lymphocyte response for pcDNA3.1-mip immunization and co-immunization were increased compared with that of pcDNA3.1(+) immunization. Furthermore, the specific antibody titer and cytotoxic T-lymphocyte response for co-immunization were increased compared with that of pcDNA3.1-mip immunization. Statistical analysis using one-way analysis of variance (ANOVA) showed that there was a significant difference between the groups (P<0.01). The results indicated that the ctxB gene enhanced the humoral and cellular immune response to the mip gene immunization. These findings provide experimental evidence to support the development of the L. pneumophila DNA vaccine.

Adjuvants, Immunologic↗

PubMed1994

Different tumours, transduced with different cytokine genes as G-CSF and IL-2, show inhibition of tumour take through neutrophil activation but differ in T cell functions.

The immune effectors and the cellular mechanisms responsible for tumour rejection of two different tumours transduced with different cytokines have been characterized by immunocytochemistry and in situ hybridization. A colon (C-26) and a mammary (TS/A) adenocarcinoma engineered to release, respectively, 90 pg/ml of G-CSF (C-26/G-CSF) and 30 U or 6000 U of IL-2 (low B1.30 and high B4.6000 level of IL-2, respectively) were compared for the type of infiltrating leucocytes and for the repertoire of secondary cytokines produced by the leucocytes recruited at the tumour site. The results indicate that in both systems tumour rejection is associated with prominent infiltration of CD45+/RB6-8C5+ polymorphonuclear (PMN) leucocytes expressing mRNA for IL-1 alpha, IL-1 beta and TNF alpha. TS/A B1.30 and B4.6000 also showed a small proportion of infiltrating T lymphocytes, expressing IFN gamma and IL-4 mRNA, which were virtually absent in the C-26/G-CSF tumour. In mice injected with C-26/G-CSF cells after 600 rad irradiation, the tumours grew to about 1.5 cm and then regressed completely. During the regression phase, T lymphocytes were recruited within C-26/G-CSF, and the infiltrating leucocytes were similar, in terms of PMN/macrophages/T lymphocytes ratio, to those found during the memory response elicited by injection of a challenging dose of parental TS/A into mice pre-immunized with B1.30 IL-2-producing cells. The memory response was characterized by a CD4/CD8 ratio of 0.4 and by IFN-gamma and IL-4 mRNA expression, whereas the T lymphocytes present within regressing C-26/G-CSF were mostly CD4 (CD4/CD8 ratio of 2.1) and expressed IFN gamma mRNA only. The gene transfers of cytokines as different as G-CSF and IL-2 are able to inhibit tumour take through a similar anti-tumour immune response mostly due to non-specific effectors (PMN), thus resembling acute inflammation phenomena. Regression of C-26/G-CSF initially established in irradiated mice as well as rejection of TS/A tumour injected into B1.30 immunized mice are similar to a chronic infection but the immune reaction elicited by C-26/G-CSF has an impaired T-lymphocyte function.

Adenocarcinoma↗

PubMed2001

Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts.

Arterial vascular cells have been successfully utilized for tissue engineering in human cardiovascular structures, such as heart valves. The present study evaluates saphenous vein-derived myofibroblasts as an alternative, easy-to-access cell source for human cardiovascular tissue engineering. Biodegradable polyurethane scaffolds were seeded with human vascular myofibroblasts. Group A consisted of scaffolds seeded with cells from ascending aortic tissue; in group B, saphenous vein-derived cells were used. Analysis included histology, electron microscopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content was comparable in both groups. Collagen and stress at maximum load was significantly higher in group B. Morphology showed viable, layered cellular tissue in all samples, with collagen fibrils most pronounced in group B. In conclusion, saphenous vein myofibroblasts cultured on biodegradable scaffolds showed excellent in vitro tissue generation. Collagen formation and mechanical properties were superior to aortic tissue derived constructs. Therefore, the easy-to-access vein cells represent a promising alternative cell source for cardiovascular tissue engineering.

Aorta↗

PubMed2004

Biomaterial-microvasculature interaction on polymers after implantation in mice.

Biomaterial research is expected to forward new materials to be used as, e.g., implant materials or as scaffolds for tissue engineering. It is central for such a scaffold material to create the track on which those cells can inhabitate the scaffold needed to rebuild functional tissue substitutes. For the biointegration of the implant with the native cellular tissue this must be able to grow on the material surface. For the elimination of the degradation products and the adeqaute transport of nutrients/gases within the newly formed tissue the angiogenesis of new blood vessels is thought to play an important role. In the present study, a new biomaterial, a non-porous polymeric AB-network based on oligo (epsilon-hydroxycaproat) and oligobutylacrylat, was implanted in animals. Male NMRI mice were implanted subcutaneously for one week to nine weeks. Immediately after the explantation, the probes were examined histologically. Already one week after implantation, there was a strong tissue-integration of the polymer. Importantly, blood vessels appeared at the polymer surface. At nine weeks after implantation the tissue integration was stronger than after one week and blood vessels were still observed in the periimplant tissue. The mechanism of the early integration of the polymer is not clear. The relationship between the new periimplant vessels and the integration of the polymer has to be studied.

Animals↗

PubMed1992

Natural genetic engineering in evolution.

The results of molecular genetics have frequently been difficult to explain by conventional evolutionary theory. New findings about the genetic conservation of protein structure and function across very broad taxonomic boundaries, the mosaic structure of genomes and genetic loci, and the molecular mechanisms of genetic change all point to a view of evolution as involving the rearrangement of basic genetic motifs. A more detailed examination of how living cells restructure their genomes reveals a wide variety of sophisticated biochemical systems responsive to elaborate regulatory networks. In some cases, we know that cells are able to accomplish extensive genome reorganization within one or a few cell generations. The emergence of bacterial antibiotic resistance is a contemporary example of evolutionary change; molecular analysis of this phenomenon has shown that it occurs by the addition rearrangement of resistance determinants and genetic mobility systems rather than by gradual modification of pre-existing cellular genomes. In addition, bacteria and other organisms have intricate repair systems to prevent genetic change by sporadic physicochemical damage or errors of the replication machinery. In their ensemble, these results show that living cells have (and use) the biochemical apparatus to evolve by a genetic engineering process. Future research will reveal how well the regulatory systems integrate genomic change into basic life processes during evolution.

Animals↗

PubMed2006

Capturing complex 3D tissue physiology in vitro.

The emergence of tissue engineering raises new possibilities for the study of complex physiological and pathophysiological processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. We discuss here some of the 'design principles' for recreating the interwoven set of biochemical and mechanical cues in the cellular microenvironment, and the methods for implementing them. We emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.

Animals↗

PubMed2002

Engineered antibody for treating lymphoma.

One approach to improving the results of antibody treatment of posttransplant and other lymphomas is to alter the geometry of the antibody molecule so as to enhance its cytotoxicity. When antibody alights on a cell, cytotoxicity can be exerted by initiation of apoptotic signals at the cell surface, and by recruitment of effectors to specific sites on the antibody Fc region. Other routes to cytotoxicity have been described but their generality is debatable. The effectors consist of complement, and a series of potentially cytotoxic cells (macrophages, NK cells, neutrophils and others) bearing Fc-receptors (FcR). Recent evidence suggests that the FcR-bearing cells are the more important in dealing with antibody-coated autologous cells, including tumor cells. If the antibody happens to be of the host IgG class then its Fc region (Fcgamma) will contain a site for attachment of a non-cytotoxic FcR known as FcRB (the Brambell receptor): it is this receptor, present on endothelial cells, which recycles the molecule intact if it happens to be endocytosed and thus prolongs its metabolic life. Replacement of the mouse Fcgamma by human Fcgamma is expected to have three beneficial effects: better recruitment of human effector cells, better persistence of the antibody in extracellular fluid, and removal of a major source of immunogenic epitopes. Chemical manipulations of Fab'gamma and Fcgamma modules, linking them in different geometries via their hinge regions, offers constructs with further enhancements of cytotoxicity. These include Fab2Fc2, in which the presence of dual Fc regions enhances recruitment of both complement and cellular effectors; and bispecific antibody of the same modular formula in which one of the Fab arms is specific for tumor while the other is specific for, and recruits powerfully, a cellular effector.

Animals↗

PubMed2005

Engineering non-natural inhibitor sensitivity in protein tyrosine phosphatase H1.

Protein tyrosine phosphatase H1, a member of the ubiquitous protein tyrosine phosphatase (PTP) superfamily of enzymes, is an important signaling molecule, mutant forms of which have been found in human colorectal cancers. Selective PTPH1 inhibitors would be valuable tools for investigating PTPH1's roles in cellular regulation. However, no PTPH1-specific inhibitors are known. To identify target-selective inhibitors of human PTPH1, we have redesigned a PTPH1/inhibitor interface. Structure-based protein design was used to identify two amino-acid residues, isoleucine 846 and methionine 883, that control PTPH1's sensitivity to oxalylaminoindole PTP inhibitors. Mutation of residues 846 and 883 to alanine and glycine, respectively, conferred novel inhibitor sensitivity onto PTPH1. From a small panel of putative inhibitors, compounds that potently and selectively target the inhibitor-sensitized PTPH1 mutants were identified.

Amino Acid Sequence↗

PubMedDate not supplied

Engineered liposomes and virosomes for delivery of macromolecules.

In order to utilize virosomes or proteoliposomes for the delivery of drugs or macromolecules to specific pathologic target cells we elaborated a system to shuttle drugs to solid tissue (liver) as well as to the macrophages, a crucial cellular compartment of the immune system. Using virosomes prepared from the P3HR1 strain of Epstein-Barr virus, we demonstrated that these particles fused with human hepatocarcinoma cell line Li7A and therefore might be used as drug vectors. Furthermore, we report that proteoliposomes prepared by reconstituting in a cocktail of phosphatidylserine-phosphatidylcholine the anion transporter band 3 protein markedly increased the phagocytic activity of macrophages in culture. This could represent a new device to be used as a drug delivery system to enhance specific macrophagic functions.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2006

Dynamic contrast-enhanced MRI to quantify VEGF-enhanced tissue-engineered bladder graft neovascularization: pilot study.

Tissue engineered organs require an immediately perfused vascular tree. Currently, neovascularization assessment requires animal sacrifice and graft harvest. In this pilot study we assess whether neovascularization of an engineered urinary bladder construct is enhanced with vascular-derived endothelial growth factor (VEGF) and assessable non-invasively with dynamic contrast-enhanced MRI (DCE-MRI). Rabbit bladder acellular matrix was hybridized with hyaluronic acid (ACM-HA), fortified with one of three concentrations (0 ng, 10 ng, or 20 ng per gram of tissue) of vascular-derived endothelial growth factor (VEGF), and grafted onto bladders in nine rabbits (3 per VEGF concentration). At 1, 2 and 3 weeks, one rabbit from each VEGF group underwent DCE-MRI and graft harvest. Microvasculature was quantified with scrial optical transverse sectioning of CD31 stained whole mounts using PCl software. Masson trichrome and H&E staining were used to assess cellular proliferation and fibrosis. There was a significant difference in mean microvascular area (MVA) between the 10 and 20 ng VEGF groups (230187 vs 477894 microm2, P=0.014) but not between the 0 and 10 ng groups (210497 microm2, P=0.21). Gadolinium uptake increased with MVA and correlated with it on linear regression analysis (Pearson's correlation coefficient r=0.71). At three weeks, stromal cellularity was greatest, and fibrosis was least, in the high VEGF group. This preliminary work demonstrates improved neovascularization of bladder constructs with VEGF fortification of ACM-HA and the feasibility of MRI as a non-invasive method to assume angiogenesis in tissue engineered constructs.

Animals↗

PubMed2004

Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line.

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.

Animals↗

PubMed2003

Tricuspid valve biopsy: a potential source of cardiac myofibroblast cells for tissue-engineered cardiac valves.

BACKGROUND AND AIMS OF THE STUDY: As progress is made in the development of a tissue-engineered cardiac valve, the need for a reliable cell source is particularly important. A technique has been developed for the reliable biopsy of tricuspid valve leaflets. Expanding the harvested cells in culture is feasible and provides a source of leaflet cells that are structurally and functionally similar to the pulmonary and aortic valve leaflet cells that they may replace. METHODS: Thirteen sheep underwent tricuspid valve biopsy. Transthoracic echocardiography (TTE) was performed to evaluate function and guide the subsequent biopsy. Myofibroblasts were isolated from the biopsy samples, expanded in culture through 10 passages, and evaluated with immunocytochemistry for valve cell markers. Two animals were sacrificed acutely, two animals died during the immediate postoperative period, and nine animals survived for four weeks or more. RESULTS: All preoperative and pre-explantation echocardiograms were normal. Both animals sacrificed acutely showed that the tricuspid valve leaflet was indeed biopsied with this technique. Two perioperative deaths occurred; one animal died secondary to injury of the chorda tendinea with subsequent destruction of the posterior leaflet; another died from disruption of the superior vena cava that led to irreversible cardiac tamponade. At sacrifice (2 to 17 weeks), all other animals showed intact tricuspid valves with normal leaflet anatomy. All cultured biopsies generated myofibroblasts that were immunocytochemically positive for alpha smooth muscle actin, chondroitin sulfate, vimentin and fibronectin. CONCLUSION: Biopsy of the tricuspid valve to obtain recipient cardiac valve leaflet cells is possible, and the technique is simple and reliable. Biopsy of the leaflet does not compromise function. Interstitial cells can be harvested and expanded in culture. Cellular structure and function is preserved and is similar to that of other cardiac leaflet cells. Tricuspid valve leaflet biopsies are a potential source for harvesting cells to be used in the development of a tissue-engineered cardiac valve.

Actins↗

PubMed1999

Frontiers in tissue engineering. In vitro modulation of chondrogenesis.

Cartilage tissue engineering can provide functional cartilaginous constructs that can be used for controlled in vitro studies of chondrogenesis and potentially for in vivo articular cartilage repair. Ideally, engineered cartilage should be indistinguishable from native articular cartilage with respect to zonal organization, biochemical composition, and mechanical properties. In the model system presented here, chondrogenic cells are expanded in vitro as required, seeded onto three-dimensional polymeric scaffolds, and cultured in bioreactor vessels. During the course of in vitro cultivation, construct cellularity plateaus at a physiologic level, fractions of glycosaminoglycan and Type II collagen increase progressively, and the scaffold biodegrades. Construct structure (composition, morphology) and function (biosynthetic activity, mechanical properties) depend on cultivation conditions. This paper reviews recent studies of in vitro modulation of chondrogenesis by: (1) cell seeding density and source; (2) the tissue regeneration template; (3) biochemical regulatory signals; (4) mixing, mass transport and hydrodynamic forces; and (5) cultivation time. Key requirements and some of the critical research needs for successful cartilage tissue engineering are discussed.

Animals↗

PubMed2005

Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.

The vascular smooth muscle cell (VSMC) is surrounded by a complex extracellular matrix that provides and modulates a variety of biochemical and mechanical cues that guide cell function. Conventional two-dimensional monolayer culture systems recreate only a portion of the cellular environment, and therefore there is increasing interest in developing more physiologically relevant three-dimensional culture systems. This review brings together recent studies on how mechanical, biochemical, and extracellular matrix stimulation can be applied to study VSMC function and how the combination of these factors leads to changes in phenotype. Particular emphasis is placed on in vitro experimental studies in which multiple stimuli are combined, especially in three-dimensional culture systems and in vascular tissue engineering applications. These studies have provided new insight into how VSMC phenotype is controlled, and they have underscored the interdependence of biochemical and mechanical signaling. Future improvements in creating more complex in vitro culture environments will lead to a better understanding of VSMC biology, new treatments for vascular disease, as well as improved blood vessel substitutes.

Adaptation, Physiological↗

PubMed2004

Development of culture techniques of keratinocytes for skin graft production.

The in vitro cultures of human tissues have attracted a great deal of medical attention as a promising technique for repairing defective tissues in vivo. In the last decade many companies have been established for supplying the regenerated grafts by means of tissue cultures of skin, cartilage, bone and so on. From the viewpoint of biochemical engineering, however, the culture systems for these tissues are not so sophisticated nor so programmed as the submerged culture systems developed for microorganisms. In manufacturing skin grafts, for instance, the raw materials of cells harvested from patients are heterogeneous, and the products of cultured tissues vary in the required size for individual epithelial sheets. Therefore, a reliable and robust process is desired for the production of cultured tissues with high reproducibility and quality. This review focuses on the strategies for developing the culture processes of keratinocytes targeting the epithelial sheet production, including (i) the introduction of culture techniques for keratinocyte cells and survey of skin graft production as it is, (ii) construction of kinetic model of cell growth, (iii) evaluation of cell properties based on image-analyzing techniques, and (iv) design of bioreactor system.

Algorithms↗