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PubMed2000

Psychosocial risk factors for myocardial infarction among Swedish railway engine drivers [corrected].

BACKGROUND: Several earlier investigations have found an increased incidence of myocardial infarction among male drivers and, not least, among railway engine drivers. In a previous study we found that increased serum cholesterol, blood pressure or tobacco smoking did not explain this increased risk. OBJECTIVE: To investigate psychosocial factors and primarily work-related factors among male engine drivers. DESIGN: Engine drivers were compared with random population samples in a cross-sectional study. METHOD: Two thousand three hundred and eighteen engine drivers aged 25-59 years were compared with 331 randomly selected men who were mainly from the Göteborg MONICA population study. Job demands and job decision latitude as well as social support were the main factors compared. RESULTS: Job demands were reported as being significantly lower by both younger and older engine drivers compared to their referents (P = 0.002 and P = 0.0001, respectively). Decision latitude was reported as being lower by both younger and older engine drivers than among controls (P = 0.0001 for both groups). Engine drivers experienced deficient support from their superiors compared to the referents (P = 0.0001 for younger as well as older engine drivers). Low decision latitude was associated with significantly higher diastolic blood pressure (r = -0.11 and P = 0.0001), but otherwise there were no significant relationships between psychosocial work characteristics and somatic risk factors. CONCLUSIONS: The combination of low decision latitude and low social support seems to be a key factor in the increased risk of myocardial infarction among railway engine drivers. They generally undertake shift work which may be an additional risk factor for myocardial infarction.

Adult↗

PubMed2004

Runx2/Cbfa1-genetically engineered skeletal myoblasts mineralize collagen scaffolds in vitro.

Genetic engineering of progenitor and stem cells is an attractive approach to address cell sourcing limitations associated with tissue engineering applications. Bone tissue engineering represents a promising strategy to repair large bone defects, but has been limited in part by the availability of a sustained, mineralizing cell source. This study examined the in vitro mineralization potential of primary skeletal myoblasts genetically engineered to overexpress Runx2/Cbfa1, an osteoblastic transcriptional regulator essential to bone formation. These cells were viable at the periphery of 3D fibrous collagen scaffolds for 6 weeks of static culture. Exogenous Runx2 expression induced osteogenic differentiation and repressed myogenesis in these constructs relative to controls. Runx2-modified cells deposited significant amounts of mineralized matrix and hydroxyapatite, as determined by microcomputed tomography, histological analysis, and Fourier transform infrared spectroscopy, whereas scaffolds seeded with control cells exhibited no mineralized regions. Although mineralization by Runx2-engineered cells was confined to the periphery of the construct, colocalizing with cell viability, it was sufficient to increase the compressive modulus of constructs 30-fold relative to controls. This work demonstrates that Runx2 overexpression in skeletal myoblasts may address current obstacles of bone tissue engineering by providing a potent cell source for in vitro mineralization and construct maturation. Additionally, the use of genetic engineering methods to express downstream control factors and transcriptional regulators, in contrast to soluble signaling molecules, represents a robust strategy to enhance cellular activities for tissue engineering applications.

Animals↗

PubMed2003

Know-how and know-why in biochemical engineering.

This contribution analyzes the position of biochemical engineering in general and bioprocess engineering particularly in the force fields between fundamental science and applications, and between academia and industry. By using culture technology as an example, it can be shown that bioprocess engineering has moved slowly but steadily from an empirical art concerned with mainly know-how to a science elucidating the know-why of culture behavior. Highly powerful monitoring tools enable biochemical engineers to understand and explain quantitatively the activity of cellular culture on a metabolic basis. Among these monitoring tools are not just semi-online analyses of culture broth by HPLC, GC and FIA, but, increasingly, also noninvasive methods such as midrange IR, Raman and capacitance spectroscopy, as well as online calorimetry. The detailed and quantitative insight into the metabolome and the fluxome that bioprocess engineers are establishing offers an unprecedented opportunity for building bridges between molecular biology and engineering biosciences. Thus, one of the major tasks of biochemical engineering sciences is not developing new know-how for industrial applications, but elucidating the know-why in biochemical engineering by conducting research on the underlying scientific fundamentals.

Biochemistry↗

PubMed2006

Challenges in tissue engineering.

Almost 30 years have passed since a term 'tissue engineering' was created to represent a new concept that focuses on regeneration of neotissues from cells with the support of biomaterials and growth factors. This interdisciplinary engineering has attracted much attention as a new therapeutic means that may overcome the drawbacks involved in the current artificial organs and organ transplantation that have been also aiming at replacing lost or severely damaged tissues or organs. However, the tissues regenerated by this tissue engineering and widely applied to patients are still very limited, including skin, bone, cartilage, capillary and periodontal tissues. What are the reasons for such slow advances in clinical applications of tissue engineering? This article gives the brief overview on the current tissue engineering, covering the fundamentals and applications. The fundamentals of tissue engineering involve the cell sources, scaffolds for cell expansion and differentiation and carriers for growth factors. Animal and human trials are the major part of the applications. Based on these results, some critical problems to be resolved for the advances of tissue engineering are addressed from the engineering point of view, emphasizing the close collaboration between medical doctors and biomaterials scientists.

Animals↗

PubMedDate not supplied

Protecting the immunocompromised patient: the role of the hospital clinical engineer.

While the discipline of clinical engineering has long been limited to the area of medical equipment management, few areas in hospital engineering practice so closely meet the literal definition of "clinical" engineering as the care of the immunocompromised patient. Although ventilation has been the domain of the plant maintenance department, the increasing numbers of clinical engineers being given responsibility for plant functions, as well as the critical nature of the topic, make the care of the bone marrow transplant (BMT) patient an appropriate area of clinical engineering practice. Further, as clinical engineering branches out of the equipment management area, the clinical engineer can be truly termed the "hospital engineer".

Air Microbiology↗

PubMed1999

Lung function disturbances and chest X-ray abnormalities among marine engineers.

BACKGROUND: The purpose of the study was to investigate to what extent marine engineers who are exposed to mineral oil mist in the engine rooms of ships have lung function disturbances and chest X-ray abnormalities. METHODS: A previous study examined a cohort of 492 seamen from which we received 169 full-scale (40 x 40 cm) chest radiographs. Among these films were 68 from marine engineers and 101 from seamen who had never worked as engineers (controls). Spirometric data were obtained from 44 engineers and from 71 of the controls. The chest X-rays were classified independently by two certified B-readers, using the ILO classification. RESULTS: Only 7% of the seamen had parenchymal or pleural changes. The only statistically significant difference between the two groups for the chest X-ray findings was for all comments made by the ILO readers which were termed "any symbol" (9.3% for the engineers and 0.6% for the controls; RR = 14.6 (95% CI 1.1-75.5)) and the FEV% (FEV1/FVC x 100) for the spirometric values (82.8 (SD = 10.8) for the engineers and 86.6 (SD = 8.0) for the controls; P = 0.03). CONCLUSIONS: The results of the study indicate a slight lung function impairment and an increase in chest X-ray abnormalities, other than small opacities, among the marine engineers.

Adult↗

PubMed2001

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures.

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.

Animals↗

PubMed2007

Mechanisms of protein evolution and their application to protein engineering.

Protein engineering holds great promise for the development of new biosensors, diagnostics, therapeutics, and agents for bioremediation. Despite some remarkable successes in experimental and computational protein design, engineered proteins rarely achieve the efficiency or specificity of natural enzymes. Current protein design methods utilize evolutionary concepts, including mutation, recombination, and selection, but the inability to fully recapitulate the success of natural evolution suggests that some evolutionary principles have not been fully exploited. One aspect of protein engineering that has received little attention is how to select the most promising proteins to serve as templates, or scaffolds, for engineering. Two evolutionary concepts that could provide a rational basis for template selection are the conservation of catalytic mechanisms and functional promiscuity. Knowledge of the catalytic motifs responsible for conserved aspects of catalysis in mechanistically diverse superfamilies could be used to identify promising templates for protein engineering. Second, protein evolution often proceeds through promiscuous intermediates, suggesting that templates which are naturally promiscuous for a target reaction could enhance protein engineering strategies. This review explores these ideas and alternative hypotheses concerning protein evolution and engineering. Future research will determine if application of these principles will lead to a protein engineering methodology governed by predictable rules for designing efficient, novel catalysts.

Binding Sites↗

PubMed2006

A team-taught interdisciplinary approach to engineering ethics.

This paper outlines the development and implementation of a new course in Engineering Ethics at the University of Tennessee. This is a three-semester-hour course and is jointly taught by an engineering professor and a philosophy professor. While traditional pedagogical techniques such as case studies, position papers, and classroom discussions are used, additional activities such as developing a code of ethics and student-developed scenarios are employed to encourage critical thinking. Among the topics addressed in the course are engineering as a profession and its role in society; ethical successes and failures; risk, safety, and the environment; professional responsibilities; credit and intellectual property; and international concerns. The most significant aspect of the course is that it brings both engineering and non-engineering points of view to the topics at hand. This is accomplished in two ways. First, as mentioned previously, it is team-taught by engineering faculty with an interest in ethical and societal issues, and by philosophy faculty with expertise in the field of professional ethics and an interest in science and technology. Second, the course is offered to both engineers and non-engineers. This mix of students requires that all students must be able to explain their technical and ethical decisions in a non-technical manner. Work teams are structured to maximize interdisciplinary interaction and to foster insights by each student into the professional commitments and attitudes of others.

Curriculum↗

PubMed2006

Structural and nanoindentation studies of stem cell-based tissue-engineered bone.

Stem cell-based gene therapy and tissue engineering have been shown to be an efficient method for the regeneration of critical-sized bone defects. Despite being an area of active research over the last decade, no knowledge of the intrinsic ultrastructural and nanomechanical properties of such bone tissue exists. In this study, we report the nanomechanical properties of engineered bone tissue derived from genetically modified mesenchymal stem cells (MSCs) overexpressing the rhBMP2 gene, grown in vivo in the thigh muscle of immunocompetent mice for 4 weeks, compared to femoral bone adjacent to the transplantation site. The two types of bone had similar mineral contents (61 and 65 wt% for engineered and femoral bone, respectively), overall microstructures showing lacunae and canaliculi (both measured by back-scattered electron microscopy), chemical compositions (measured by energy dispersive X-ray analysis), and nanoscale topographical morphologies (measured by tapping-mode atomic force microscopy imaging or TMAFM). Nanoindentation experiments revealed that the small length scale mechanical properties were statistically different with the femoral bone (indented parallel to the bone long axis) being stiffer and harder (apparent elastic modulus, E approximately 27.3+/-10.5 GPa and hardness, H approximately 1.0+/-0.7G Pa) than the genetically engineered bone (E approximately 19.8+/-5.6 GPa, H approximately 0.9+/-0.4G Pa). TMAFM imaging showed clear residual indents characteristic of viscoelastic plastic deformation for both types of bone. However, fine differences in the residual indent area (smaller for the engineered bone), pile up (smaller for the engineered bone), and fracture mechanisms (microcracks for the engineered bone) were observed with the genetically engineered bone behaving more brittle than the femoral control.

Biomechanical Phenomena↗

PubMed2005

Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells.

OBJECTIVE: Prosthetic and bioprosthetic materials currently in use lack growth potential and therefore must be repeatedly replaced in pediatric patients as they grow. Tissue engineering is a new discipline that offers the potential for creating replacement structures from autologous cells and biodegradable polymer scaffolds. In May 2000, we initiated clinical application of tissue-engineered vascular grafts seeded with cultured cells. However, cell culturing is time-consuming, and xenoserum must be used. To overcome these disadvantages, we began to use bone marrow cells, readily available on the day of surgery, as a cell source. The aim of the study was to assess the safety and feasibility of this technique for creating vascular tissue under low-pressure systems such as pulmonary artery or venous pressure. METHODS: Since September 2001, tissue-engineered grafts seeded with autologous bone marrow cells have been implanted in 42 patients. The patients or their parents were fully informed and had given consent to the procedure. A 5-mL/kg specimen of bone marrow was aspirated with the patient under general anesthesia before the skin incision. The polymer tube serving as a scaffold for the cells was composed of a copolymer of l -lactide and -caprolactone (50:50). This copolymer is degraded by hydrolysis. The matrix is more than 80% porous, and the diameter of each pore is 20 to 100 microm. Polyglycolic acid woven fabric with a thickness of 0.5 mm was used for reinforcement. Twenty-three tissue-engineered conduits (grafts for extracardiac total cavopulmonary connection) and 19 tissue-engineered patches were used for the repair of congenital heart defects. The patients' ages ranged from 1 to 24 years (median 5.5 years). All patients underwent a catheterization study, computed tomographic scan, or both, for evaluation after the operation. The patients received anticoagulation therapy for 3 to 6 months after surgery. RESULTS: Mean follow-up after surgery was 490 +/- 276 days (1.3-31.6 months, median 16.7 months). There were no complications such as thrombosis, stenosis, or obstruction of the tissue-engineered autografts. One late death at 3 months after total cavopulmonary connection was noted in patient with hypoplastic left heart syndrome; this was unrelated to the tissue-engineered graft function. There was no evidence of aneurysm formation or calcification on cineangiography or computed tomography. All tube grafts were patent, and the diameter of the tube graft increased with time (110% +/- 7 % of the implanted size). CONCLUSION: Biodegradable conduits or patches seeded with autologous bone marrow cells showed normal function (good patency to a maximum follow-up of 32 months). As living tissues, these vascular structures may have the potential for growth, repair, and remodeling. The tissue-engineering approach may provide an important alternative to the use of prosthetic materials in the field of pediatric cardiovascular surgery. Longer follow-up is necessary to confirm the durability of this approach.

Adolescent↗

PubMed2003

Cardiac tissue engineering for replacement therapy.

Cell therapy is a new concept to repair diseased organs. For patients with myocardial infarction, heart failure, and congenital heart diseases cell based therapies might represent a potential cure. The field can be subdivided into two principally different approaches: (1) Implantation of isolated cells and (2) implantation of in vitro engineered tissue constructs. This review will focus on the latter approach. Cardiac tissue engineering comprises the fields of material sciences and cell biology. In general, scaffold materials such as gelatin, collagen, alginate, or synthetic polymers and cardiac cells are utilized to reconstitute tissue-like constructs in vitro. Ideally, these constructs display properties of native myocardium such as coherent contractions, low diastolic tension, and syncytial propagation of action potentials. To be applicable for surgical repair of diseased myocardium engineered tissue constructs should have the propensity to integrate and remain contractile in vivo. Size and mechanical properties of engineered constructs are critical for surgical repair of large tissue defects. Successful application of tissue engineering in men will depend on the utilization of an autologous or non-immunogeneic cell source and scaffold material to avoid life long immunosuppression. This review will give an overview of recent approaches in cardiac tissue engineering and its first applications in vivo. We will discuss materials and cell sources for cardiac tissue engineering. Further, principle obstacles will be addressed. Cardiac tissue engineering for replacement therapy has an intriguing perspective, but is in its early days. Its true value remains to be thoroughly evaluated.

Animals↗

PubMed2001

Bronchial mucosa produced by tissue engineering: a new tool to study cellular interactions in asthma.

BACKGROUND: The use of fiberoptic bronchial biopsies has improved our understanding of the immunopathology of asthma. However, this approach offers a limited ability to perform mechanistic studies observing cell-cell and cell-matrix interactions, which are a key issue in the study of airway remodeling. Tissue engineering is a technique that combines the use of biology and engineering expertise to generate a limitless amount of tissue from small samples. This technology allows for the study of cell interactions under conditions as close as possible to the natural environment. OBJECTIVE: The aim of this study was to evaluate the feasibility of an engineered human bronchial mucosa as a model to study cellular interactions in asthma. METHODS: Human bronchial fibroblasts from normal and asthmatic donors were incorporated into collagen gel. Bronchial epithelial cells were seeded over this gel and then cultured in an air-liquid interface in the presence or the absence of T lymphocytes. Biopsy specimens from these engineered mucosa were taken for structural and ultrastructural analysis, and T lymphocytes were harvested and used to localize IL-5. RESULTS: Histologic analysis showed that engineered mucosa with normal bronchial cells presented a pseudostratified ciliated epithelium with the presence of mucus secretory cells. The electron microscopy analysis confirmed these histologic results. These features were comparable with those observed in normal bronchial tissues. However, in engineered mucosa from asthmatic subjects, the tissue structure was disorganized, particularly the epithelial cell arrangement. The percentage of IL-5(+) lymphocytes was significantly (P =.03) higher in engineered bronchial mucosa from asthmatic subjects (87% +/- 2%) compared with mucosa from normal volunteers (2% +/- 0.3%). CONCLUSION: Using tissue engineering, we produced an in vitro model of bronchial mucosa from normal and asthmatic subjects. These models could be a valuable tool to better understand key mechanisms involved in inflammation and airway repair.

Asthma↗

PubMed2006

Tissue Engineering of the TMJ disc: a review.

The potential impact of a tissue-engineered temporomandibular joint (TMJ) disc is immense. Currently, patients suffering from a severely dysfunctional TMJ have few options. Facing the general lack of safe, effective TMJ disc implants, many patients undergo discectomy, a procedure that removes the injured TMJ disc in hopes of reducing debilitating symptoms associated with severe TMJ disorders. This procedure may not be ideal as the TMJ is left without an important functional component. Tissue engineering is a promising approach for the creation of viable, effective implants. The first attempt to investigate TMJ disc cells on a biomaterial was conducted in 1991. The first TMJ tissue-engineered constructs to be tested biochemically and biomechanically were formed in 1994; however, in examining this study in retrospect, it is clear how little TMJ knowledge was available at that time. Within the last 10 to 15 years, multiple studies have investigated critical TMJ disc characteristics, and while this characterization is not complete, these data have created a solid foundation for tissue-engineering research. Thus, the last 5 years have yielded core studies investigating the principal elements of tissue engineering: scaffold, cell source, and biological/biomechanical stimuli. Although TMJ disc tissue engineering is still in its formative years, its future is quite promising. Key studies are now being conducted that will assist in the establishment of a solid TMJ disc tissue-engineering approach. As the challenges of tissue engineering are faced and met, the ultimate goal of creating a functional biological implant nears.

Animals↗

PubMed2006

Methodological issues when comparing hearing thresholds of a group with population standards: the case of the ferry engineers.

OBJECTIVES: To discuss appropriate and inappropriate methods for comparing distributions of hearing thresholds of a study group with distributions in population standards and to determine whether the thresholds of Washington State Ferries engineers are different from those of men in the general population, using both frequency-by-frequency comparisons and analysis of audiometric shape. DESIGN: The most recent hearing conservation program audiograms of 321 noise-exposed engineers, ages 35 to 64, were compared with the predictions of Annexes A, B, and C from ANSI S3.44. There was no screening by history or otoscopy; all audiograms were included. 95% confidence intervals (95% CIs) were calculated for the engineers' median thresholds for each ear, for the better ear (defined two ways), and for the binaural average. For Annex B, where 95% CIs are also available, it was possible to calculate z scores for the differences between Annex B and the engineers' better ears. Bulge depth, an audiometric shape statistic, measured curvature between 1 and 6 kHz. RESULTS: Engineers' better-ear median thresholds were worse than those in Annex A but (except at 1 kHz) were as good as or better than those in Annexes B and C, which are more appropriate for comparison to an unscreened noise-exposed group like the engineers. Average bulge depth for the engineers was similar to that of the Annex B standard (no added occupational noise) and was much less than that of audiograms created by using the standard with added occupational noise between 90 and 100 dBA. CONCLUSIONS: Audiograms from groups that have been selected for a particular exposure, but, without regard to severity, can appropriately be compared with population standards, if certain pitfalls are avoided. For unscreened study groups with large age-sex subgroups, a simple method to assess statistical significance, taking into consideration uncertainties in both the study group and the comparison standard, is the calculation of z scores for the proportion of better-ear thresholds above the Annex B median. A less powerful method combines small age-sex subgroups after age correction. Small threshold differences, even if statistically significant, may not be due to genuine differences in hearing sensitivity between study group and standard. Audiometric shape analysis offers an independent dimension of comparison between the study group and audiograms predicted from the ANSI S3.44 standard, with and without occupational noise exposure. Important pitfalls in comparison to population standards include nonrandom selection of study groups, inappropriate choice of population standard, use of the right and left ear thresholds instead of the better-ear threshold for comparison to Annex B, and comparing means with medians. The thresholds of the engineers in this study were similar to published standards for an unscreened population.

Adult↗

PubMed2006

Cartilage tissue engineering: its potential and uses.

PURPOSE OF REVIEW: The prevalent nature of osteoarthritis, a cartilage degenerative disease that results in the erosion of joint surfaces and loss of mobility, underscores the importance of developing functional articular cartilage replacement. Recent research efforts have focused on tissue engineering as a promising approach for cartilage regeneration and repair. Tissue engineering is a multidisciplinary research area that incorporates both biological and engineering principles for the purpose of generating new, living tissues to replace the diseased/damaged tissue and restore tissue/organ function. This review surveys and highlights the current concepts and recent progress in cartilage tissue engineering, and discusses the challenges and potential of this rapidly advancing field of biomedical research. RECENT FINDINGS: Cartilage tissue engineering is critically dependent on selection of appropriate cells (differentiated or progenitor cells); fabrication and utilization of biocompatible and mechanically suitable scaffolds for cell delivery; stimulation with chondrogenically bioactive molecules introduced in the form of recombinant proteins or via gene transfer; and application of dynamic, mechanical loading regimens for conditioning of the engineered tissue constructs, including the design of specialized biomechanically active bioreactors. SUMMARY: Cell selection, scaffold design and biological stimulation remain the challenges of function tissue engineering. Successful regeneration or replacement of damaged or diseased cartilage will depend on future advances in our understanding of the biology of cartilage and stem cells and technological development in engineering.

Biocompatible Materials↗

PubMed2005

Tissue-engineered flexible ear-shaped cartilage.

BACKGROUND: Previous attempts to engineer human ear-shaped constructs mimicked human shape but lacked the flexibility and size of a human ear. Recently, the authors engineered flexible cartilage by incorporating a perichondrium-like layer into the construct. In this study, they used lyophilized swine perichondrium as a pseudoperichondrium, examined its ability to confer flexibility to tissue-engineered cartilage, and used it to engineer flexible cartilage in the shape and size of a human ear. METHODS: Auricular chondrocytes and perichondrium were isolated from swine. Chondrocytes were mixed with fibrin polymer and gelled to form 5 x 20-mm constructs. Constructs alone (control, n = 6) or constructs sandwiched between two layers of lyophilized swine perichondrium (experimental, n = 6) were implanted into athymic mice. Auricular chondrocytes in fibrin polymer and lyophilized perichondrium were also used to form a tri-layer, ear-shaped construct, which was implanted into an athymic rat and externally stented for 6 weeks (n = 1). At 12 weeks, constructs were analyzed with histology and gross mechanical testing. RESULTS: New cartilaginous tissue was engineered in both the experimental and control groups. In samples laminated with lyophilized swine perichondrium, the intimate integration of the laminate with the neocartilage closely resembled the histoarchitecture of the native swine ear. Experimental constructs had mechanical properties similar to those of the native swine ear, while control constructs fractured with similar testing. The engineered ear could not be fractured with gross mechanical testing, and its size, shape, and flexibility remained stable. CONCLUSIONS: This study demonstrates that it is possible to engineer a cartilage construct that resembles the human ear not only in shape but also in size and flexibility. This study also confirms that lamination is a reliable method to confer elastic-like flexibility to an engineered cartilage construct.

Animals↗

PubMed1995

Hydrophobicity engineering to increase solubility and stability of a recombinant protein from respiratory syncytial virus.

Site-directed mutagenesis has been employed to engineer the hydrophobic properties of a 101-amino-acid fragment from the human respiratory syncytial virus (RSV) major glycoprotein (G protein). When this protein was produced in Escherichia coli, more than 70% of the gene product was found as inclusion bodies, and the product recovered from the soluble fraction was severely degraded. Substitution of two cysteine residues for serine residues, did not significantly change the solubility or stability of the gene product. In contrast, a dramatic increase in both solubility and stability was achieved by multiple engineering of hydrophobic phenylalanine residues. As compared to the non-engineered protein, the fraction of soluble protein in vivo could be increased from 27% to 75%. Surprisingly, this effect was accompanied by a remarkable increase in stability. The in vitro solubility of the purified gene products was similarly increased approximately fivefold. Structural studies using circular dichroism suggest that the two engineered fragments have a distribution of secondary-structure elements similar to the non-engineered fragment. In addition, the two engineered G-protein variants were demonstrated to be at least in part antigenically authentic to the non-engineered gene product. These results demonstrate that engineering of hydrophobic residues can be used as a tool to increase the solubility and proteolytic stability of poorly soluble and labile proteins.

Amino Acid Sequence↗