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At least 1,117 records · Page 62Linked to original sources
PubMed2003

Human arteries engineered in vitro.

There is a pressing need to develop methods to engineer small-calibre arteries for bypass surgery. We hypothesized that the rate-limiting step that has thwarted previous attempts to engineer such vessels from non-neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non-neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.

Aorta↗
PubMed2003

Role of immunotherapy in stem cell transplantation.

Relapse of the underlying malignancy continues to be a major problem after both autologous and allogeneic stem cell transplantation. Over the years, it has been recognized that immune-mediated graft-versus-tumor effects are crucially involved in eliminating minimal disease and controlling its recurrence after stem cell transplantation. This recognition has led to a number of studies that have attempted to stimulate a cellular immune response in the recipient, especially after allogeneic transplantation. Immunotherapy after autologous transplantation has to take into consideration the fact that patients' immune cells frequently are compromised and tolerance to the host tumor may have developed. Hence, trials involving the administration of cytokines (such as with interleukin and interferon) have shown limited benefits. This situation is different for allogeneic transplantation for which the infusion of donor lymphocytes has shown disease regression, especially in patients with chronic leukemias. However, such treatment is effective only if the patient has limited disease, and severe graft-versus-host disease frequently has to be accepted as a complication. This fact has led investigators to pursue the generation of specific lymphocytes that can recognize tumor antigens but not necessarily induce graft-versus-host disease. Such studies are in the early stages, and although some promising results have been observed, it is unclear at this point if the antitumor effect can be separated sufficiently from the graft-versus-host disease mediated by allogeneic lymphocytes. More recently, it has been shown that natural killer (NK) cells can have an antitumor effect in myeloid malignancies, particularly if the cells are allogeneic and do not recognize self-HLA antigens. At this point, it appears that engineered T-lymphocytes and allogeneic NK cells may be useful in preventing or treating relapse after allogeneic transplantation. It remains to be seen if such novel cellular therapies can also be implemented after autologous transplantation via the use of engineered allogeneic immune cells.

Cytokines↗
PubMed2004

Analysis of the dynamic permeation experiment with implication to cartilaginous tissue engineering.

In the present study, a I-D dynamic permeation of a monovalent electrolyte solution through a negatively charged-hydrated cartilaginous tissue is analyzed using the mechano-electrochemical theory developed by Lai et al. (1991) as the constitutive model for the tissue. The spatial distributions of stress, strain, fluid pressure, ion concentrations, electrical potential, ion and fluid fluxes within and across the tissue have been calculated. The dependencies of these mechanical, electrical and physicochemical responses on the tissue fixed charge density, with specified modulus, permeability, diffusion coefficients, and frequency and magnitude of pressure differential are determined. The results demonstrate that these mechanical, electrical and physicochemical fields within the tissue are intrinsically and nonlinearly coupled, and they all vary with time and depth within the tissue.

Animals↗
PubMed2000

Transient expression of human telomerase extends the life span of normal human fibroblasts.

We utilized the Cre/lox recombination system to transiently express the catalytic subunit of telomerase (hTERT) in normal diploid foreskin fibroblasts (BJ cells). A retroviral construct containing an hTERT cDNA, flanked by loxP-sites was introduced into near senescent BJ cells (population doubling 85). At population doubling (PD) 92, which exceeds the typical life span of these cells, we excised the gene via Cre-mediated recombination. All clones lost telomerase activity and showed telomere shortening over an additional 50 PDs. Interestingly, the average telomere length in these cells became shorter than in untreated BJ cells at senescence. This may be due to hTERT preferentially elongating the shortest telomeres, leading to greater length uniformity. In summary, transient telomerase expression and only a very small average telomere elongation by hTERT resulted in a 50% increase in life span of human fibroblasts. This suggests a potentially safe use of hTERT in tissue engineering.

Catalytic Domain↗
PubMed2005

The development of structural and mechanical anisotropy in fibroblast populated collagen gels.

An in vitro model system was developed to study structure-function relationships and the development of structural and mechanical anisotropy in collagenous tissues. Fibroblast-populated collagen gels were constrained either biaxially or uniaxially. Gel remodeling, biaxial mechanical properties, and collagen orientation were determined after 72 h of culture. Collagen gels contracted spontaneously in the unconstrained direction, uniaxial mechanical constraints produced structural anisotropy, and this structural anisotropy was associated with mechanical anisotropy. Cardiac and tendon fibroblasts were compared to test the hypothesis that tendon fibroblasts should generate greater anisotropy in vitro. However, no differences were seen in either structure or mechanics of collagen gels populated with these two cell types, or between fibroblast populated gels and acellular gels. This study demonstrates our ability to control and measure the development of structural and mechanical anisotropy due to imposed mechanical constraints in a fibroblast-populated collagen gel model system. While imposed constraints were required for the development of anisotropy in this system, active remodeling of the gel by fibroblasts was not. This model system will provide a basis for investigating structure-function relationships in engineered constructs and for studying mechanisms underlying the development of anisotropy in collagenous tissues.

Animals↗
PubMed2004

Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression.

We report here the genetic engineering of dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The parental HT-1080 and the derived dual-color clones had similar cell proliferation rates, suggesting that expression of GFP and/or RFP does not affect cell cycle progression. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse brain where extreme elongation of the cell body as well as the nucleus occurred. Dual-color cells in various positions of the cell cycle were visualized in excised mouse lungs after tail-vein injection of the dual-color cells. In the lung, the dual-color cells were observed frequently juxtaposing their nuclei, suggesting a potential novel form of cell-cell communication. The dual-color cells thus are a useful tool for visualizing living-cell dynamics in vivo as well as in vitro. Drugs that could specifically perturb these processes can now be readily screened in real time in vivo.

Animals↗
PubMed1994

Problems in the introduction of genetically engineered microorganisms into the environment.

The use and release of genetically engineered microorganisms (GEMs) into the environment, usually the agricultural environment, is increasing exponentially. Potential applications of GEMs include crop production, pest management, degradation of environmental pollutants, mining and mineral recovery, and others. Several strategies of molecular and cellular biotechnology, such as recombinant DNA techniques, nuclear microinjection and cell fusion may be used to modify bacteria and fungi for useful purposes. The benefits expected from release of genetically engineered microorganisms, if safely applied, might be substantial in various fields. However, a safe introduction of GEMs into the environment requires full environmental and ecological risks assessment. Because of the wide scope of genetic engineering targets this review will focus on the application of GEMs potentially useful in agricultural practices (crop nutrition, pest and disease control) and ecological problems associated with the introduction of alien microorganisms into the environment.

Agriculture↗
PubMed2004

Decellularized vein as a potential scaffold for vascular tissue engineering.

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.

Antigens, Surface↗
PubMed2006

Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes.

Long-term function of three-dimensional (3D) tissue constructs depends on adequate vascularization after implantation. Accordingly, research in tissue engineering has focused on the analysis of angiogenesis. For this purpose, 2 sophisticated in vivo models (the chorioallantoic membrane and the dorsal skinfold chamber) have recently been introduced in tissue engineering research, allowing a more detailed analysis of angiogenic dysfunction and engraftment failure. To achieve vascularization of tissue constructs, several approaches are currently under investigation. These include the modification of biomaterial properties of scaffolds and the stimulation of blood vessel development and maturation by different growth factors using slow-release devices through pre-encapsulated microspheres. Moreover, new microvascular networks in tissue substitutes can be engineered by using endothelial cells and stem cells or by creating arteriovenous shunt loops. Nonetheless, the currently used techniques are not sufficient to induce the rapid vascularization necessary for an adequate cellular oxygen supply. Thus, future directions of research should focus on the creation of microvascular networks within 3D tissue constructs in vitro before implantation or by co-stimulation of angiogenesis and parenchymal cell proliferation to engineer the vascularized tissue substitute in situ.

Animals↗
PubMed2004

Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants.

Cartilage engineering is a strategic experimental goal for the treatment of multiple joint diseases. Based on the process of embryonic chondrogenesis, we hypothesized that cartilage could be engineered by condensing chondrocytes in pellet culture and, in the present study, examined the quality of regenerated cartilage in direct comparison with native cartilage. Chondrocytes isolated from the sterna of chick embryos were cultured in pellets (4 x 10(6) cells per pellet) for 2 weeks. Cartilage explants from the same source were cultured as controls. After 2 weeks, the regenerated cartilage from pellet culture had a disc shape and was on average 9 mm at the longest diameter. The chondrocyte phenotype was stabilized in pellet culture as shown by the synthesis of type II collagen and aggrecan, which was the same intensity as in the explant after 7 days in culture. During culture, chondrocytes also continuously synthesized type IX collagen. Type X collagen was negatively stained in both pellets and explants. Except for fibril orientation, collagen fibril diameter and density in the engineered cartilage were comparable with the native cartilage. In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure.

Aggrecans↗
PubMed2004

Preparation of porcine carotid arteries for vascular tissue engineering applications.

Biomaterials derived from tissue continue to offer viable alternatives to synthetic materials when autologous materials are unavailable for transplantation due to their unique chemical and mechanical properties. Tissue processing aims to stabilize the material against host degradation and render it immunologically inert by removing cellular material and crosslinking the structural proteins. It is clear that different approaches taken to achieve these goals have very different chemical and mechanical effects on the material. We describe herein the development of a tissue processing methodology to generate acellular scaffolds for tissue engineering small-diameter vascular grafts. Carotid arteries were isolated from Great White pigs and exposed to various solvent treatments, xylene, butanol, and ethanol to determine optimal parameters for the extraction of host lipids. The tissue was then exposed to a limited proteolysis with trypsin to disrupt cellular protein. This resulted in a controlled digestion that disrupted porcine nuclear DNA and cleared bulk cellular protein, leaving the more resistant structural proteins largely intact and retaining the bulk mechanical properties of the matrix. Histological analysis and scanning electron microscopy illustrated the complete removal of intact cells and nuclear material. The decellularized graft was stabilized by crosslinking with the photooxidative dye methylene green in the presence of 30,000 LUX of broad-band light energy. High-performance liquid chromatography analysis showed that the crosslinked tissue yielded 78.6% less hydroxyproline, compared with control tissue, after 20 h incubation with pepsin. Analysis of the crosslinked vessels' burst-pressure and stress-strain characteristics have shown comparable mechanical properties to those of control vessels. Assessment of in vitro cell adhesion and compatibility was conducted by seeding primary human umbilical vein endothelial cells and adult human vascular smooth muscle cells onto the lumenal and ablumenal surfaces, respectively; these cells were shown to adhere and proliferate under traditional static culture conditions.

Animals↗
PubMed2006

Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.

Animals↗
PubMed2005

Antenna modeling considerations for accurate SAR calculations in human phantoms in close proximity to GSM cellular base station antennas.

International bodies such as International Commission on Non-Ionizing Radiation Protection (ICNIRP) and the Institute for Electrical and Electronic Engineering (IEEE) make provision for human exposure assessment based on SAR calculations (or measurements) and basic restrictions. In the case of base station exposure this is mostly applicable to occupational exposure scenarios in the very near field of these antennas where the conservative reference level criteria could be unnecessarily restrictive. This study presents a variety of critical aspects that need to be considered when calculating SAR in a human body close to a mobile phone base station antenna. A hybrid FEM/MoM technique is proposed as a suitable numerical method to obtain accurate results. The verification of the FEM/MoM implementation has been presented in a previous publication; the focus of this study is an investigation into the detail that must be included in a numerical model of the antenna, to accurately represent the real-world scenario. This is accomplished by comparing numerical results to measurements for a generic GSM base station antenna and appropriate, representative canonical and human phantoms. The results show that it is critical to take the disturbance effect of the human phantom (a large conductive body) on the base station antenna into account when the antenna-phantom spacing is less than 300 mm. For these small spacings, the antenna structure must be modeled in detail. The conclusion is that it is feasible to calculate, using the proposed techniques and methodology, accurate occupational compliance zones around base station antennas based on a SAR profile and basic restriction guidelines.

Cell Phone↗
PubMed2006

Effect of photo-immobilization of epidermal growth factor on the cellular behaviors.

We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg(2+) although integrin-mediated cell adhesion to natural ECMs is dependent on Mg(2+). Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF.

Adsorption↗
PubMed2004

Cellular and molecular mechanisms of atherosclerosis with mouse models.

Recently, there has been an explosion in the number of in vivo studies using genetically engineered mouse models. Atherosclerosis research using mice began with the invention of traditional atherosclerotic mice including low-density lipoprotein receptor knockout (LDLR(-/-)) and apolipoprotein E knockout (apoE(-/-)) mice, which provided tremendous progress in atherosclerosis research. Since then, a number of modified atherosclerotic mouse models have been reported to generate lesions that more closely characterize human atherosclerotic lesions. Those modifications include inflammation, hypertension, proteinases and extracellular matrix, glucose metabolism, and immune systems. This article focuses on various kinds of mouse models with atherosclerosis and their contributions to the current advances of research.

Animals↗
PubMed2003

An engineer's approach to the development of real anti-aging medicine.

In this Viewpoint, I list the various age-related molecular and cellular changes that are thought to limit mammalian life-span, and I outline a problem-solving approach to reversing these detrimental changes. This approach should help to prevent the development of these age-related changes into life-threatening pathologies and possibly, in due course, allow a large increase in healthy human life expectancy.

Aging↗