SEARCH · PubMed Health

Results for “cellular engineering”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,747 records · Page 97Linked to original sources

PubMed2005

Adjacent tissues (cartilage, bone) affect the functional integration of engineered calf cartilage in vitro.

OBJECTIVE: An in vitro model was used to test the hypothesis that culture time and adjacent tissue structure and composition affected chondrogenesis and integrative repair in engineered cartilage. METHOD: Engineered constructs made of bovine calf chondrocytes and hyaluronan benzyl ester non-woven mesh were press-fitted into adjacent tissue rings made of articular cartilage (AC), devitalized bone (DB), or vital bone (VB) and cultured in rotating bioreactors for up to 8 weeks. Structure (light and electron microscopy), biomechanical properties (interfacial adhesive strength, construct compressive modulus), biochemical composition (construct glycosaminoglycans (GAG), collagen, and cells), and adjacent tissue diffusivity were assessed. RESULTS: Engineered constructs were comprised predominately of hyaline cartilage, and appeared either closely apposed to adjacent cartilage or functionally interdigitated with adjacent bone due to interfacial deposition of extracellular matrix. An increase in culture time significantly improved construct adhesive strength (P<0.001), modulus (P=0.02), GAG (P=0.04) and cellularity (P<0.001). The type of adjacent tissue significantly affected construct adhesion (P<0.001), modulus (P<0.001), GAG (P<0.001) and collagen (P<0.001). For constructs cultured in rings of cartilage, negative correlations were observed between ring GAG content (log transformed) and construct adhesion (R2=0.66, P<0.005), modulus (R2=0.49, P<0.05) and GAG (R2=0.44, P<0.05). Integrative repair was better for constructs cultured adjacent to bone than cartilage, in association with its solid architectural structure and high GAG content, and best for constructs cultured adjacent to DB, in association with its high diffusivity. CONCLUSIONS: Chondrogenesis and integrative repair in engineered cartilage improved with time and depended on adjacent tissue architecture, composition, and transport properties.

Animals↗

PubMed2026

Periplasmic SacB as a robust counter-selection tool for genome engineering in the polyploid bacterium Zymomonas mobilis.

UNLABELLED: The alpha-proteobacterium Zymomonas mobilis exhibits exceptional ethanologenic physiology, which makes it a traditional alcoholic beverage producer and a promising chassis for biofuel production. Although genetic tools for this organism have expanded in recent years, a fundamental aspect of its chromosome organization remains to be understood. In particular, Z. mobilis has been suggested to exhibit polyploidy, but this feature is not fully confirmed because of discrepancies among studies reporting the copy number of chromosomes. Here, we tagged the chromosome-partitioning protein ParB with a fluorescent marker to visualize its cellular localization and estimate chromosome copy number in individual cells. Imaging showed that Z. mobilis exhibits several distinctive ParB foci throughout the cytoplasm and an accumulated focus at the pole, indicating that a single Z. mobilis cell contains >5 copies of the chromosome at the oriC regions. We then sought to establish an efficient counter-selection system, which is crucial for engineering multiple copies of the chromosome. We assessed the efficacy of levan-sucrase (SacB) toxicity in Z. mobilis. We found that, despite Z. mobilis secreting a native extracellular sucrase SacB, heterologous periplasmically localized Bacillus subtilis SacB rendered Z. mobilis cells sensitive to sucrose. We successfully used this effect for counter-selection when deleting and inserting targeted DNA sequences into the Z. mobilis genome. Together, this work provides important insights and tools for advancing Z. mobilis genetics and its biotechnological applications. IMPORTANCE: Zymomonas mobilis is a promising industrial bacterium with the capacity to convert sugars into ethanol at nearly maximum theoretical yield. With its expanding use in industrial applications, it is crucial to clarify if individual Z. mobilis cells carry multiple copies of the chromosome, as this has important implications for genome engineering. Two previous studies have used quantitative PCR to address this question, but their reported chromosome copy numbers varied widely from 20 to 100. Here, we used a cell biological approach to estimate the copy number and confirmed that a single Z. mobilis cell possesses multiple copies. In addition, we show that a SacB-based counter-selection works in Z. mobilis, enabling efficient and complete mutation of all chromosome copies.

Zymomonas↗

PubMed1997

The in vivo and in vitro characterisation of an engineered human antibody to E-selectin.

BACKGROUND: E-selectin is an endothelial cell specific adhesion molecule that is believed to play an important role in the early stages of leukocyte extravasation. OBJECTIVES: Here we describe the construction and evaluation of an engineered human monoclonal antibody that blocks E-selectin function. RESULTS: SPLAT-1 is an engineered human monoclonal antibody that has a very similar affinity for E-selectin as its murine parent antibody. In vitro SPLAT-1 blocks the binding of human leukocytes to E-selectin and does not mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated lysis of endothelial cells. In vivo, SPLAT-1 inhibits the recruitment of leukocytes to cytokine-inflamed human skin grafted on to SCID mice and has a long circulating half-life in primates. It does not appear to provoke an immune response in primates even on repeat administration. CONCLUSIONS: SPLAT-1 has the characteristics of a antibody suitable for human therapy studies.

Animals↗

PubMed2004

[Plant volatiles and their metabolic engineering].

Plant volatiles mainly include terpenoid, benzenoid/phenylpropanoid and fatty-acid derivatives, and they play diverse roles in plant-plant and plant-insect communications. In recent years, great progress has been made in biochemical and molecular characterization of the formation and release of these volatiles, and this make it possible to dissect their biological functions. More than 30 kinds of genes related with biosynthesis of these volatiles have been cloned. The pathways leading to synthesis of these metabolites can be controlled by enzyme activity and the substrates available as well as gene architecture. Plant volatiles can serve as attractants for specific pollinators, important cellular regulators in developmental processes and protection against environmental stress, whereas those released after herbivory or pathogen attacks can induce the expression a set of defensive genes or attraction of predators of the herbivores. An attractive prospect in this area is to design scent spectrum of plant with genetic and molecular technology. Two alternative approaches are used to genetically engineer these plant volatiles. One is based on the introduction of foreign genes encoding enzymes with activities that are missing in target plant, while the other is focused on modulating the expression of native genes.

Cloning, Molecular↗

PubMed1988

Rift Valley fever virus M segment: cellular localization of M segment-encoded proteins.

The Phlebovirus Rift Valley fever virus (RVFV), like other members of the Bunyaviridae family, matures intracellularly at the smooth-surfaced vesicles in the Golgi region of infected cells. Here we show that in cultured cells the RVFV glycoproteins G2 and G1 accumulate and are retained at this site. To investigate the parameters governing this subcellular localization, we have engineered portions of the cloned RVFV M segment (which encodes a 14- and a 78-kDa protein, in addition to glycoproteins G2 and G1) into vaccinia virus. Immunofluorescent analysis of cells infected with a vaccinia virus recombinant containing the entire open reading frame of the RVFV M segment revealed Golgi localization for glycoproteins G2, G1, the 78-kDa protein, and Golgi as well as some reticular distribution for the 14-kDa protein. These distributions paralleled those seen in authentic RVFV-infected cells. RVFV-vaccinia virus recombinants possessing progressive deletions within the 152 amino acid preglycoprotein sequence of the M segment were analyzed for possible effects on the cellular distribution of G2 and G1. Removal of the first 130 amino acids of the open reading frame amino-terminal to the mature glycoprotein coding sequences, while abolishing production of the 78- and 14-kDa proteins, did not alter the Golgi location of G2 and G1. The data suggest that Golgi-specific signals reside within the G2 and/or G1 glycoprotein sequences. The use of vaccinia virus recombinants provides a genetically manipulable expression system with which to further investigate the sequences involved in the intracellular localization of these Phlebovirus proteins.

Animals↗

PubMed2003

Encephalomyocarditis virus (EMCV) proteins 2A and 3BCD localize to nuclei and inhibit cellular mRNA transcription but not rRNA transcription.

We have followed the viral processing cascade and polyprotein precursor fates during encephalomyocarditis virus (EMCV) infection of HeLa cells using a panel of monoclonal antibodies (mAbs). Within the first 2-4 h of infection, signals of antibodies specific for the 2A, 3B(VPg), 3C(pro) and 3D(pol) proteins were found to co-localize in nucleoli at the rRNA synthesis and cellular protein B23 (nucleophosmin) sites. Cellular fractionation identified viral protein precursor 3BCD as the common source of the P3-region antibody signals. Previously thought to be a minor product of the polymerase region cleavage pathways, the nuclear targeting of this precursor was localized with engineered mutations to five P2 and P3 region polyprotein processing sites. A nuclear localization motif (NLS), similar to that in many yeast ribosomal proteins, was identified near the N-terminus of the 3D(pol) sequence. Point mutations within this motif prevented nuclear and nucleolar localization by all forms of 3B(VPg), 3C(pro) and 3D(pol), and were lethal to the virus because they also prevented genome replication. However, viral RNA synthesis was not required for nucleolar transport and 3BCD was found in nuclei, even when the 3D(pol) was inactivated. Co-immunoprecipitation experiments showed a tight association between 3BCD and B23 (nucleophosmin), suggesting a possible ribosomal protein-like mechanism for nuclear transport. Infected cell extracts analyzed with microarrays, quantitative slot-blots and pulse-labeling experiments confirmed a nearly complete shutoff of host pol-II-dependent mRNA synthesis during EMCV infection, in reactions that depended on wild-type 2A protein. In contrast to human rhinovirus-16 infection, rRNA synthesis by pol-I and pol-III were not turned off by EMCV, although the cellular concentration of rRNA decreased during infection, relative to control samples. The data suggest that nuclear targeting by 2A and 3BCD may be responsible for regulating cellular mRNA and rRNA transcription during infection, perhaps via a proteolytic mechanism catalyzed by the endogenous 3C(pro) sequence.

3C Viral Proteases↗

PubMed2003

Biochemical engineering: cues from cells.

Engineering principles are used in the exploitation of biocatalysts derived from cells. The purity of reagents, catalysts and maintenance of operation variables are extremely important for bioengineering systems. Any change in the purity of reagents or in operation variables usually leads to a dramatic decrease in productivity. Cellular systems, however, are able to work with relatively high impure conditions and increase their productivity in response to external signals. Thus the seemingly disordered 'bag of juice' or cytoplasm has more order and much higher order of integration than first appears. Learning the semantics of this paradoxical ability of order and integration would help bioengineers to understand and enhance productivity even using impure reagents.

Biomedical Engineering↗

PubMed2005

Culture of mouse prostatic epithelial cells from genetically engineered mice.

BACKGROUND: The lack of appropriate prostate cancer models is a major problem for prostate cancer research. Progress has been made towards the development of better in vivo rodent genetic models for prostatic disease. However, an in vitro model is often preferred for the elucidation of cellular mechanisms involved in the disease. METHODS: We microdissected the four prostatic lobes from young male mice, harvested the epithelial components, and grew epithelial cells from these tissues. We maintained the growth of these cells in long-term and three-dimensional culture. RESULTS: We have reproducibly harvested and cultured for extended passages mouse prostatic epithelial cells (MPECs) from a variety of mouse genetic strains. These cells express luminal and basal epithelial markers as well as the androgen receptor. Additionally, MPECs form classic branching structures in a three-dimensional collagen matrix. CONCLUSIONS: We have developed a novel culture system to harvest and grow MPECs in long-term culture. These cells will serve as a useful in vitro complement to studies using mouse genetic models for prostatic disease.

Animals↗

PubMed2005

Analysis of mesenchymal stem cells grown on a three-dimensional HYAFF 11-based prototype ligament scaffold.

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.

Animals↗

PubMed2005

Bioresorbable composites prepared by supercritical fluid foaming.

Bone is a natural composite construct, with a gradient structure going from a loose interconnected cellular core to an outer dense wall, thus minimizing bone weight while keeping a high mechanical resistance. Due to this unique and complex structure, bone defects are difficult to replace or repair. Tissue engineering aims at providing artificial bone grafts. Several techniques have been proposed to produce porous structures or scaffolds, but, as yet, with no optimal solutions. This article focuses on bioresorbable ceramic-polymer composite foams obtained by supercritical fluid foaming. This flexible technique enables an adequate morphology and suitable properties for bone tissue engineering to be obtained. Composite scaffolds are biocompatible, allowing cell proliferation and differentiation.

Absorbable Implants↗

PubMed2006

Characterization of emulsified chitosan-PLGA matrices formed using controlled-rate freezing and lyophilization technique.

This study evaluated the formation of chitosan-50:50 poly-lactic-co-glycolic acid (PLGA) blend matrices using controlled-rate freezing and lyophilization technique (CRFLT). An emulsion system was used in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), a cellular component, as a stabilizer. Blended scaffolds showed an open pore morphology and homogenous interdispersion of PLGA and chitosan. Forming emulsions after dissolving PLGA in chloroform, benzene, or methylene chloride indicated better emulsion stability with benzene and chloroform. Scaffolds formed by freezing at -20, -78, and -196 degrees C from these emulsions showed significant influence of the solvent and freezing temperature on the microarchitecture of the scaffold. By controlling the concentration of chitosan, scaffolds with greater than 90% porosity were attained. Since the two polymers degrade by different mechanisms, formed scaffolds were analyzed for degradation characteristics for 4 weeks in presence of 10 mg/L lysozyme. These results showed no significant difference in the weight loss and dimension changes, as all scaffolds showed significant (a) weight loss and (b) nearly 60% reduction in volume. Further, pH of the incubation media decreased in all the samples. When cellular activity of green fluorescence protein-transfected smooth muscle cells was analyzed, no apparent cytotoxicity was observed. However, the cell spreading area decreased. In summary, these results show promising potential in tissue engineering and drug delivery applications.

Biocompatible Materials↗

PubMed2005

Heterologous protein production using the Pichia pastoris expression system.

The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

Bioreactors↗

PubMed2006

Should human chondrocytes fly? The impact of electromagnetic irradiation on chondrocyte viability and implications for their use in tissue engineering.

A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplete.

Apoptosis↗

PubMed2005

Total internal reflection fluorescence microscopy: technical innovations and novel applications.

Recent years have seen the introduction of novel techniques and applications of total internal reflection fluorescence microscopy (TIRFM). Key technical achievements include miniaturization, enhanced depth resolution, reduction of detection volumes and the combination of TIRFM with other microscopic techniques. Novel applications have concentrated on single-molecule detection (e.g. of cellular receptors), imaging of exocytosis or endocytosis, measurements of adhesion foci of microtubules, and studies of the localization, activity and structural arrangement of specific ion channels. In addition to conventional fluorescent dyes, genetically engineered fluorescent proteins are increasingly being used to measure molecular conformations or intermolecular distances by fluorescence resonance energy transfer.

Endocytosis↗

PubMed2002

Why mechanobiology? A survey article.

The central paradigm of skeletal mechanobiology is that mechanical forces modulate morphological and structural fitness of the skeletal tissues-bone, cartilage, ligament and tendon. Traditionally, skeletal biomechanics has focussed on how these tissues perform the structural and locomotory functions of the vertebrate skeleton. In mechanobiology the central question is how these same load-bearing tissues are produced, maintained and adapted by cells as an active response to biophysical stimuli in their environment. The idea that 'form follows function' is not new, but we now believe that the scientific community has the knowledge and tools to prove, understand and use functional adaptation to benefit medicine and human health. In this Survey Article the philosophy and progress of skeletal mechanobiology are discussed. The revival of this science, with roots dating back to the 19th Century, is now driven by new developments in cellular, molecular and computational technologies. These developments are still in an early stage of application, but if modern mechanobiology fulfills the promises of its ambitions, the results will bring great benefits to tissue engineering and to the treatment and prevention of skeletal conditions such as congenital deformities, osteoporosis, osteoarthritis and bone fractures.

Animals↗

PubMed1999

DNA inoculations with HIV-1 recombinant genomes that express cytokine genes enhance HIV-1 specific immune responses.

Vaccination with HIV-1 DNA sequences induce both humoral and cellular immune responses in experimental animals. However, these responses are relatively weak and are often only transient in their nature. In order to enhance the level of HIV-1 specific immunity, we have engineered HIV-1 DNA constructs which contained various cytokine genes such as interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) gene. These constructs have deleted the tat and nmf genes of HIV-1 to eliminate their immunosuppressive effects. Immunizations with these recombinant constructs elicited moderate proliferative T cell responses but poor antibody responses in rats. However, inoculations of HIV-1 DNA that contained the GM-CSF or the IL-2 gene significantly enhanced humoral and proliferative T cell responses, respectively. Thus, recombinant HIV-1 genomes such as those described here may increase the efficacy of DNA vaccination.

AIDS Vaccines↗

PubMed1999

Actions of intrathecal diphtheria toxin-substance P fusion protein on models of persistent pain.

Substance P (SP) plays a central role in the transduction of second messenger signals from primary afferent nociceptive terminals to second-order neurons in the spinal cord. We have tested a recombinant engineered diphtheria toxin/SP fusion protein (DAB389SP) in acute and chronic pain models in the rat. DAB389SP binds to the SP receptor (SPR) and is internalized and kills SPR-expressing cells by blocking cellular protein synthesis. DAB389SP delivery was by intrathecal infusion, of varying duration, at the lumbar level. In the chronic constriction injury model of neuropathic pain a significant reduction in mechanically induced hyperalgesia was obtained. This effect was less marked in an acute carageenan inflammation model. Although other pain characteristics (mechano-allodynia, cold-allodynia, and heat-hyperalgesia) showed some improvement, these were less pronounced. Immunocytochemistry revealed a toxin-induced reduction in lamina I, of SPR and of NMDA NR1 subunit receptor expressing neurons, and of c-Fos, an inducible molecular marker of persistent nociceptive activity. The use of cytotoxic fusion proteins to target specific cell types may be of considerable benefit in the study of nociception and the treatment of chronic pain.

Acute Disease↗

PubMed1997

Allogenic transplantation of mobilized peripheral blood progenitor cells: towards tailored cell therapy.

Mobilized peripheral blood prognitor cells (PBPC) are increasingly being used instead of bone marrow for allogeneic transplantation. The present article will briefly review important aspects of allogeneic PBPCT including, donor safety, timing of leukapheresis, factors influencing the yield, cellular composition of PBPC allografts, hematopoietic capacity of allogeneic PBPC, and graft-versus-host and graft-versus-leukemia activities. It will particularly focus on the perspectives opened by PBPC for graft engineering and cell therapy.

Adolescent↗