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PubMed2004

Incorporating environmental ethics into the undergraduate engineering curriculum.

The design and economic realities associated with Personal Computers (PCs) was used as a model for implementing ethical issues into the core-engineering curriculum. Historically, products have not been designed to be recycled easily. By incorporating environmental ethics into our classrooms and industries, valuable materials can be recovered and harmful materials can be eliminated from our waste stream. Future engineers must consider the economic cost-benefit analysis of designing a product for easy material recovery and recycling versus the true cost of the disposal and continued use of virgin materials. A three hour unit on the economic and environmental impacts of product design is proposed for inclusion in the ABET accredited engineering program.

Curriculum↗
PubMed2006

The complex challenges of ethical choices by engineers in public service.

This paper proposes that engineers in public service are confronted with unavoidable complexity in their ethical considerations. The complexity begins with interactions among venues of ethical choices. Engineers must make ethical choices simultaneously at the individual, professional, organizational and societal levels. These ethical domains often conflict. The complexity also stems from situations in which physical properties may remain stable, but important social, economic, institutional and political conditions can change substantially. The paper proposes that the reflective learning approach of pragmatism can help with these challenging situations. This approach depends upon employing Dewey's five stage process of inquiry to engage the ethical complexity inherent in the practice of engineering in the public service.

Conflict, Psychological↗
PubMed2006

Moral creativity in science and engineering.

Creativity in science and engineering has moral significance and deserves attention within professional ethics, in at least three areas. First, much scientific and technological creativity constitutes moral creativity because it generates moral benefits, is motivated by moral concern, and manifests virtues such as beneficence, courage, and perseverance. Second, creativity contributes to the meaning that scientists and engineers derive from their work, thereby connecting with virtues such as authenticity and also faults arising from Faustian trade-offs. Third, morally creative leadership is important at all levels of science and engineering.

Creativity↗
PubMed1995

Engineered idiotypes. Immunochemical analysis of antigenized antibodies expressing a conformationally constrained Arg-Gly-Asp motif.

We report on the immunochemical characterization of two antibodies engineered to express RGD, a peptide from adhesive proteins of the extracellular matrix. One or three RGD motifs were introduced in the third complementarity-determining region (CDR) of a murine heavy (H) chain variable (V) region gene yielding two antibodies, gamma 1RGD and gamma 1(RGD)3. A murine monoclonal antibody (mAb) raised against an RGD-containing synthetic peptide bound in Western blot the H chain of both gamma 1RGD and gamma 1(RGD)3. Pronectin F, a genetically-engineered polymer containing RGD, abrogated this binding. Anti-idiotypic antibodies against the (RGD)3 loop were generated in a rabbit by immunization with gamma 1(RGD)3. Anti-idiotype antibodies purified by affinity-chromatography on the synthetic peptide GRGDSPC reacted in ELISA with gamma 1(RGD)3 and human fibronectin. Adhesive proteins, unlike RGD-containing synthetic peptides, were able to interfere with the interaction between gamma 1(RGD)3 and the anti-idiotypic antibodies. These results suggest that it is possible to genetically engineer the hypervariable loops of immunoglobulins and confer them new idiotypic characteristics. These results support the concept of antibody mimicry.

Amino Acid Sequence↗
PubMed1994

The blind watchmaker and rational protein engineering.

In the present review some scientific areas of key importance for protein engineering are discussed, such as problems involved in deducting protein sequence from DNA sequence (due to posttranscriptional editing, splicing and posttranslational modifications), modelling of protein structures by homology, NMR of large proteins (including probing the molecular surface with relaxation agents), simulation of protein structures by molecular dynamics and simulation of electrostatic effects in proteins (including pH-dependent effects). It is argued that all of these areas could be of key importance in most protein engineering projects, because they give access to increased and often unique information. In the last part of the review some potential areas for future applications of protein engineering approaches are discussed, such as non-conventional media, de novo design and nanotechnology.

Animals↗
PubMed1996

Protection of non-murine mammals against encephalomyocarditis virus using a genetically engineered Mengo virus.

Genetically engineered Mengo viruses with artificial deletions in the 5' noncoding poly(C) tracts are highly attenuated for pathogenicity when introduced as live vaccines into the natural murine host. Inoculation produces lifelong protective immunity without disease or viral persistence. This report extends the vaccination studies to non-murine hosts, including baboons, macaques and domestic pigs, all of which are susceptible to severe cardiovirus epizootics. All animals of these species that were inoculated with vMC24, an engineered strain of Mengo, seroconverted. When the immunized animals were challenged, they were protected against lethal doses of encephalomyocarditis virus (EMCV) derived from currently circulating epizootic strains. In baboons, the neutralizing antibody titers induced by vMC24 were significantly higher than from an inactivated EMCV vaccine. Moreover, terminal histopathology on baboons (inoculated intramuscularly), macaques (inoculated intracerebrally), and pigs (inoculated intramuscularly) showed few, if any, gross lesions characteristic of EMCV-like disease, in the vMC24 vaccinates. We suggest that genetically engineered, short poly(C) Mengo viruses may be universally potent attenuated vaccines for many types of animals and can possibly provide safe, efficacious protection against all cardioviruses of the EMCV serotype.

Animals↗
PubMed1990

Small-scale field test of the genetically engineered lacZY marker.

Commercial genetic engineering is advancing into areas that require the small-scale introduction of genetically engineered microorganisms (GEMs) to better quantify variables that affect microorganism distribution and survival and to document potential long-term consequences. A recombinant DNA marker system, the lacZY marker, developed by the Monsanto Agricultural Co., enables the distribution and fate of marked fluorescent pseudomonad organisms to be monitored under actual field conditions. Critical evaluation of GEMs under field conditions is imperative if plant-beneficial effects are to be correlated with organism release. This paper evaluates the effectiveness of this marker system and its ability to facilitate the assessment of risks associated with deliberate environmental introductions of genetically engineered microorganisms. Results of prerelease contained growth chamber and field experiments demonstrated that: (1) the scientific risk assessment methodology adopted by Monsanto and approved by the U.S. Environmental Protection Agency was appropriate and comprehensive; (2) the deliberate introduction of a GEM did not pose unacceptable or unforeseen risks to human health or the environment; (3) the lacZY marker is an effective environmental tracking tool; and (4) regulatory oversight should reflect the expected risk and not be excessively burdensome for all GEMs.

DNA, Recombinant↗
PubMed1991

Custom polymerase-chain-reaction engineering of a plant expression vector.

Polymerase-chain-reaction (PCR) amplification combined with custom-synthesized oligodeoxyribonucleotide (oligo) primers can be used to make complex genetic engineering steps (e.g., translational fusions) easy. Much of the complexity of the engineering steps can be incorporated into the custom oligo primers. Using this technique, a plant constitutive expression vector, pUC18cpexp, was constructed. This vector is based on the cauliflower mosaic virus 35S gene-regulatory elements and the cucumber mosaic virus coat protein-encoding gene (cp) 5'-untranslated region. Use of this vector is demonstrated by modifying the cp genes of several plant viruses and cloning them into pUC18cpexp. Because the construction and use of this vector system require custom oligo primer synthesis and PCR amplification, the technique is referred to as custom PCR engineering.

Amino Acid Sequence↗
PubMed1993

Cytogenetic effects of promutagens in genetically engineered V79 Chinese hamster cells expressing cytochromes P450.

V79 Chinese hamster cell lines genetically engineered to express rat CYP2B1, CYP1A1, CYP1A2, and their parental cell lines V79-MZ, without acetyltransferase, and V79-NH, with acetyltransferase, were studied for chromosome aberrations and sister chromatid exchange induced by aflatoxin B1, cyclophosphamide, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The parental V79 cell lines did not show clastogenic effects. Significant clastogenic effects were observed after an 18 h exposure to aflatoxin B1 and cyclophosphamide in CYP2B1 expressing cells, to benzo[a]pyrene in CYP1A1 and CYP1A2 expressing cells, to 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine in cells, expressing CYP1A2 with or without acetyltransferase, and to cyclophosphamide in cells expressing both CYP1A2 and acetyltransferase. A significant sister chromatid exchange inducing effect was found after a 24 h exposure in each of the genetically engineered cell lines, except for benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in CYP2B1 expressing cells, and for benzo[a]pyrene in cells expressing both CYP1A2 and acetyltransferase. Thus, a battery of cell lines genetically engineered for metabolic competence may serve as a tool for investigating chromosomal changes induced by activated xenobiotics.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

Engineering membrane proteins.

Much of the research on integral membrane proteins mirrors that on soluble proteins; however, membrane protein engineering also has its own ends and means, many of which take advantage of the peculiar situation of membrane proteins, whose chains are distributed between one lipidic and two aqueous phases. Extramembrane loops have been shortened, cut, or elongated with segments forming proteolytic cleavage sites, foreign epitopes, extra transmembrane segments, or even whole proteins, with the aim of facilitating purification, biochemical/biophysical studies, or crystallogenesis. Transmembrane alpha-helices have been deleted, duplicated, exchanged, transported into a foreign context or replaced with synthetic peptides, in order to both understand their integration into, and assembly in, the membrane and unravel their functional role. Insertion of cysteine residues has been the basis for a great diversity of experiments, ranging from the exploration of secondary, tertiary and quaternary structures of the transmembrane region to the creation of anchoring points for reporter molecules. Chemical engineering--the synthesis of protein fragments or even of whole proteins--offers particularly exciting new prospects, given the small size of folding domains in alpha-helical membrane proteins. Membrane protein engineering is rapidly developing its own agenda of questions and tool chest of techniques.

Bacteriorhodopsins↗
PubMed2006

The side chain interaction index as a tool for predicting fast-folding elements and the structure and stability of engineered peptides.

The side chain interaction index (SCII) is a method of calculating the propensity for short-range interactions among side chains within a peptide sequence. Here, it is shown that the SCII values of secondary structure elements that have been shown to fold early and independently cluster separately from those of structures that fold later and/or are dependent on long-range interactions. In addition, the SCII values of engineered peptides that spontaneously adopt a particular desired fold in solution are significantly different from those of engineered peptides that fail to exhibit a stable conformation. Thus, the SCII, as a measure of local structural stability, constitutes a useful tool in folding prediction and in protein/peptide engineering. A program that allows rapid calculation of SCII values is presented.

Drug Stability↗
PubMed2006

Viral vectors for gene delivery in tissue engineering.

The goal of tissue engineering is the production of functional, biocompatible tissues by seeding cells within biological or synthetic scaffolds. One tissue engineering approach involves the genetic modification of cells that are seeded onto (or into) scaffolds prior to implantation. The genetic modification is achieved through gene delivery, with can utilize viral transduction or non-viral transfection systems. Although novel non-viral systems have continued to emerge as innovative vehicles for controlled gene delivery, viruses remain the most efficient means by which exogenous genes can be introduced into and expressed by mammalian cells. Retrovirus, adenovirus, adeno-associated virus and herpes virus are widely studied viral gene transfer systems and have attracted the most attention in the field of transduction. This review thoroughly discusses the genomic structures of each virus type, along with the advantages and disadvantages of their use in tissue engineering applications.

Adenoviridae↗
PubMed2006

Tissue engineering for anterior cruciate ligament reconstruction: a review of current strategies.

The anterior cruciate ligament (ACL) is one the most commonly injured ligaments of the knee. Chronic ACL insufficiency can result in episodic instability, chondral and meniscal injury, and early osteoarthritis. The intra-articular environment of the ligament precludes normal healing and surgical replacement of the injured ligament is often mandated to restore stability. Current surgical strategies include the use of local autograft or allograft tissues for ligament reconstruction. These procedures have yielded superior long-term clinical results yet have the potential for serious associated morbidities. Existing limitations have prompted ongoing research designed to engineer a replacement ligament that will parallel the native ACL in both its biologic properties and mechanical durability. Ligament engineering necessitates the use of appropriate source cells and a growth matrix to support cell proliferation and collagen synthesis. The identification of appropriate growth modulators including both biochemical factors and mechanical stimuli are requisites for successful tissue growth. The characterization of the elements essential for successful graft development represents a significant challenge for investigators. This review examines the current literature regarding the potential and limitations of ligament engineering and describes the development of a novel 3-dimensional scaffold and bioreactor system at our institution.

Animals↗
PubMed2005

Novel tissue-engineered biodegradable material for reconstruction of vascular wall.

BACKGROUND: To solve several problems with artificial grafts, we sought to develop a novel bioengineered material that can promote tissue regeneration without ex vivo cell seeding and that has sufficient durability to be used for artery reconstruction. Here, we tested whether this biodegradable material could accelerate the in situ regeneration of autologous cardiovascular tissue, especially of the arterial wall, in various models of cardiovascular surgeries. METHODS: The tissue-engineered patch was fabricated by compounding a collagen-microsponge with a biodegradable polymeric scaffold composed of polyglycolic acid knitted mesh, reinforced on the outside with woven polylactic acid. Tissue-engineered patches without precellularization were grafted into the porcine descending aorta (n = 5), the porcine pulmonary arterial trunk (n = 8), or the canine right ventricular outflow tract (as the large graft model; n = 4). Histologic and biochemical assessments were performed 1, 2, and 6 months after the implantation. RESULTS: There was no thrombus formation in any animal. Two months after grafting, all the grafts showed good in situ cellularization by hematoxylin/eosin and immunostaining. The quantification of the cell population by polymerase chain reaction showed a large number of endothelial and smooth muscle cells 2 months after implantation. In the large graft model, the architecture of the patch was similar to that of native tissue 6 months after implantation. CONCLUSIONS: A tissue-engineered patch made of our biodegradable polymer and collagen-microsponge provided good in situ regeneration at both the venous and arterial wall, suggesting that this patch can be used as a novel surgical material for the repair of the cardiovascular system.

Absorbable Implants↗
PubMed2006

Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors.

BACKGROUND: Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells. METHODS: Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase). RESULTS: Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression. CONCLUSIONS: These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.

Biomechanical Phenomena↗
PubMed2006

Novel chitosan/collagen scaffold containing transforming growth factor-beta1 DNA for periodontal tissue engineering.

The current rapid progression in tissue engineering and local gene delivery system has enhanced our applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-beta1 (TGF-beta1). These scaffolds were evaluated in vitro by analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen scaffold. The scaffold containing Ad-TGF-beta1 exhibited the highest proliferation rate, and the expression of type I and type III collagen up-regulated in Ad-TGF-beta1 scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-TGF-beta1 as a good substrate candidate in periodontal tissue engineering.

Adenoviridae↗
PubMed2006

Engineering of beta-propeller protein scaffolds by multiple gene duplication and fusion of an idealized WD repeat.

The ability to design specific amino acid sequences that fold into desired structures is central to engineering novel proteins. Protein design is also a good method to assess our understanding of sequence-structure and structure-function relationships. While beta-sheet structures are important elements of protein architecture, it has traditionally been more difficult to design beta-proteins than alpha-helical proteins. Taking advantage of the tandem repeated sequences that form the structural building blocks in a group of beta-propeller proteins; we have used a consensus design approach to engineer modular and relatively large scaffolds. An idealized WD repeat was designed from a structure-based sequence alignment with a set of structural guidelines. Using a plasmid sequential ligation strategy, artificial concatemeric genes with up to 10 copies of this idealized repeat were then constructed. Corresponding proteins with 4 through to 10 WD repeats were soluble when over-expressed in Escherichia coli. Notably, they were sufficiently stable in vivo surviving attack from endogenous proteases, and maintained a homogeneous, non-aggregated form in vitro. The results show that the beta-propeller scaffold is an attractive platform for future engineering work, particularly in experiments in which directed evolution techniques might improve the stability of the molecules and/or tailor them for a specific function.

Amino Acid Motifs↗
PubMed2004

PDLLA/Bioglass composites for soft-tissue and hard-tissue engineering: an in vitro cell biology assessment.

The aim of this study was to examine the effect of increased content of 45S5 Bioglass (0-40 wt%) in poly(dl-lactic acid) (PDLLA) porous foams on the behaviour of MG-63 (human osteosarcoma cell line) and A549 cells (human lung carcinoma cell line). The ability of these cell lines to grow on bioactive composites was quantitatively investigated in order to assess the potentiality for their use in hard and soft-tissue engineering. Two hours after cell seeding, an increase of cell adhesion according to the increased content of Bioglass((R)) present in the foams for both cell types was observed. Cell proliferation studies performed over a period of 4 weeks showed a better aptitude of the A549 cells to proliferate on PDLLA foams containing 5 wt% Bioglass when compared to the proliferation on foams with 40 wt% Bioglass. A lower proliferation rate was obtained for cells on pure PDLLA. Scanning electron microscopy analysis showed for both cell types the presence of cells inside the porous structure of the foams. These results confirmed the biocompatibility of PDLLA/Bioglass composite foams and the positive effect of Bioglass on MG-63 cell behaviour and also showed for the first time the possibility for human lung epithelial type II cells to adhere and proliferate on these porous scaffolds. In addition, we describe a positive effect of 45S5 Bioglass on A549 cell behaviour in a dose-dependent manner, indicating the potential of using PDLLA/Bioglass composites with an optimal concentration of 45S5 Bioglass not only in bone tissue engineering but also in lung tissue engineering.

Biocompatible Materials↗