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PubMedDate not supplied

Exploration of sequence space for protein engineering.

The process of protein engineering is currently evolving towards a heuristic understanding of the sequence-function relationship. Improved DNA sequencing capacity, efficient protein function characterization and improved quality of data points in conjunction with well-established statistical tools from other industries are changing the protein engineering field. Algorithms capturing the heuristic sequence-function relationships will have a drastic impact on the field of protein engineering. In this review, several alternative approaches to quantitatively assess sequence space are discussed and the relatively few examples of wet-lab validation of statistical sequence-function characterization/correlation are described.

Algorithms↗
PubMed2006

Nondestructive evaluation of osteogenic differentiation in tissue-engineered constructs.

Conventional measurements of osteogenesis in tissue-engineered constructs are destructive to living cells and incapable to provide three-dimensional information. In the present study, noninvasive magnetic resonance (MR) microscopy was used to evaluate osteogenic differentiation in vitro in human mesenchymal stem cell-based tissue-engineered constructs. The constructs were prepared by seeding the cells (10(6)cells/ml) on 4 x 4 x 4 mm gelatin sponge cubes and subsequently exposing them to osteogenic differentiation or basic medium. During the 4-week experiment, alkaline phosphatase (ALP) activity and calcium content of differentiated constructs were significantly increased compared to the basic medium controls. The T1, T2, and apparent diffusion coefficient (ADC) of differentiated constructs were significantly lower than those of the control group at each time point (p < 0.05). The MR parameters of constructs were significantly correlated to their ALP activities (r to T1, T2, and ADC = -0.57, -0.78, and -0.81, respectively) and calcium content (r to T1, T2, and ADC = 0.48, 0.90, and 0.92, respectively) measured by biochemical techniques. MR microscopy can be a promising tool for noninvasive assessment of osteogenic differentiation and to provide three-dimensional information about tissue-engineered constructs.

Alkaline Phosphatase↗
PubMed1989

Should there be a formal mechanical-optic-electronic biomedical-engineering program?

To progress in the development of laser medicine and laser surgery, multidisciplinary efforts are required. One important phase in its development is the laser biomedical engineering program being offered following the basic laboratory research. Some of this type of programming is present today, but more development is needed for a formal program offering crossfertilization between engineering and medical uses. Therefore, a formal program is being proposed, which will be reviewed by mixed groups over a period of time. A final recommendation will indicate whether to go ahead with this revision of the program, to wait for further development of the program, or to maintain the status quo. The relationship of technical training for technicians in laser biomedical engineering is also reviewed.

Biomedical Engineering↗
PubMed2004

Hepatic tissue engineering for adjunct and temporary liver support: critical technologies.

The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the "proof of principle" test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist.

Biomedical Technology↗
PubMed2004

Thermally produced biodegradable scaffolds for cartilage tissue engineering.

A novel process was developed to fabricate biodegradable polymer scaffolds for tissue engineering applications, without using organic solvents. Solvent residues in scaffolds fabricated by processes involving organic solvents may damage cells transplanted onto the scaffolds or tissue near the transplantation site. Poly(L-lactic acid) (PLLA) powder and NaCl particles in a mold were compressed and subsequently heated at 180 degrees C (near the PLLA melting temperature) for 3 min. The heat treatment caused the polymer particles to fuse and form a continuous matrix containing entrapped NaCl particles. After dissolving the NaCl salts, which served as a porogen, porous biodegradable PLLA scaffolds were formed. The scaffold porosity and pore size were controlled by adjusting the NaCl/PLLA weight ratio and the NaCl particle size. The characteristics of the scaffolds were compared to those of scaffolds fabricated using a conventional solvent casting/particulate leaching (SC/PL) process, in terms of pore structure, pore-size distribution, and mechanical properties. A scanning electron microscopic examination showed highly interconnected and open pore structures in the scaffolds fabricated using the thermal process, whereas the SC/PL process yielded scaffolds with less interconnected and closed pore structures. Mercury intrusion porosimetry revealed that the thermally produced scaffolds had a much more uniform distribution of pore sizes than the SC/PL process. The utility of the thermally produced scaffolds was demonstrated by engineering cartilaginous tissues in vivo. In summary, the thermal process developed in this study yields tissue-engineering scaffolds with more favorable characteristics, with respect to, freedom from organic solvents, pore structure, and size distribution than the SC/PL process. Moreover, the thermal process could also be used to fabricate scaffolds from polymers that are insoluble in organic solvents, such as poly(glycolic acid). Cartilage tissue regenerated from thermally produced PLLA scaffold.

Animals↗
PubMed2004

Bone tissue engineering: state of the art and future trends.

Although several major progresses have been introduced in the field of bone regenerative medicine during the years, current therapies, such as bone grafts, still have many limitations. Moreover, and in spite of the fact that material science technology has resulted in clear improvements in the field of bone substitution medicine, no adequate bone substitute has been developed and hence large bone defects/injuries still represent a major challenge for orthopaedic and reconstructive surgeons. It is in this context that TE has been emerging as a valid approach to the current therapies for bone regeneration/substitution. In contrast to classic biomaterial approach, TE is based on the understanding of tissue formation and regeneration, and aims to induce new functional tissues, rather than just to implant new spare parts. The present review pretends to give an exhaustive overview on all components needed for making bone tissue engineering a successful therapy. It begins by giving the reader a brief background on bone biology, followed by an exhaustive description of all the relevant components on bone TE, going from materials to scaffolds and from cells to tissue engineering strategies, that will lead to "engineered" bone. Scaffolds processed by using a methodology based on extrusion with blowing agents.

Biocompatible Materials↗
PubMed2005

Microrobotics and MEMS-based fabrication techniques for scaffold-based tissue engineering.

Scaffold based tissue engineering strategies use cells, biomolecules and a scaffold to promote the repair and regeneration of tissues. Although scaffold-based tissue engineering approaches are being actively developed, most are still experimental, and it is not yet clear what defines an ideal scaffold/cell construct. Solid free form fabrication (SFF) techniques can precisely control matrix architecture (size, shape, interconnectivity, branching, geometry and orientation). The SFF methods enable the fabrication of scaffolds with various designs and material compositions, thus providing a control of mechanical properties, biological effects and degradation kinetics. This paper reviews the application of micro-robotics and MEMS-based fabrication techniques for scaffold design and fabrication. It also presents a novel robotic technique to fabricate scaffold/cell constructs for tissue engineering by the assembly of microscopic building blocks.

Biocompatible Materials↗
PubMed2004

De novo adipose formation in a vascularized engineered construct.

While the field of tissue engineering is a burgeoning one, progress with fat engineering has lagged, due in large part to problems associated with nurturing and sustaining this unique tissue in vivo. In a pilot study using an experimental rat model, we induced liponeogenesis with a combination of Matrigel and basic fibroblast growth factor in an fibrovascular scaffold, isolating the construct to a pedicled blood supply (the superficial inferior epigastric vessels) via a silicone housing, creating an engineered three-dimensional adipose tissue construct. Adipose tissue and vascular ingrowth were assessed histologically and followed by serial study at 4-week intervals for 16 weeks. We demonstrated persistence of shaped adipose tissue constructs over time, and postulate that incorporating a vascular supply may enhance the durability of experimentally induced fat constructs and potentially provide a means for microsurgical transfer of the construct.

Adipose Tissue↗
PubMed2002

Fabricating autologous tissue to engineer artificial nerve.

This study reports on the successful fabrication of artificial nerves with tissue engineering methods. Schwann cells were cultured for 2 weeks, seeded on polyglactin 910 scaffolds, and biomembrane-coated with rat-tail glue and laminin. Observation of the scaffolds' adsorptivity to Schwann cells, and of the growth and migration of Schwann cells, was made using a light microscope, and by scanning and transmission electron microscopy. The Schwann cells were able to migrate and proliferate on the polyglactin 910 fiber. Schwann cells were well-distributed, and formed a Büngner band on which the Schwann cells produced more matrices. Schwann cells on the biomembrane also grew well. We investigated the role of the tissue engineering conduit guide in vivo, using an established rabbit peripheral nerve regeneration model. At 8 weeks, axonal regeneration was observed in the distal nerve stump. Adult Schwann cells can be produced on the coated fiber and the biomembrane. Three-dimensional scaffolds with Schwann cells had the basic characteristics of the artificial nerve. These findings will provide a practical method for fabricating tissue-engineered artificial nerve to repair long nerve defects.

Animals↗
PubMed2002

Influence of insulin-like growth factor-I (IGF-I) on nerve autografts and tissue-engineered nerve grafts.

To overcome the problems of limited donor nerves for nerve reconstruction, we established nerve grafts made from cultured Schwann cells and basal lamina from acellular muscle and used them to bridge a 2-cm defect of the rat sciatic nerve. Due to their basal lamina and to viable Schwann cells, these grafts allow regeneration that is comparable to autologous nerve grafts. In order to enhance regeneration, insulin-like growth factor (IGF-I) was locally applied via osmotic pumps. Autologous nerve grafts with and without IGF-I served as controls. Muscle weight ratio was significantly increased in the autograft group treated with IGF-I compared to the group with no treatment; no effect was evident in the tissue-engineered grafts. Autografts with IGF-I application revealed a significantly increased axon count and an improved g-ratio as indicator for "maturity" of axons compared to autografts without IGF-I. IGF-I application to the engineered grafts resulted in a decreased axon count compared to grafts without IGF-I. The g-ratio, however, revealed no significant difference between the groups. Local administration of IGF-I improves axonal regeneration in regular nerve grafts, but not in tissue-engineered grafts. Seemingly, in these grafts the interactive feedback mechanisms of neuron, glial cell, and extracellular matrix are not established, and IGF-I cannot exert its action as a pleiotrophic signal.

Animals↗
PubMed2003

Proteomic changes in Escherichia coli TG1 after metabolic engineering for enhanced trichloroethene biodegradation.

Through metabolic engineering, new enzymatic pathways can be introduced into cells to enable or enhance production or biotransformation of chemicals. However, these changes have physiological consequences that can be important but are not well understood. Here we describe the use of two-dimensional gel electrophoresis (2-DE) to detect changes in the proteome of Escherichia coli cells that have been engineered to transform the pollutant trichloroethene (TCE) with the enzyme toluene o-monooxygenase (TOM). Comparison of 2-DE gels (isoelectric point range 4-7) for E. coli cells with and without the ability to synthesize TOM revealed 31 new proteins in TOM-containing cells as well as nine proteins not detected in those cells but present in the plasmid control strain. Exposure of TOM-containing cells to TCE led to the synthesis of four new proteins and the loss of only one protein. Thus, this example of metabolic engineering has a substantial and complex impact on the physiology of these cells that was clearly revealed using a proteomic approach.

Bacterial Proteins↗
PubMed2001

Anhydrobiotic engineering of bacterial and mammalian cells: is intracellular trehalose sufficient?

Anhydrobiotic engineering aims to confer a high degree of desiccation tolerance on otherwise sensitive living organisms and cells by adopting the strategies of anhydrobiosis. Nonreducing disaccharides such as trehalose and sucrose are thought to play a pivotal role in resistance to desiccation stress in many microorganisms, invertebrates, and plants, and in vitro trehalose is known to confer stability on dried biomolecules and biomembranes. We have therefore tested the hypothesis that intracellular trehalose (or a similar molecule) may be not only necessary for anhydrobiosis but also sufficient. High concentrations of trehalose were produced in bacteria by osmotic preconditioning, and in mammalian cells by genetic engineering, but in neither system was desiccation tolerance similar to that seen in anhydrobiotic organisms, suggesting that trehalose alone is not sufficient for anhydrobiosis. In Escherichia coli such desiccation tolerance was achievable, but only when bacteria were dried in the presence of both extracellular trehalose and intracellular trehalose. In mouse L cells, improved osmotolerance was observed with up to 100 mM intracellular trehalose, but desiccation was invariably lethal even with extracellular trehalose present. We conclude that anhydrobiotic engineering of at least some microorganisms is achievable with present technology, but that further advances are needed for similar desiccation tolerance of mammalian cells.

Animals↗
PubMed1993

Transplantation of a polymer-encapsulated cell line genetically engineered to release NGF.

The delivery of nerve growth factor (NGF) to the lateral ventricle of a fimbria-fornix-lesioned rat prevents the lesion-induced reduction in choline acetyltransferase (ChAT) expression by medial septal cells. Although delivery has been achieved through neural grafting of genetically engineered cell lines which release NGF, transplanted cells have grown beyond the implantation site and formed tumors. The encapsulation of cells within a permselective polymer capsule prior to transplantation allows cell growth only within the capsule space, while allowing molecular exchange between the host tissue and enclosed cells. Rat fibroblasts from the parent cell line (R208F) or fibroblasts genetically modified to produce NGF (R208N.8) were loaded within a thermoplastic hollow fiber-based capsule. Only the capsules loaded with the genetically engineered cells released measurable amounts of NGF in culture. Adult rats received unilateral aspirative fimbria-fornix lesions, followed by intraventricular implantation of a R208F capsule (n = 6) or a R208N.8 capsule (n = 6). After 2 weeks, rats receiving encapsulated cells showed no undue reaction to the implants. With both cell types, the cells remained viable and confined to the capsule space. R208N.8 capsules released sufficient NGF to prevent the lesion-induced loss of septal ChAT expression, whereas R208F capsules did not. This study suggests that encapsulated genetically engineered cells can provide an efficient means for future applications involving delivery of neurotrophic factors.

Acetylcholinesterase↗
PubMed1999

Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins.

High-alkaline serine proteases have been successfully applied as protein degrading components of detergent formulations and are subject to extensive protein engineering efforts to improve their stability and performance. Dynamics has been suggested to play an important role in determining enzyme activity and specificity and it is therefore of interest to establish how local changes in internal mobility affect protein stability, specificity and performance. Here we present the dynamic properties of the 269 residue serine proteases subtilisin PB92 (Maxacal(TM)) and subtilisin BLS (Savinase(TM)), secreted by Bacillus lentus, and an engineered quadruple variant, DSAI, that has improved washing performance. T1, T2 and heteronuclear NOE measurements of the 15N nuclei indicate that for all three proteins the majority of the backbone is very rigid, with only a limited number of residues being involved in local mobility. Many of the residues that constitute the S1 and S4 pockets, determining substrate specificity, are flexible in solution. In contrast, the backbone amides of the residues that constitute the catalytic triad do not exhibit any motion. Subtilisins PB92, BLS and DSAI demonstrate similar but not identical NMR relaxation rates. A detailed analysis of local flexibility indicates that the motion of residues Thr143 and Ala194 becomes more restricted in subtilisin BLS and DSAI. Noteworthy, the loop regions involved in substrate binding become more structured in the engineered variant as compared with the two native proteases, suggesting a relation between altered dynamics and performance. Similar conclusions have been established by X-ray crystallograpic methods, as shown in the accompanying paper.

Bacillus↗
PubMed1999

Metabolic fluxes and metabolic engineering.

Metabolic engineering is the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology. As such, metabolic engineering emphasizes metabolic pathway integration and relies on metabolic fluxes as determinants of cell physiology and measures of metabolic control. The combination of analytical methods to quantify fluxes and their control with molecular biological techniques to implement genetic modifications is the essence of metabolic engineering. Strategies for metabolic flux determination are reviewed in this paper and it is shown how metabolic fluxes can be used in the systematic elucidation of metabolic control in the framework of reaction grouping and top-down metabolic control analysis.

Animals↗
PubMed2000

Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway.

The biosynthesis of the osmoprotectant glycine betaine (GlyBet) is a target for metabolic engineering to enhance stress resistance in crops. Certain plants synthesize GlyBet in chloroplasts via a two-step oxidation of choline (Cho). In previous work, a chloroplastic GlyBet synthesis pathway was inserted into tobacco (which lacks GlyBet) by expressing spinach choline monooxygenase (CMO). The transformants had low CMO enzyme activity, and produced little GlyBet (less than or = 70 nmol g(-1) fresh wt). In this study, transformants with up to 100-fold higher CMO activity showed no further increase in GlyBet. In contrast, tobacco expressing a cytosolic GlyBet synthesis pathway accumulated significantly more GlyBet (430 nmol g(-1) fresh wt), suggesting that subcellular localization influences pathway flux. Modeling of the labeling kinetics of Cho metabolites observed when [14C]Cho was supplied to engineered plants demonstrated that Cho import into chloroplasts indeed limits the flux to GlyBet in the chloroplastic pathway. A high-activity Cho transporter in the chloroplast envelope may therefore be an integral part of the GlyBet synthesis pathway in species that accumulate GlyBet naturally, and hence a target for future engineering.

Alcohol Oxidoreductases↗
PubMed2001

Metabolic engineering of isoprenoids.

The metabolic engineering of natural products has begun to prosper in the past few years due to genomic research and the discovery of biosynthetic genes. While the biosynthetic pathways and genes for some isoprenoids have been known for many years, new pathways have been found and known pathways have been further investigated. In this article, we review the recent advances in metabolic engineering of isoprenoids, focusing on the molecular genetics that affects pathway engineering the most. Examples in mono- sequi-, and diterpenoid synthesis as well as carotenoid production are discussed.

Carbohydrate Sequence↗
PubMed2001

Biochemical engineering of natural product biosynthesis pathways.

Metabolic engineering of natural products is a science that has been built on the goals of traditional strain improvement with the availability of modern molecular biological technologies. In the past 15 years, the state of the art in metabolic engineering of natural products has advanced from the first proof-of-principle experiment based on minimal known genetics to a commonplace event using highly specific and sophisticated gene manipulation methods. With the availability of genes, host organisms, vector systems, and standard molecular biological tools, it is expected that metabolic engineering will be translated into industrial reality.

Bacteria↗