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PubMed2004

Tracheal reconstruction using tissue-engineered cartilage.

OBJECTIVES: To determine whether rabbit cartilage can be tissue engineered using a polyglycolic acid (PGA) construct composed of PGA mesh, autologous chondrocytes, and alginate covalently linked with the cell adhesion sequence arginine-glycine-aspartic acid (RGD), and to investigate the feasibility of reconstructing tracheal defects using the PGA construct in conjunction with a bioabsorbable intratracheal stent. METHODS: Nineteen New Zealand White rabbits were used. Nine rabbits underwent subcutaneous implantation of 3 different PGA construct combinations: (1) PGA, autologous chondrocytes, and RGD-modified alginate; (2) PGA, autologous chondrocytes, and unmodified alginate; and (3) PGA and RGD-modified alginate. The remaining 10 animals underwent anterior tracheal reconstruction using fascia lata grafts and the complete PGA construct (PGA, autologous chondrocytes, and RGD-modified alginate). At the time of tracheal reconstruction, a poly-l-lactic acid intratracheal stent was placed in 5 of these latter animals. Rates of tracheal stenosis and mortality were compared with those of historical control animals. Histologic analysis was performed on the PGA constructs. RESULTS: In the subcutaneous implants, the PGA constructs made with chondrocytes (with and without RGD) demonstrated mature cartilage formation in 7 (78%) of the 9 animals. No cartilage was seen in PGA constructs made without chondrocytes. Two of the 10 animals that underwent tracheal reconstruction with the complete PGA construct survived to 20 weeks and demonstrated patent airways, 1 with a stent and 1 without a stent (80% overall mortality). Histologic analysis showed mature cartilage formation at the tracheal reconstruction site. Historical control animals that underwent reconstruction with fascia lata alone demonstrated the lowest overall mortality. CONCLUSIONS: Cartilage can be tissue engineered in rabbits using PGA mesh embedded with alginate-encapsulated autologous chondrocytes. It is also possible to reconstruct tracheal defects with this method of cartilage engineering, although the mortality rate in this study is high.

Alginates↗
PubMed2004

Tissue-engineered cartilage as a graft source for laryngotracheal reconstruction: a pig model.

OBJECTIVE: To evaluate the feasibility of using tissue-engineered cartilage for laryngotracheal reconstruction in the pig model. DESIGN: Auricular cartilage was harvested from 3 young swine. The cartilage was digested, processed, and suspended and a cell culture was obtained. The cells were then suspended in 3 mL of a 30% solution of a biodegradable polymer (Pluronic F-127) (polyethylene oxide/polypropylene oxide copolymer) at a cellular concentration of 50 x 10(6) cells/mL. This suspension was then implanted subcutaneously into each pig's dorsum. Eight weeks after implantation, the cartilage was harvested with the surrounding perichondrial capsule. An anterior cartilage graft laryngotracheal reconstruction was performed. Bronchoscopy was performed at 3 postoperative weeks to demonstrate airway patency. The animals were killed at 3 months, and specimens were obtained for histological analysis. SETTING: An animal research facility. SUBJECTS: Three young Yorkshire swine. RESULTS: All 3 pigs survived to the 3-month postoperative interval with no evidence of stridor or airway distress. Interval bronchoscopy revealed a normal patent airway with a mucosalized graft. Histopathologic analysis revealed incorporation of the tissue-engineered cartilage graft in the cricoid area, which correlated with results of bronchoscopic evaluation. CONCLUSION: Tissue-engineered auricular cartilage served as a viable graft in the pig model and might be an alternative cartilage source for laryngotracheal reconstruction.

Animals↗
PubMed1997

Proportionate mortality among unionized construction operating engineers.

This report presents the results of proportionate mortality ratios (PMR) and proportionate cancer mortality ratios (PCMR) among 15,843 members of the International Union of Operating Engineers who had died between 1988-1993. Operating engineers represent one of the 15 unions in the Building and Construction Trades Department and are responsible for the operation and maintenance of heavy earthmoving equipment used in the construction of buildings, bridges, roads, and other facilities. Using U.S. proportionate cancer mortality as the referent, statistically significant elevated mortality was observed for cancers of the lung (PCMR = 1.14, 95% confidence interval (CI) = 1.09-1.19) and bone (PCMR = 2.14, CI = 1.19-3.52). Using U.S. proportionate mortality as the referent, statistically significant elevated mortality was observed for other benign and unspecified neoplasms (PMR = 1.54, CI = 1.09-2.13), emphysema (PMR = 1.37, CI = 1.20-1.55), other injuries (PMR = 1.43, CI = 1.20-1.70) (which included crushing under/in machinery, tractor rollover, run over by crane), and suicide (PMR = 1.22, CI = 1.06-1.40). The PMR for leukemia, and aleukemia (PMR = 1.19, CI = 1.02-1.37), but not the PCMR (1.07, CI = 0.92-1.24), was also significantly elevated. Some of the occupational exposures that may have contributed to these excesses include diesel exhaust, asphalt and welding fumes, silica dust, ionizing radiation, and coal tar pitch. The present study underscores the need to control airborne exposures to these substances and for injury prevention efforts aimed at operating engineers in the construction industry.

Accidents, Occupational↗
PubMedDate not supplied

How will bioinformatics influence metabolic engineering?

Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development.

Computational Biology↗
PubMed1998

Principles of tissue engineering applied to programmable osteogenesis.

This article presents a strategy for design, engineering, and fabrication of a bioresorbable, manufactured bone graft substitute (BGS) device. The approach is based on established precepts of osteogenesis, molecular biology of hyaluronic acid and osteoinductive proteins, and theoretical preformance criteria for such a device collated from the literature of 1991 to 1996. Application of this design and engineering strategy results in a composite device consisting of a D,D-L,L-polylactic acid macrostructure optimized to the architecture of cancellous bone, a microstructure composed of a filamentous velour of hyaluronan and a recombinant human bone morphogenetic protein 2 (rhBMP-2). The performance of this construct was tested in vivo in the dog, intertransverse process, spinal fusion model and in a critical sized defect of the rabbit radius. Data from these studies are used to illustrate principle points of the design and engineering concept.

Animals↗
PubMedDate not supplied

Adipose tissue engineering: the future of breast and soft tissue reconstruction following tumor resection.

Reconstructive surgeons have always been at the forefront of medical technology. The history of reconstructive surgery began with ablative surgery, which was followed by tissue and organ transplantation, leading to contemporary tissue reconstruction. The field of reconstructive surgery is poised at the next stage of its evolution, namely tissue regeneration. The field of tissue engineering has largely defined this evolutionary leap. One active area of investigation is the development of tissue engineering strategies for adipose tissue. Bioengineers, life scientists, and reconstructive surgeons are synergistically coupling expertise in areas such as cell culture technology, tissue transfer, cell differentiation, angiogenesis, computer modeling, and polymer chemistry to regenerate adipose tissue de novo for breast replacement and soft-tissue augmentation following tumor resection. This work presents the current state of the art in adipose tissue engineering, as well the clinically translatable strategies currently under development. Semin. Surg. Oncol. 19:302-311, 2000.

Adipose Tissue↗
PubMed1991

Catalytic antibodies: contributions from engineering and expression in Escherichia coli.

Antibodies have been raised against the transition state of many reactions and shown to catalyse the relevant reaction. Their moderate catalytic efficiencies can be increased by protein engineering, if ways can be found to express the engineered antibody. We have developed a system by which fully functional Fv and Fab fragments can be expressed in Escherichia coli. The Fv fragment dissociates at low concentrations; we therefore devised methods to stabilize the fragment. We showed that the Fv fragment of the antibody McPC603, a phosphorylcholine-binding immunoglobulin A, binds the antigen with the same affinity as does the intact antibody isolated from mouse ascites. Phosphorylcholine is an analogue of the transition state for the hydrolysis of choline carboxylate ester. The Fv fragment of McPC603 catalysed this hydrolysis. Mutational analysis of the residues in the binding site of the antibody has shown which are essential for binding and for catalysis, and the importance of charged residues in certain positions. The E. coli expression system combined with protein engineering and screening methods will facilitate understanding of enzyme catalysis and the development of new catalytic antibodies.

Antibodies↗
PubMed2001

Injectable gels for tissue engineering.

Recently, tissue engineering approaches using injectable, in situ gel forming systems have been reported. In this review, the gelation processes and several injectable systems that exhibit in situ gel formation at physiological conditions are discussed. Applications of selected injectable systems (alginate, chitosan, hyaluronan, polyethylene oxide/polypropylene oxide) in tissue engineering are also described. Injectable polymer formulation can gel in vivo in response to temperature change (thermal gelation), pH change, ionic cross-linking, or solvent exchange. Kinetics of gelation is directly affected by its mechanism. Injectable formulations offer specific advantages over preformed scaffolds such as: possibility of a minimally invasive implantation, an ability to fill a desired shape, and easy incorporation of various therapeutic agents. Several factors need to be considered before an injectable gel can be selected as a candidate for tissue engineering applications. Apart from tissue-specific cell-matrix interactions, the following gel properties need to be considered: gelation kinetics, matrix resorption rate, possible toxicity of degradation products and their elimination routes, and finally possible interference of the gel matrix with histogenesis.

Alginates↗
PubMed2005

Suicidal genetically engineered microorganisms for bioremediation: need and perspectives.

In the past few decades, increased awareness of environmental pollution has led to the exploitation of microbial metabolic potential in the construction of several genetically engineered microorganisms (GEMs) for bioremediation purposes. At the same time, environmental concerns and regulatory constraints have limited the in situ application of GEMs, the ultimate objective behind their development. In order to address the anticipated risks due to the uncontrolled survival/dispersal of GEMs or recombinant plasmids into the environment, some attempts have been made to construct systems that would contain the released organisms. This article discusses the designing of safer genetically engineered organisms for environmental release with specific emphasis on the use of bacterial plasmid addiction systems to limit their survival thus minimizing the anticipated risk. We also conceptualize a novel strategy to construct "Suicidal Genetically Engineered Microorganisms (SGEMs)" by exploring/combining the knowledge of different plasmid addiction systems (such as antisense RNA-regulated plasmid addiction, proteic plasmid addiction etc.) and inducible degradative operons of bacteria.

Bacteria↗
PubMed2003

Surface engineering of living myoblasts via selective periodate oxidation.

Cell surface molecules are vital for normal cell activity. To study the functions of these molecules or manipulate cell behavior, the ability to decorate cell surfaces with bioactive molecules of our choosing is a potentially powerful technique. Here, we describe the molecular engineering of living L6 myoblast monolayers via selective periodate oxidation of sialic acid residues and the application of this surface modification in the artificial aggregation of cells. The aldehyde groups generated by this reaction were used to selectively ligate a model molecule, biotin hydrazide, to the cell surfaces. Flow cytometry analysis after staining with fluorescently conjugated avidin revealed a concentration-dependent increase in fluorescence compared to untreated cells with a maximal shift of 345.1 +/- 27.4-fold and an EC(50) of 17.4 +/- 1.1 microM. This mild oxidation reaction did not affect cell number, viability, or morphology. We then compared this chemical technique with the metabolic incorporation of reactive cell surface ketone groups using N-levulinoylmannosamine (ManLev). In this cell line, only a 22.3-fold fluorescence shift was observed compared to untreated cells when myoblasts were incubated with a high concentration of ManLev for 48 hours. Periodate oxidation was then used to modify myoblast surfaces to induce cell aggregation. Crosslinking biotinylated myoblasts, which do not spontaneously aggregate in culture, with avidin resulted in the rapid formation of millimeter-sized, multicellular structures. These data indicate that sodium periodate treatment is an effective, noncytotoxic method for the in vitro molecular engineering of living cell surfaces with the potential for cell biology and tissue engineering applications.

Aldehydes↗
PubMed2003

A perspective of metabolic engineering strategies: moving up the systems hierarchy.

Metabolic engineering has been established as an important field in biotechnology. It involves the analysis, design, and alteration of the stoichiometric network using sophisticated mathematical and molecular biology techniques. It allows for improvement of pathway kinetics by removing flux bottlenecks, balancing precursors, and recycling cofactors used to increase product formation. The next step in the systems hierarchy is the constructive manipulation of regulatory networks. As our understanding of regulation continues to expand rapidly, engineering of intracellular regulation will become an integral aspect of metabolic engineering.

Cloning, Molecular↗
PubMed2004

Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli.

L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)). Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc).

Directed Molecular Evolution↗
PubMed2005

Laser-guided direct writing for three-dimensional tissue engineering.

One of the principal limitations to the size of an engineered tissue is oxygen and nutrient transport. Lacking a vascular bed, cells embedded in an engineered tissue will consume all available oxygen within hours while out branching blood vessels will take days to vascularize the implanted tissue. One possible solution is to directly write vascular structures within the engineered tissue prior to implantation, reconstructing the tissue according to its native architecture. The cell patterning technique, laser-guided direct writing (LGDW), can pattern multiple cells types with micrometer resolution on arbitrary surfaces, including biological gels. Here we show that LGDW can pattern human umbilical vein endothelial cells (HUVEC) in two- and three-dimensions with micrometer accuracy. By patterning HUVEC on Matrigel, we can direct their self-assembly into vascular structures along the desired pattern. Finally, co-culturing the vascular structures with hepatocytes resulted in an aggregated tubular structure similar in organization to a hepatic sinusoid. This capability can facilitate studies of tissue architecture at the single cell level, and of heterotypic interactions underlying processes such as liver and pancreas morphogenesis, differentiation, and angiogenesis.

Animals↗
PubMed2006

Tissue engineering of blood vessels.

BACKGROUND: Tissue engineering techniques have been employed successfully in the management of wounds, burns and cartilage repair. Current prosthetic alternatives to autologous vascular bypass grafts remain poor in terms of patency and infection risk. Growing biological blood vessels has been proposed as an alternative. METHODS: This review is based on a literature search using Medline, PubMed, ISIS and CAS of original articles and reviews, and unpublished material and abstracts. RESULTS AND CONCLUSIONS: Complete incorporation into host tissues and the maintenance of a viable and self-renewing endothelial layer are the fundamental goals to be achieved when developing a tissue-engineered blood vessel. Sourcing of cells and modulating their interaction with extracellular matrix and supporting scaffold have been the focus of intense research. Although the use of tissue-engineered blood vessels in humans is so far limited, advances in our knowledge of stem cell precursors and the development of new biomaterials should enable this technology to reach routine clinical practice within a decade.

Biotechnology↗
PubMed2002

Presentation of native TROP-2 tumor antigens to human cytotoxic T lymphocytes by engineered antigen-presenting cells.

Professional antigen-presenting cells (APC), e.g. dendritic cells, express immuno-proteasome components and process proteins for MHC presentation differently from non-immune cells. Thus, they induce reactivities against sets of peptides that do not overlap with those generated by non-professional APC, i.e., tumor cells, and stimulate cytotoxic T lymphocytes (CTL) that may not recognize them. The goal of this work was to establish a system for antigen presentation and in vitro stimulation of human CTL using "tumor-cell-like" engineered APC. Murine fibroblasts were transfected with human HLA Class I alleles, together with the B7.1, ICAM-1 and germ-line TROP2 genes. The last encodes a cell surface glycoprotein widely expressed by human cancers. Unseparated peripheral blood mononuclear cells from HLA Class I-matched individuals were stimulated in vitro by the engineered APC. These efficiently induced the activation and proliferation of antigen-specific HLA-restricted CTL lines and clones. The Trop-2-specific CTL demonstrated high specific cytotoxicity against the appropriate transfected target cells. They also efficiently lysed MCF-7 human tumor cells expressing endogenous HLA-A2.1, Trop-2 together with ICAM-1. These results demonstrate that Trop-2 is a target molecule recognized by human CTL. Moreover, they demonstrate that non-immune engineered APC efficiently process and present native tumor-specific proteins in the context of human MHC Class I, and stimulate the growth and cytotoxicity of specific anti-tumor CTL.

Animals↗
PubMed2002

Application of magnetic resonance microscopy to tissue engineering: a polylactide model.

Absorbable polymers are unique materials that find application as temporary scaffolds in tissue engineering. They are often extremely sensitive to histological processing and, for this reason, studying fragile, tissue-engineered constructs before implantation can be quite difficult. This research investigates the use of noninvasive imaging using magnetic resonance microscopy (MRM) as a tool to enhance the assessment of these cellular constructs. A series of cellular, polylactide constructs was developed and analyzed using a battery of tests, including MRM. Distribution of rat aortic smooth muscle cells within the scaffolds was compared as one example of a tissue engineering MRM application. Cells were loaded in varying amounts using static and dynamic methods. It was found that the cellular component was readily identified and the polymer microstructure readily assessed. Specifically, the MRM results showed a heterogeneous distribution of cells due to static loading and a homogenous distribution associated with dynamic loading, results that were not visible through biochemical tests, scanning electron microscopy, or histological evaluation independently. MRM also allowed differentiation between different levels of cellular loading. The current state of MRM is such that it is extremely useful in the refinement of polymer processing and cell seeding methods. This method has the potential, with technological advances, to be of future use in the characterization of cell-polymer interactions.

Absorbable Implants↗
PubMed2001

New pulsatile bioreactor for fabrication of tissue-engineered patches.

To date, one approach to tissue engineering has been to develop in vitro conditions to ultimately fabricate functional cardiovascular structures prior to final implantation. In our current experiment, we developed a new pulsatile flow system that provides biochemical and biomechanical signals to regulate autologous patch-tissue development in vitro. The newly developed patch bioreactor is made of Plexiglas and is completely transparent (Mediport Kardiotechnik, Berlin). The bioreactor is connected to an air-driven respirator pump, and the cell culture medium continuously circulates through a closed-loop system. We thus developed a closed-loop, perfused bioreactor for long-term patch-tissue conditioning, which combines continuous, pulsatile perfusion and mechanical stimulation by periodically stretching the tissue-engineered patch constructs. By adjusting the stroke volume, the stroke rate, and the inspiration/expiration time of the ventilator, it allows various pulsatile flows and different levels of pressure. The whole system is a highly isolated cell culture setting, which provides a high level of sterility, gas supply, and fits into a standard humidified incubator. The bioreactor can be sterilized by ethylene oxide and assembled with a standard screwdriver. Our newly developed bioreactor provides optimal biomechanical and biodynamical stimuli for controlled tissue development and in vitro conditioning of an autologous tissue-engineered patch.

Biocompatible Materials↗
PubMed2002

Matrix engineering for osteogenic differentiation of rabbit periosteal cells using alpha-tricalcium phosphate particles in a three-dimensional fibrin culture.

Tissue engineering using periosteal cells is a promising approach for bioactive bone repair. Of central importance in tissue engineering is the cell-matrix interaction. In the present study we tested in vitro the influence of alpha-tricalcium phosphate (alpha-TCP) particles on the expression of osteogenic markers in rabbit periosteal cells embedded in specially manufactured fibrin beads. After cell isolation from tibial periosteum of New Zealand White rabbits, and following monolayer culture, cells were embedded in alginate-fibrin beads containing 7.5% alpha-TCP particles and, as a control group, in beads without particles. The alginate was extracted immediately after polymerization. The beads were cultivated for at least 53 days. The DNA content, alkaline phosphatase activity, and osteocalcin level were determined. In monolayer culture the number of cells increased 6.5-fold. DNA content increased in both three-dimensional culture groups but was significantly higher in the beads containing alpha-TCP. Alkaline phosphatase activity increased in both groups without significant differences. Osteocalcin content was significantly higher in the beads containing alpha-TCP than it was in those without alpha-TCP. These observations indicate that matrix engineering using inorganic particles in fibrin culture can influence the osteogenic differentiation of mesenchymal cells. The three-dimensional culture system presented here facilitates the preparation of grafts for bone reconstruction.

Alkaline Phosphatase↗