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Quantitative histological evaluation of early fracture healing of cortical bones immobilized by stainless steel and composite plates.

Internal fixation devices of less bending stiffness than conventional plates made of stainless steel or vitallium were compared with conventional plates in a study of fracture healing. The material for this investigation was a fine graphite fiber reinforced methyl methacrylate resin composite with a modulus of elasticity approximately ten times less than that of stainless steel. Osteotomies were performed on canine radii. Internal fixation was accomplished by means of a composite plate on the left side, and a stainless steel plate on the right. Clinical assessment, as well as biomechanical and quantitative histological techniques, were used to compare osteotomy healing of the two sides. At four months, all osteotomies had healed and the bioengineering tests showed radii from the two sides had equivalent strength. However, significantly less cortical porosity was found in the side with the composite plate (6.8 per cent), as compared to that of the stainless steel plated side (14 per cent). These results suggest that a less stiff fixation plate may have some advantage in the treatment of long bone fracture if there is no implant failure, and if union rates are equivalent.

Animals↗

Skin equivalent produced with human collagen.

Several studies have recently been conducted on cultured skin equivalent (SE), prepared using human keratinocytes seeded on various types of dermal equivalents (DE). We previously showed the advantages of our anchorage method in preventing the severe surface reduction of DE due to fibroblast contractile properties in vitro. A new anchored human SE was established in our laboratory in order to obtain a bioengineered tissue that would possess the appropriate histological and biological properties. In order to compare the effects of different collagen origins on the evolution of SE in vitro, human keratinocytes were seeded on three types of anchored DE. A comparative study was carried out between bovine SE (bSE), human SE (hSE), and human skin equivalent containing additional dermal matrix components (hSE+). Immunohistological analysis showed that hSE and hSE+ presented good structural organization, including the deposition of several basement membrane constituents. Higher amounts of transglutaminase, ceramides, and keratin 1 were detected in the epidermal layers of all SE when cultured at the air-liquid interface. However, a 92 kDa gelatinase activity was higher in bovine skin equivalent (bSE) compared to hSE cultures. The use of human collagens comparatively to bovine collagen as SE matricial component delayed the degradation of the dermal layer in culture.

Animals↗

Determination of the optimal feeding regime during biosynthesis of erythromycin.

A systems approach to the analysis of the process of fermentaton of erythromycin in demonstrated. On the basis of biochemical and bioengineering data obtained in hydrodynamic tests and pilot plant fermentations of erythromycin a mathematical model for optimal technological parameters of growth of Streptomyces erythreus and product formation was postulated. The optimal regime was simulated and subsequently verified. The production of erythromycin increased to 112%.

Culture Media↗

New trends in microbial technology.

Microbial technology includes not only the production of materials in bioreactors, or the production of new catalysts by genetic engineering but extends to aspects of both human and animal health care, waste and pollution management, enhanced oil recovery, mineral leaching, advanced plant breeding, diagnostics and analytical equipment, biosensors, bioelectronics and renewable energy system based on biomass feedstocks. National strategies of industrialized countries are being developed which identify microbial technology as a substantial factor in the attainment of industrial and economic goals. Although extremely promising microbial technology is not a quick fix and its application will only arise as a result of systematic programme of research and development. Such programme requires a broad base of disciplinary underpinning in molecular biology, genetics and bioengineering. The development of expertise of this kind in the tertiary educational institutions is the essential starting point. It should be developed by appropriate programmes and networking systems.

Animals↗

Use of regulated secretion in protein production from animal cells: an overview.

Traditional industrial cell culture processes require extensive downstream product refining due to low product titer and purity in the spent growth medium. A controlled secretion process incorporating cells derived from endocrine or exocrine organs could potentially alleviate this processing burden by dynamically decoupling product recovery from cell growth and product biosynthesis. In addition, such specialized secretory cells may be uniquely capable of performing desirable post-translational processing of the secretory product. We briefly review the biology of regulated protein secretion as well as the biology and biochemistry of the signal transduction mechanisms by which regulated systems respond to environmental stimuli. Drawing on these and other basic principles from cell biology and bioengineering, we describe the important features of a controlled secretion process. Among other issues we discuss the choice of cell lines, expression systems, cell culture methods, and bioreactor configurations. We extensively analyze the kinetics of regulated secretion in the context of a controlled secretion process. This discussion is illustrated with experimental results from two model cell lines, recombinant AtT-20 and beta TC3, expressing recombinant human endocrine hormones or native murine insulin respectively.

Animals↗

In vivo stationary flux analysis by 13C labeling experiments.

Stationary flux analysis is an invaluable tool for metabolic engineering. In the last years the metabolite balancing technique has become well established in the bioengineering community. On the other hand metabolic tracer experiments using 13C isotopes have long been used for intracellular flux determination. Only recently have both techniques been fully combined to form a considerably more powerful flux analysis method. This paper concentrates on modeling and data analysis for the evaluation of such stationary 13C labeling experiments. After reviewing recent experimental developments, the basic equations for modeling carbon labeling in metabolic systems, i.e. metabolite, carbon label and isotopomer balances, are introduced and discussed in some detail. Then the basics of flux estimation from measured extracellular fluxes combined with carbon labeling data are presented and, finally, this method is illustrated by using an example from C. glutamicum. The main emphasis is on the investigation of the extra information that can be obtained with tracer experiments compared with the metabolite balancing technique alone. As a principal result it is shown that the combined flux analysis method can dispense with some rather doubtful assumptions on energy balancing and that the forward and backward flux rates of bidirectional reaction steps can be simultaneously determined in certain situations. Finally, it is demonstrated that the variant of fractional isotopomer measurement is even more powerful than fractional labeling measurement but requires much higher numerical effort to solve the balance equations.

Biological Transport↗

[Regenerative medicine and plastic surgery].

Regenerative medicine recently evolved as a new medical field that includes tissue engineering, cell/system biology, nanotechnology, pharmacology, stem-cell biology, and bioengineering. Regenerative medicine targets new forms of therapy to promote and support the intrinsic, autologous, regenerative potential of human biological systems. All fields of surgery have profited from these developments, and spectacular experimental results and clinical benefits have been obtained. Plastic surgery has shown interest in regenerative medicine due to its focus on reconstructive surgery. Early on, several interdisciplinary experimental working groups were founded including plastic surgery. This overview takes a closer look at common experimental and clinical results of regenerative medicine and plastic surgery.

Animals↗

[Effect of urea nail lacquer on nail quality. Clinical evaluation and biophysical measurements].

BACKGROUND: Urea is a well-known humectant. The aim of our study was to evaluate the in vivo effect of 10% urea lacquer on nail quality. METHODS: 60 healthy probands with brittle nails were included in a randomized, controlled study. The nail lacquer was applied once (n=30) or twice daily (n=30) on one hand for 28 days, while the other hand served as untreated control. Assessments included clinical evaluations and bioengineering measurements. RESULTS: Application of the nail lacquer was well-tolerated and led to a significant clinical improvement of nail surface morphology, nail plate consistency, reflection of the nail plate surface, appearance of the cuticle and the nail fold. Sonographic nail density was slightly increased. No relevant changes were found in nail thickness and transonychial water loss. CONCLUSIONS: Nail lacquer containing urea improves the cosmetic appearance of fingernails.

Adult↗

Nucleic acid based strategies as potential therapeutic tools: mechanistic considerations and implications to restenosis.

The capacity of DNA to bind RNA via Watson-Crick base-pairing is fundamental to antisense oligonucleotide strategies to inhibit gene expression, and is a property that has been exploited by bioengineers in the generation of catalytic molecules such as ribozymes, ribozyme subtypes, and more recently DNAzymes. This review describes the evolution of these gene-specific agents and summarizes recent efforts to inhibit smooth muscle cell growth with these molecules as candidate therapeutic tools in restenosis.

Animals↗

Importance of the GLUT2 glucose transporter for pancreatic beta cell toxicity of alloxan.

AIMS/HYPOTHESIS: We investigated the importance of the low affinity GLUT2 glucose transporter in the diabetogenic action of alloxan in bioengineered RINm5F insulin-producing cells with different expressions of the transporter. METHODS: GLUT2 glucose transporter expressing RINm5F cells were generated through stable transfection of the rat GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Cells expressing the GLUT2 transporter were susceptible to alloxan toxicity due to the uptake of alloxan by this specific glucose transporter isoform. The extent of the toxicity of alloxan was dependent upon the GLUT2 protein expression in the cells. The lipophilic alloxan derivative, butylalloxan, was toxic also to non-transfected control cells. Expression of the GLUT2 glucose transporter caused only a marginal increase in the toxicity of this substance. Butylalloxan, unlike alloxan itself, is not diabetogenic in vivo although, like the latter substance, it is beta-cell toxic in vitro through its ability to generate free radicals during redox cycling with glutathione. CONCLUSION/INTERPRETATION: Our results are consistent with the central importance of selective uptake of alloxan through the low affinity GLUT2 glucose transporter for the pancreatic beta-cell toxicity and diabetogenicity of this substance. Redox cycling and the subsequent generation of oxygen free radicals leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as alloxan diabetes in experimental diabetes research.

3-O-Methylglucose↗

Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin.

AIMS/HYPOTHESIS: The role of selective uptake and alkylation in the diabetogenic action of streptozotocin was investigated in bioengineered RINm5F insulin-producing cells, with different expression levels of the glucose transporter GLUT2, by comparing the toxicity of streptozotocin with that of four chemically related alkylating compounds, N-methyl-N-nitrosourea (MNU), N-ethyl-N nitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). METHODS: GLUT2 expressing RINm5F cells were generated through stable transfection of the rat glucose transporter GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Cells expressing the glucose transporter GLUT2 were much more susceptible to streptozotocin toxicity than control cells due to the uptake of streptozotocin by this specific glucose transporter. In contrast, the GLUT2 expression had no effect upon the toxicity of MNU, ENU, MMS or EMS. Although the latter substances are, like streptozotocin, cytotoxic through their ability to cause DNA alkylation, they are not diabetogenic because they are not taken up through the glucose transporter GLUT2. CONCLUSION/INTERPRETATION: Our results are consistent with the central importance of selective uptake and alkylating activity in the mechanism of streptozotocin diabetogenicity. Alkylation of DNA leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as streptozotocin diabetes in experimental diabetes research.

3-O-Methylglucose↗

Surface perspectives in the biomedical applications of poly(alpha-hydroxy acid)s and their associated copolymers.

Impressive advances in biotechnology, bioengineering, and biomaterials with unique properties have led to increased interest in polymers and other novel materials in biological and biomedical research and development over the past two decades. Although biomaterials have already made an enormous impact in biomedical research and clinical practice, there is a need for better understanding of the surface and interfacial chemistry between tissue (or cells) and biomedical materials. This is because the detailed physicochemical events related to the biological response to the surface of materials still often remain obscure, even though surface properties are important determinants of biomedical material function. In this regard, data available in the literature show the complexity of the interactions (surface reorganization, non-specific/specific protein adsorption, and chemical reactions such as acid-base, ion pairing, ion exchange, hydrogen bonding, divalent-ion bridging) and the interrelationship between biological environments, interfacial properties, and surface functional groups responsible for the biological responses. Because of the multidisciplinary nature of surface and interfacial phenomena at the surface of biomedical polymers, this review focuses on several aspects of current work published on poly(alpha-hydroxy acid)s and their associated copolymers:surface structure-biomedical function relationships;physicochemical strategies for surface modification; and, finally,synthetic strategies to increase biocompatibility for specific in-vivo and/or in-vitro biomedical applications.

Biocompatible Materials↗

Bioglass 45S5 stimulates osteoblast turnover and enhances bone formation In vitro: implications and applications for bone tissue engineering.

We investigated the concept of using bioactive substrates as templates for in vitro synthesis of bone tissue for transplantation by assessing the osteogenic potential of a melt-derived bioactive glass ceramic (Bioglass 45S5) in vitro. Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric analysis of the cell cycle suggested that the bioactive glass-ceramic substrate induced osteoblast proliferation, as indicated by increased cell populations in both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical analysis of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin indicated that the bioactive glass-ceramic substrate augmented osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning electron microscopic observations of discrete bone nodules over the surface of the bioactive material, from day 6 onward, further supported this notion. A combination of fluorescence, confocal, transmission electron microscopy, and X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of cell aggregates which produced mineralized collagenous matrix. Control substrates did not exhibit mineralized nodule formation at any point studied up to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability to stimulate the growth and osteogenic differentiation of human primary osteoblasts. These findings have potential applications for tissue engineering where this bioactive glass substrate could be used as a template for the formation of bioengineered bone tissue.

Alkaline Phosphatase↗

Biorefinery.

The biorefinery produces fuels, solvents, plastics and food for human beings. In some countries, these biorefinery products are made from waste biomass. The main processes in the biorefinery involve ethanol fermentation and lactic acid fermentation. For the biorefinery, many hybrid technologies were developed from different fields, such as bioengineering, polymer chemistry, food science and agriculture.

Bacillus↗

Yeast cell-surface display--applications of molecular display.

In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.

Agglutinins↗

Construction of a recA mutant of Burkholderia (formerly Pseudomonas), cepacia.

A recA mutant was constructed of a soil isolate of Burkholderia cepacia, strain ATCC 17616. Prior to mutagenesis, the recA gene was cloned from this strain by its ability to complement the methyl methanesulfonate sensitivity of an Escherichia coli recA mutant. Sequence analysis of the strain showed high sequence similarity (94% nucleic acid and 99% amino acid identity) with the recA gene previously cloned from a clinical isolate of B. cepacia, strain JN25. The subcloned recA gene from B. cepacia ATCC 17616 restored UV resistance and recombination proficiency to recA mutants of E. coli and Pseudomonas aeruginosa, as well as restoring the ability of D3 prophages to be induced to lytic growth from a RecA- strain of P. aeruginosa. The recA mutant of B. cepacia ATCC 17616 was constructed by lambda-mediated Tn5 mutagenesis of the cloned recA gene in E. coli, followed by replacement of the Tn5-interrupted gene for the wild-type allele in the chromosome of B. cepacia by marker exchange. The RecA- phenotype of the mutant was demonstrated by the loss of UV resistance as compared to the parental strain. Southern hybridization analysis of chromosomal DNA from the mutant indicated the presence of Tn5 in the recA gene, and the location of the Tn5 insertion in the recA allele was identified by nucleotide sequence analysis. A test using the recA mutant to see if acquired resistance to D-serine toxicity in B. cepacia might be a result of RecA-mediated activities proved negative; nevertheless, RecA activity potentially contributes to the overall genomic plasticity of B. cepacia and a recA mutant will be useful in bioengineering of this species.

Amino Acid Sequence↗

Taxol: biosynthesis, molecular genetics, and biotechnological applications.

Over the past decade, Taxol and its closely related structural analogue Taxotere have emerged as very important antitumor agents. Their widespread use in the treatment of a variety of cancer types, their likely approval for the treatment of additional forms of cancer, and their use at earlier stages of intervention will lead to increased demand for these drugs in the future. Because of yield considerations, Taxol and Taxotere are currently derived via semisynthesis from the advanced taxoid 10-deacetylbaccatin III, which must be isolated from yew (Taxus) trees. Thus, efforts are underway to produce Taxol (and other advanced taxoids for use in semisynthesis) by alternate, biotechnological means. This article provides a current overview of research on taxoid biosynthesis and an assessment of bioengineering applications for taxoid production in yew cell culture.

Biotechnology↗

Functional genomics and proteomics--the role of nuclear medicine.

Now that the sequencing of the human genome has been completed, the basic challenges are finding the genes, locating their coding regions and predicting their functions. This will result in a new understanding of human biology as well as in the design of new molecular structures as potential novel diagnostic or drug discovery targets. The assessment of gene function may be performed using the tools of the genome program. These tools represent high-throughput methods used to evaluate changes in the expression of many or all genes of an organism at the same time in order to investigate genetic pathways for normal development and disease. This will lead to a shift in the scientific paradigm: In the pre-proteomics era, functional assignments were derived from hypothesis-driven experiments designed to understand specific cellular processes. The new tools describe proteins on a proteome-wide scale, thereby creating a new way of doing cell research which results in the determination of three-dimensional protein structures and the description of protein networks. These descriptions may then be used for the design of new hypotheses and experiments in the traditional physiological, biochemical and pharmacological sense. The evaluation of genetically manipulated animals or newly designed biomolecules will require a thorough understanding of physiology, biochemistry and pharmacology and the experimental approaches will involve many new technologies, including in vivo imaging with single-photon emission tomography and positron emission tomography. Nuclear medicine procedures may be applied for the determination of gene function and regulation using established and new tracers or using in vivo reporter genes such as enzymes, receptors, antigens or transporters. Pharmacogenomics will identify new surrogate markers for therapy monitoring which may represent potential new tracers for imaging. Also, drug distribution studies for new therapeutic biomolecules are needed, at least during preclinical stages of drug development. Finally, new biomolecules will be developed by bioengineering methods which may be used for isotope-based diagnosis and treatment of disease.

Animals↗