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 379 records · Page 21Linked to original sources

PubMed2002

Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells.

CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+ (BM-CD34+) or granulocyte-colony-stimulating factor-mobilized peripheral blood CD34+ (PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle-initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+ cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.

Antigens, CD34↗

PubMed2004

Algorithmic self-assembly of DNA Sierpinski triangles.

Algorithms and information, fundamental to technological and biological organization, are also an essential aspect of many elementary physical phenomena, such as molecular self-assembly. Here we report the molecular realization, using two-dimensional self-assembly of DNA tiles, of a cellular automaton whose update rule computes the binary function XOR and thus fabricates a fractal pattern--a Sierpinski triangle--as it grows. To achieve this, abstract tiles were translated into DNA tiles based on double-crossover motifs. Serving as input for the computation, long single-stranded DNA molecules were used to nucleate growth of tiles into algorithmic crystals. For both of two independent molecular realizations, atomic force microscopy revealed recognizable Sierpinski triangles containing 100-200 correct tiles. Error rates during assembly appear to range from 1% to 10%. Although imperfect, the growth of Sierpinski triangles demonstrates all the necessary mechanisms for the molecular implementation of arbitrary cellular automata. This shows that engineered DNA self-assembly can be treated as a Turing-universal biomolecular system, capable of implementing any desired algorithm for computation or construction tasks.

Algorithms↗

PubMed2006

Module-based analysis of robustness tradeoffs in the heat shock response system.

Biological systems have evolved complex regulatory mechanisms, even in situations where much simpler designs seem to be sufficient for generating nominal functionality. Using module-based analysis coupled with rigorous mathematical comparisons, we propose that in analogy to control engineering architectures, the complexity of cellular systems and the presence of hierarchical modular structures can be attributed to the necessity of achieving robustness. We employ the Escherichia coli heat shock response system, a strongly conserved cellular mechanism, as an example to explore the design principles of such modular architectures. In the heat shock response system, the sigma-factor sigma32 is a central regulator that integrates multiple feedforward and feedback modules. Each of these modules provides a different type of robustness with its inherent tradeoffs in terms of transient response and efficiency. We demonstrate how the overall architecture of the system balances such tradeoffs. An extensive mathematical exploration nevertheless points to the existence of an array of alternative strategies for the existing heat shock response that could exhibit similar behavior. We therefore deduce that the evolutionary constraints facing the system might have steered its architecture toward one of many robustly functional solutions.

Computer Simulation↗

PubMed2004

p53-Dependent activation of a molecular beacon in tumor cells following exposure to doxorubicin chemotherapy.

In an effort to begin developing a non-invasive strategy for in-vivo detection of the cellular DNA damage response, we engineered a molecular beacon to detect expression of p21(WAF1/CIP1), a gene whose transcription is directly activated by the p53 tumor suppressor protein. Introduction of a phosphorothioate-modified p21-beacon by transfection in human tumor cells led to a slight background signal that increased in a dose dependent manner between 50 and 400 nM beacon. Strong nuclear signal was observed following treatment of wild-type p53-expressing human H460 lung cancer cells for 8 hours with the chemotherapeutic agent doxorubicin (adriamycin). Similar induction was observed in wild-type p53-expressing HCT116 cells. Interestingly, following doxorubicin exposure, there was activation of the p21-beacon in p21-null HCT116 cells, which was not observed in p53-null HCT116, or mutant p53-expressing DLD1 cells that are either wild-type or p21-null. Increased signal from the phosphorothioate-modified p21-beacon in doxorubicin-treated cells likely resulted from sequence-specific hybridization as well as sequence-independent cleavage that may occur due to p53-dependent activation of endonucleases during apoptosis. We conclude that activation of p53 by chemotherapy leads to a strong signal from a p21-beacon that may be useful in further testing both in vitro and in vivo. Strategies need to be developed to optimize the gene or damage specificity as well as the sensitivity to therapeutic response of this non-invasive imaging approach.

Antibiotics, Antineoplastic↗

PubMed2001

TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function.

BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle fabrication and after growth factor release. At an optimal TGF-beta1 dosage of 1.0 ng/ml after 3 days, the released TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells over 21 days of culture, with increased total cell number, alkaline phosphatase activity, and osteocalcin production. CONCLUSIONS: PLGA/PEG blend microparticles can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitro. CLINICAL RELEVANCE: Controlled release of TGF-beta1 from PLGA/PEG microparticles is representative of emerging tissue engineering technologies that may modulate cellular responses to encourage bone regeneration at a skeletal defect site.

Animals↗

PubMedDate not supplied

Generalizations regarding the process and phenomenon of osseointegration. Part II. In vitro studies.

In this review, the appropriate use of cell culture to evaluate substrate effects on osteoblast behavior during the process of osseointegration has been considered in the context of existing reports. The interactions of osteoblasts with different substrates can be measured in terms of cytotoxicity, attachment, proliferation, and differentiation. The osteoblast culture systems that produce an osteoblast matrix opposing implant material substrates provide one model for evaluating the implant-bone interface. Alterations in osteoblast behavior at different culture substrates may reflect clinical determinants of bone formation at these substrates in vivo; however, cell responses in vitro have not been compared or correlated with in vivo outcomes. Legitimate interpretations of in vitro experiments are discussed in terms of practical, technical, and biologic limitations presented by the cell culture approach. Cell culture provides access to molecular and cellular information that fosters nanostructural engineering approaches to implant design and significant hypotheses to be tested in vivo. In this way, cell culture offers unique insights into the process and phenomenon of osseointegration.

Animals↗

PubMed1999

Metabolic flux analysis: a powerful tool for monitoring tissue function.

In recent years, metabolic flux analysis has been widely used in bioprocess engineering to monitor cell viability and improve strain activity. Metabolic flux analysis refers to a methodology for investigating cellular metabolism whereby intracellular fluxes are calculated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. A powerful feature of this methodology is its ability to consider cellular biochemistry in terms of reaction networks. By considering the stoichiometry of biochemical reactions, it is possible to estimate the degree of engagement of each pathway participating in overall cellular activity, and hence obtain a comprehensive view of a cell s metabolic state. Given the potential impact of cellular energy metabolism on the function of engineered tissues, such comprehensive analysis of metabolic activity can be an extremely useful tool for tissue engineers. Estimates of intracellular fluxes under various environmental conditions could be used to optimize function in vivo as well as culture conditions in vitro. In this review, we provide a brief theoretical background of metabolic flux analysis and summarize the most widely used experimental approaches to obtain flux data. This review is intended as an overview of the field and as a starting point for tissue engineers wishing to learn about and eventually employ this methodology.

Animals↗

PubMed2005

Improved effector functions of a therapeutic monoclonal Lewis Y-specific antibody by glycoform engineering.

The aim of the present study was to produce glycosylation variants of the therapeutic Lewis Y-specific humanized IgG1 antibody IGN311 to enhance cell-killing effector function. This was achieved via genetic engineering of the glycosylation machinery of the antibody-producing host. Antibody genes were transiently cotransfected with acetyl-glycosaminyltransferase-III genes into human embryonic kidney-EBV nuclear antigen cells. A control wild-type antibody, IGN311wt, was expressed in the same host using identical expression vectors, but without cotransfection of genes for acetyl-glycosaminyltransferase-III expression. Both expression products were purified to homogeneity and characterized. The glyco-engineered expression product (IGN312-Glyco-I) showed a remarkably homogenous N-linked glycosylation pattern consisting of one major hybrid-type, non-fucosylated and agalactosylated form carrying a bisecting GlcNAc-group. Wild-type expression product (IGN311wt) on the other hand was glycosylated by a multitude of different core-fucosylated complex-type structures of variable degrees of galactosylation. Target affinity of the glyco-engineered antibody as well as heavy and light chain assembly were not affected by acetyl-glycosaminyltransferase-III expression. In vitro experiments showed a approximately 10-fold increase of antibody-dependent cellular cytotoxicity of the glyco-engineered antibody using different Lewis Y-positive target cancer cell lines (SK-BR-3, SK-BR-5, OVCAR-3, and Kato-III). Complement-mediated cytotoxicity of IGN312-Glyco-I was 0.4-fold reduced using SK-BR-5 as target cell line. The reduction of complement activation could be prevented and even converted into a slight increase of activity by using a different molecular-biological approach directing the glycosylation towards increased levels of complex N-linked oligosaccharides of bisected, non-fucosylated type, as a result of cotransfection of mannosidase II together with acetyl-glycosaminyltransferase-III.

Antibodies, Monoclonal↗

PubMedDate not supplied

Cellular utilization determines viability and matrix distribution profiles in chondrocyte-seeded alginate constructs.

The long-term success of any cellular construct used for cartilage tissue engineering is dependent on the maintenance of cell viability throughout the construct thickness. Furthermore, the cells must continue to be metabolically active in order to synthesize a mechanically functional extracellular matrix (ECM). In the present study, a live-dead staining technique and systematic profiling procedure enabled the spatial and temporal distribution of chondrocyte viability to be characterized within 4-mm-thick alginate scaffolds. ECM distribution after 14 days of culture is described both biochemically and histologically and the mechanical functionality of the constructs was assessed by an unconfined compression test. Parameters investigated included alginate permeability, cell-seeding density, and volume of culture medium. Nonhomogeneity of cell and matrix distribution was evident, with greater densities of both parameters in the periphery of the constructs. The culture time preceding central viability loss was inversely related to cell density but relatively independent of scaffold density. However, homogeneity could be attained with increasing medium volume, as evidenced with cell and matrix distribution for cultures in 6.4 mL of medium per 10(6) cells. Moreover, the mechanical properties of the construct were enhanced by culture in increasing volumes of medium. This work indicates that cellular utilization determines the nonhomogeneous nature of cartilage formation in three-dimensional constructs and presents a guide to nonlimiting medium volumes for static culture conditions.

Adaptation, Physiological↗

PubMed2004

Impact of 'ome' analyses on inverse metabolic engineering.

Genome-wide or large-scale methodologies employed in functional genomics such as DNA sequencing, transcription profiling, proteomics, and metabolite profiling have become important tools in many metabolic engineering strategies. These techniques allow the identification of genetic differences and insight into their cellular effects. In the field of inverse metabolic engineering mapping of differences between strains with different degree of a certain desired phenotype and subsequent identification of factors conferring that phenotype are an essential part. Therefore, the tools of functional genomics in particular have the potential to promote and expand inverse metabolic engineering. Here, we review the use of functional genomics methods in inverse metabolic engineering, examples are presented, and we discuss the identification of targets for metabolic engineering with low fold changes using these techniques.

Animals↗

PubMed2006

Direct self-assembly of hepatocytes spheroids within hollow fibers in presence of collagen.

Opposite to the established view that collagen is an extracellular substratum for only dispersed hepatocyte culture, hepatocyte spheroids were directly formed within hollow fibers by addition of moderate concentrations of soluble collagen. Morphologically, these spheroids indicated a close relationship with their in vivo structure of liver. The albumin and urea synthetic profiles confirmed that those spheroids maintained liver-specific functions for at least 8 days. Spheroid formation by addition of collagen not only presents a potential methodology for clinical use of spheroids in bioartificial liver device but also indicates a likely function of collagen for self-assembly of primary cells in tissue engineering.

Animals↗

PubMed2006

Engineering a biospecific communication pathway between cells and electrodes.

Methods for transducing the cellular activities of mammalian cells into measurable electronic signals are important in many biotechnical applications, including biosensors, cell arrays, and other cell-based devices. This manuscript describes an approach for functionally integrating cellular activities and electrical processes in an underlying substrate. The cells are engineered with a cell-surface chimeric receptor that presents the nonmammalian enzyme cutinase. Action of this cell-surface cutinase on enzyme substrate self-assembled monolayers switches a nonelectroactive hydroxyphenyl ester to an electroactive hydroquinone, providing an electrical activity that can be identified with cyclic voltammetry. In this way, cell-surface enzymatic activity is transduced into electronic signals. The development of strategies to directly interface the activities of cells with materials will be important to enabling a broad class of hybrid microsystems that combine living and nonliving components.

Amino Acid Sequence↗

PubMed2001

[Symptoms reported by mobile cellular telephone users].

A survey study, using questionnaire, was conducted in 161 students and workers in a French engineering school on symptoms experienced during use of digital cellular phones. A significant increase in concentration difficulty (p < 0.05) was reported by users of 1800-MHz (DCS) cellular phones compared to 900-MHz (GSM) phone users. In users of cellular phones, women significantly (p < 0.05) complained more often of sleep disturbance than men. This sex difference for sleep complaint is not observed between women and men non-users of cellular phone. The use of both cellular phones and VDT significantly (p < 0.05) increased concentration difficulty. Digital cellular phone users also significantly (p < 0.05) more often complained of discomfort, warmth, and picking on the ear during phone conversation in relation with calling duration per day and number of calls per day. The complaint of warmth on the ear might be a signal to users for stopping the call.

Academies and Institutes↗

PubMed2005

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation.

Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH- and NH(2)-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH(3) groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against beta(1) integrin inhibited matrix mineralization on the OH and NH(2) surfaces, whereas function-perturbing antibodies specific for beta(3) integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.

3T3 Cells↗

PubMed2007

Electrospun fibrinogen: feasibility as a tissue engineering scaffold in a rat cell culture model.

Fibrinogen has a well-established tissue engineering track record because of its ability to induce improved cellular interaction and scaffold remodeling compared to synthetic scaffolds. While the feasibility of electrospinning fibrinogen scaffolds of submicron diameter fibers and their mechanical properties have been demonstrated, in vitro cellular interaction has not yet been evaluated. The goal of this study was to demonstrate, based on cellular interaction and scaffold remodeling, that electrospun fibrinogen can be used successfully as a tissue engineering scaffold. Electrospun fibrinogen scaffolds were disinfected, seeded with neonatal rat cardiac fibroblasts, and cultured for 2, 7, and 14 days. Cultures were treated to regulate scaffold degradation by either supplementing serum-containing media with aprotinin or crosslinking the scaffolds with glutaraldehyde vapor. Biocompatibility was assessed through a WST-1 cell proliferation assay. Postculture scaffolds were evaluated by scanning electron microscopy and histology. Cell culture demonstrated that fibroblasts readily migrate into and remodel electrospun fibrinogen scaffolds with deposition of native collagen. Supplementation of culture media with different concentrations of aprotinin-modulated scaffold degradation in a predictable fashion, but glutaraldehyde vapor fixation was less reliable. Based on the observed cellular interactions, there is tremendous potential for electrospun fibrinogen as a tissue engineering scaffold.

Animals↗

PubMed2006

Design of custom-shaped vascularized tissues using microtissue spheroids as minimal building units.

Tissue engineering strategies are gathering clinical momentum in regenerative medicine and are expected to provide excellent opportunities for therapy for difficult-to-treat human pathologies. Being aware of the requirement to produce larger artificial tissue implants for clinical applications, we used microtissues, produced using gravity-enforced self-assembly of monodispersed primary cells, as minimal tissue units to generate scaffold-free vascularized artificial macrotissues in custom-shaped agarose molds. Mouse myoblast, pig and human articular-derived chondrocytes, and human myofibroblast (HMF)-composed microtissues (microm3 scale) were amalgamated to form coherent macrotissue patches (mm3 scale) of a desired shape. Macrotissues, assembled from the human umbilical vein endothelial cell (HUVEC)-coated HMF microtissues, developed a vascular system, which functionally connected to the chicken embryo's vasculature after implantation. The design of scaffold-free vascularized macrotissues is a first step toward the scale-up and production of artificial tissue implants for future tissue engineering initiatives.

Animals↗

PubMed2003

Human hepatocytes as a tool for studying toxicity and drug metabolism.

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.

Cells, Cultured↗

PubMed2003

Biomechanical strategies for articular cartilage regeneration.

Two major contributions to the development of articular cartilage are growth factors and mechanical loading. Growth factors have long been used to modulate the secretion of certain molecules from different cells. The TGF-beta superfamily, specifically the BMPs, CDMPs, OPs, and GDFs, have a dramatic effect on the development of bone and cartilage tissue. These growth factors help produce an extracellular matrix that can withstand extreme loading conditions in the body. In addition to growth factors, it is known that mechanical forces stimulate the synthesis of extracellular proteins in vitro and in vivo and can affect the tissue's overall structure. Load-bearing tissue, such as articular cartilage, will atrophy in the absence of mechanical forces, and this observation has caused researchers to incorporate mechanical stimulation into the tissue engineering process. This article focuses on the importance of mechanical forces in tissue engineering of articular cartilage and the growth factors that help stimulate the formation of load-bearing tissue.

Cartilage, Articular↗