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At least 613 records · Page 34Linked to original sources

PubMed2006

Antibody-based metabolic engineering in plants.

Genetic engineering is a powerful tool for the manipulation of cellular metabolism and the development of plant varieties with enhanced biological and nutrional functions. Several strategies are available for the in vivo modulation of enzymatic activities, allowing metabolic flux to be directed towards desired biochemical products. Such strategies include the simultaneous expression and/or suppression of multiple genes encoding rate-limiting enzymes, ectopic expression of transcription factors, and the RNA-based inhibition of catabolic enzymes. As an alternative approach, recombinant antibodies expressed in plants have been used to inactivate or sequestrate specific host proteins or compounds, resulting in significant changes to metabolic pathways. The impact of this approach depends on prudent selection of the target antigen, careful antibody design, appropriate subcellular targeting and stable accumulation of the recombinant antibodies in planta. Here, we describe the current status of antibody-based metabolic engineering in plants, discuss procedures for the optimisation of this technology and consider the remaining challenges to its widespread use.

Antibodies↗

PubMed1999

Electrical, chemical, and topological addressing of mammalian cells with microfabricated systems.

This communication describes our work in electrical, topological, and chemical micromodification of surfaces to modulate cellular form and function. We have addressed the surface physico-chemico-mechano properties of cell culture substrates that play a role in modulating cellular behavior. Single factorial model systems have been built using techniques adapted from microlithography. The tools and techniques of microfabrication, if harnessed and used correctly, can be enabling in elucidating the underlying principles and fundamental forces driving the cell-substrate interface. Additionally, the long-term practical applications of microfabrication in medicine and biomaterial/tissue engineering lie in enabling "communication" with living cells/tissues at the cellular and subcellular levels.

Animals↗

PubMedDate not supplied

Artificial design of three-dimensional retina-like tissue from dissociated cells of the mammalian retina by rotation-mediated cell aggregation.

The goal of this study was to establish a reliable three-dimensional culture system for the mammalian retina that allows the analysis of retinal function and dysfunction. To produce three-dimensional retinal tissues in vitro, dissociated retinal cells of neonatal rats were maintained in culture dishes on a self-made orbital shaker. On the basis of well-defined rotation conditions, dissociated free-floating cells reaggregate in the center of the culture dish to form a multicellular cluster. Subsequently, cells begin to proliferate, whereby they form spherelike retinal tissues that grow to a size of 180-210 microm. Immunohistochemical characterization of mature retinal spheres revealed the presence of ganglion cells, amacrine cells, Müller cells, and rod photoreceptors, which are arranged in different retina-like layers. Although a small number of cells undergo programmed cell death, retinal spheres remain viable for at least 35 days in culture as revealed by fluorescein diacetate and TUNEL staining. Because most biological processes involved in tissue organization such as proliferation, differentiation, apoptosis, and survival are also observable in retinal spheres, the presented novel mammalian three-dimensional culture system is not only an outstanding model for basic research but may also be of great benefit for stem cell tissue engineering and the pharmaceutical industry.

Animals↗

PubMed2004

Mechanotransduction of bone cells in vitro: mechanobiology of bone tissue.

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E2 values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6 +/- 0.3 Pa). Cyclic strain (1000 microstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E2. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.

Bone Remodeling↗

PubMed2026

Geometric mechanogenomics: engineering boundary conditions for deterministic cell fate control.

In tissue development and regeneration, cellular behavior has traditionally been interpreted through biochemical signaling frameworks. However, cells exist within physically defined environments, where geometric boundary conditions - including confinement, curvature, anisotropy, and multicellular architecture - define the mechanical state space in which mechanical forces are generated, transmitted, and interpreted. Here, we introduce geometric mechanogenomics, a conceptual framework that positions geometry as an upstream spatial regulator linking tissue-scale boundary conditions to nuclear mechanics, chromatin organization, and genome regulation. We propose a boundary-to-nucleus axis through which geometric information is decoded by adhesion-mediated mechanotransduction, cytoskeletal force transmission, and nuclear mechanoregulation to regulate chromatin accessibility, epigenetic remodeling, and transcriptional programs. Rather than introducing new mechanotransduction pathways, this framework emphasizes that geometry spatially organizes conserved mechanotransductive machinery to generate context-dependent mechanogenomic outcomes. We further discuss how engineered geometries reduce morphogenetic stochasticity, coordinate multicellular organization, and establish mechanical memory that influences long-term cell fate. Finally, we highlight current challenges in establishing predictive geometry-to-genome relationships and discuss emerging opportunities enabled by spatial omics, artificial intelligence-assisted inverse design, and dynamic biomaterials for programmable mechanobiology, regenerative medicine, developmental biology, and disease modeling.

genome organization↗

PubMed2004

Metabolic functionalization of recombinant glycoproteins.

Glycoproteins are essential for cellular communication and are the most rapidly growing class of therapeutic agents. Chemical modification of glycoproteins has been employed to improve their in vivo efficacy or to label them for detection. Methods for the controlled derivatization of glycoproteins are presently limited by the repertoire of natural amino acid side chain and carbohydrate functionalities. Here, we use metabolic oligosaccharide engineering to introduce a bioorthogonal functional group, the azide, into cellular and recombinant glycoproteins for subsequent chemical elaboration via Staudinger ligation. As most therapeutic glycoproteins are sialylated and require this saccharide for optimal pharmacokinetics, we targeted sialic acid as a host for azides using N-azidoacetylmannosamine (ManNAz) as a biosynthetic precursor. Metabolic conversion of ManNAz to N-azidoacetylsialic acid (SiaNAz) within membrane-bound and secreted glycoproteins was quantified in a variety of cell types. SiaNAz was found to comprise between 4% and 41% of total sialosides, depending on the system. Metabolic labeling of recombinant interferon-beta and GlyCAM-Ig was achieved, demonstrating the utility of the method for functionalizing N-linked and O-linked glycoproteins of therapeutic interest. More generally, the generation of recombinant glycoproteins containing chemical handles within their glycans provides a means for studying their behavior and for improving their in vivo efficacy.

Animals↗

PubMed2001

Tissue engineering of cardiac valves on the basis of PGA/PLA Co-polymers.

The limitations of currently used heart valve devices are well known. For prosthetic valves they include infection risk and thrombembolic complications; biologic devices have limited durability. Particularly for pediatric cardiac patients the problem of a lack of growth potential remains a serious issue. The multidisciplinary field of tissue engineering potentially offers an attractive pathway to overcome these disadvantages. The basic concept of tissue engineering is to build a new "tissue" from individual cellular components in vitro using a scaffold to provide an architecture upon which the cells can organize and develop into the desired "tissue" prior to implantation. The scaffold provides the biomechanical profile for the replacement tissue until the cells produce their own extracellular matrix. This newly generated matrix would then ultimately provide the structural integrity and biomechanical profile for the newly developed tissue structure. This work focuses on the concept of using a synthetically produced co-polymer (polyglycolic acid/polylactid acid) as the scaffold for the development of a new generation of heart valves.

Biomechanical Phenomena↗

PubMed2003

[Tissue engineering in urology. Basic principles and application].

Tissue engineering is a rather new field of science. Despite this fact, some experimental investigations have already been applied in clinical studies. Compared to other medical fields, tissue engineering in urology is well established. Tissue-engineered bulking agents and tissue-engineered bladder augments are being investigated in clinical trials. Even though the knowledge gained in recent years is promising, the results of cellular therapies need to be critically judged before being finally applied in patients. Genetic engineering and stem cell research (adult undifferentiated cells) have had major impact on the field of tissue engineering over the past 2 years. By using the technology of genetic engineering, biochemical and functional qualities of tissues may be modified. Adult stem cells may help to substitute lost tissue in an autologous fashion by isolating undifferentiated cells from the body and by differentiating them into a desired cell type. These cells may be used to form native functional tissue to replace a diseased organ or organ part.

Adult↗

PubMed2002

Genetic immunization using nanoparticles engineered from microemulsion precursors.

PURPOSE: Genetic immunization using "naked" plasmid DNA (pDNA) has been shown to elicit broad humoral and cellular immune responses. However, more versatile and perhaps cell-targeted delivery systems are needed. To this end, a novel process to engineer cationic nanoparticles coated with pDNA for genetic immunization was explored. METHODS; Cationic nanoparticles were engineered from warm oil-in-water microemulsion precursors composed of emulsifying wax as the oil phase and cetyltrimethylammonium bromide (CTAB) as the cationic surfactant. Plasmid DNA was coated on the surface of the cationic nanoparticles to produce pDNA-coated nanoparticles. An endosomolytic lipid and/or a dendritic cell-targeting ligand (mannan) were incorporated in or deposited on the nanoparticles to enhance the in vitro cell transfection efficiency and the in vivo immune responses after subcutaneous injection to Balb/C mice. The IgG titer to expressed beta-galactosidase and the cytokine release from isolated splenocytes after stimulation were determined on 28 days. RESULTS: Cationic nanoparticles (around 100 nm) were engineered within minutes. The pDNA-coated nanoparticles were stable at 37 degrees C over 30 min in selected biologic fluids. Transmission electron microscopy showed the nanoparticles were spherical. Plasmid DNA-coated nanoparticles. especially those with both an endosomolytic lipid and dendritic cell-targeting ligand. resulted in significant enhancement in both IgG titer (over 16-fold) and T-helper type-1 (Th1-type) cytokine release (up to 300% increase) over "naked" pDNA. CONCLUSION: A novel method to engineer pDNA-coated nanoparticles for enhanced in vitro cell transfection and enhanced in vivo immune responses was reported.

Animals↗

PubMed2005

Cryoconserved shielded and EGF receptor targeted DNA polyplexes: cellular mechanisms.

Recently, cryoconservable polyethylene glycol (PEG)-shielded and epidermal growth factor receptor (EGFR)-targeted polyplexes (EGF+ polyplexes) were engineered in our laboratory for tumor-directed transfer and expression of DNA. Here, we further analyzed specificity and kinetics of EGFR-mediated cellular uptake of these polyplexes. Similar to our previous results, EGF+ polyplexes significantly enhanced the transfection efficiency as compared to polyplexes without EGF (EGF- polyplexes) in HUH-7 hepatoma cells and Renca-EGFR renal carcinoma cells. EGF+ polyplexes rapidly associated with the cells within 30 min of exposure, and binding of EGF+ polyplexes to the cells after 4 h was significantly higher than that of EGF- polyplexes. In the presence of free EGF, both cell association and transfection efficiency of EGF+ polyplexes were markedly reduced indicating that these effects were primarily mediated via ligand receptor interaction. Fluorescence microscopy revealed that the cell-associated EGF+ polyplexes aggregated to micrometer sized clusters, resembling typical clustering of receptors upon ligand binding. In conclusion, EGFR-targeting enhances transfection efficiency due to accelerated and increased cell association followed by aggregation of the bound EGF+ polyplexes.

Cells, Cultured↗

PubMed2003

Mechanical characterization of collagen fibers and scaffolds for tissue engineering.

Engineered tissues must utilize scaffolding biomaterials that support desired cellular functions and possess or can develop appropriate mechanical characteristics. This study assessed properties of collagen as a scaffolding biomaterial for ligament replacements. Mechanical properties of extruded bovine achilles tendon collagen fibers were significantly affected by fiber diameter, with smaller fibers displaying higher tangent moduli and peak stresses. Mechanical properties of 125 micrometer-diameter extruded fibers (tangent modulus of 359.6+/-28.4MPa; peak stress of 36.0+/-5.4MPa) were similar to properties reported for human ligaments. Scaffolds of extruded fibers did not exhibit viscoelastic creep properties similar to natural ligaments. Collagen fibers from rat tail tendon (a well-studied comparison material) displayed characteristic strain-softening behavior, and scaffolds of rat tail fibers demonstrated a non-intuitive relationship between tangent modulus and specimen length. Composite scaffolds (extruded collagen fibers cast within a gel of Type I rat tail tendon collagen) were maintained with and without fibroblasts under standard culture conditions for 25 days; cell-incorporated scaffolds displayed significantly higher tangent moduli and peak stresses than those without cells. Because tissue-engineered products must possess appropriate mechanical as well as biological/chemical properties, data from this study should help enable the development of improved tissue analogues.

Animals↗

PubMed2005

Building in vitro models of organs.

Tissue-engineering techniques are being used to build in vitro models of organs as substitutes for human donor organs for transplantation as well as in vitro toxicology testing (as alternatives to use of animals). Tissue engineering involves the fabrication of scaffolds from materials that are biologically compatible to serve as cellular supports and microhabitats in order to reconstitute a desired tissue or organ. Three organ systems that are currently the foci of tissue engineering efforts for both transplantation and in vitro toxicology testing purposes are discussed. These are models of the cornea, nerves (peripheral nerves specifically), and cardiovascular components. In each of these organ systems, a variety of techniques and materials are being used to achieve the same end results. In general, models that are designed with consideration of the developmental and cellular biology of the target tissues or organs have tended to result in morphologically and physiologically accurate models. Many of the models, with further development and refinement, have the potential to be useful as functional substitute tissues and organs for transplantation or for in vitro toxicology testing.

Animals↗

PubMed2004

Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.

Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.

Animals↗

PubMed2006

Analysis of local tissue-specific gene expression in cellular micropatterns.

While cellular micropatterning approaches are employed extensively in cell biology and tissue engineering, only a limited number of methods for analysis of local function in the context of a complex, microfabricated environment are currently available. The present study develops a novel strategy for analysis of local tissue-specific function in cellular micropatterns. Model hepatocytes (HepG2 cells) were seeded onto silane-modified glass slides containing robotically printed arrays of collagen type I. These model hepatocytes formed cell arrays with individual cell cluster dimensions (150 or 500 microm) corresponding in size to the printed collagen spots. Non-parenchymal cells (3T3 fibroblasts) were added to hepatocellular micropatterns to create heterotypic cocultures. Expression of hepatic phenotype in HepG2 cells was first verified by traditional techniques including intracellular staining and ELISA for albumin. In order to evaluate local liver function in the cellular microarray, individual array members composed of approximately 400 hepatocytes were retrieved using laser capture microdissection and analyzed with real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). Hepatic function was assessed based on expression of four genes associated with differentiated liver phenotype: albumin, transferrin, alpha-fetoprotein, and alpha1-antitrypsin. "Titration" experiments, carried out to identify the smallest population of HepG2 cells yielding detectable mRNA levels and RT-PCR signals, showed that extraction area of 12,500 microm2 (corresponding to approximately 70 cells) provided detectable gene expression signals. All four liver-specific genes were routinely evaluated after extraction of approximately 400 HepG2 from the micropatterned surfaces. Significantly, selective retrieval and subsequent analysis of tissue-specific function was demonstrated for hepatic cells micropatterned alone and in coculture with non-parenchymal cells. In the future, methods described in this study will offer the possibility to investigate dynamic and reciprocal interactions between two or more cell types positioned on a microfabricated cell culture surface. We also envision the proposed approaches to be ideally suited for cell analysis in the context of combinatorial microenvironment.

3T3 Cells↗

PubMed2002

Tissue engineering: advances in in vitro cartilage generation.

Damaged or diseased articular cartilage frequently leads to progressive debilitation resulting in a marked decrease in the quality of life. Tissue engineering, a budding field in modern biomedical sciences, promises creation of viable substitutes for failing organs or tissues. It represents the amalgamation of rapid developments in cellular and molecular biology on the one hand and material, chemical and mechanical engineering on the other. Current tissue engineering approaches are mainly focused on the restoration of pathologically altered tissue structure based on the transplantation of cells in combination with supportive matrices and biomolecules. The ability to manipulate and reconstitute tissue structure and function in vitro has tremendous clinical implications and is likely to have a key role in cell and gene therapies in coming years.

Biocompatible Materials↗

PubMed2004

The application of recombinant human collagen in tissue engineering.

Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions. The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct, growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.

Animals↗

PubMed2006

Thermoreversible laminin-functionalized hydrogel for neural tissue engineering.

Traumatic injury to the central nervous system (CNS) triggers cell death and deafferentation, which may activate a cascade of cellular and network disturbances. These events often result in the formation of irregularly shaped lesions comprised of necrotic tissue and/or a fluid-filled cavity. Tissue engineering represents a promising treatment strategy for the injured neural tissue. To facilitate minimally invasive delivery of a tissue engineered system, a thermoreversible polymer is an attractive scaffold candidate. We have developed a bioactive scaffold for neural tissue engineering by tethering laminin-1 (LN) to methylcellulose (MC), a thermoresponsive hydrogel. The base MC chain was oxidized via sodium m-periodate to increase MC tethering capacity. Protein immobilization was facilitated by a Schiff base reaction between primary amine groups on LN and the carbonyl groups of the oxidized MC chain. Immunoassays demonstrated tethering of LN at 1.6 +/- 0.5 ng of LN per milligram of MC. Rheological measurements for different MC-LN constructs indicated MC composition- and MC treatment-dependent effects on solution-gelation transition temperature. Cellular assays with primary rat cortical neurons demonstrated enhanced cell adhesion and viability on LN-functionalized MC when compared with base and oxidized MC. This bioadhesive thermoresponsive scaffold may provide a robust delivery vehicle to injured CNS tissue for neural cell transplantation strategies.

Animals↗

PubMedDate not supplied

Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.

The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and mut(s) phenotypes, intracellular expression and folding pathways is examined. The roles of pre-pro signal sequences such as the alpha mating factor (alpha-MF) and the Glu-Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered. Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described. Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered. Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic and site-directed mutagenesis methods are also discussed.

Gene Expression↗