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Results for “reverse engineering”

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PubMed2005

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

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

Absorbable Implants↗

PubMed2005

Schwann cell behavior after nerve repair by means of tissue-engineered muscle-vein combined guides.

Schwann cells play a critical role in peripheral nerve regeneration. When a non-nervous conduit is used to bridge a nerve defect, the conduit is soon colonized by a number of Schwann cells that make a pathway for regrowing axons. By using electron microscopy, immunohistochemistry, and reverse transcriptase-polymerase chain reaction analysis, we have investigated the behavior of migratory glial cells along a particular type of autologous tissue-engineered conduit made of a vein filled with fresh skeletal muscle, using the rat sciatic nerve model. With this particular type of autograft, our data show that many Schwann cells soon take up a close relationship with grafted muscle fibers, and especially with their basal lamina, which appears to serve as a migration pathway for them. The early and massive colonization of the conduit is sustained by both Schwann cell migration and proliferation, as demonstrated by PCNA immunostaining. Later, as they meet regenerating axons, Schwann cells become closely associated with them and eventually lose their connections with grafted muscle fibers because of the formation of perineurial envelopes. Because previous studies showed that alpha(2a-2b) NRG1 is overexpressed at early stages along the muscle-vein combined tubes, we have also investigated mRNA expression of its two receptors, erbB2 and erbB3. Both messengers are overexpressed, although with different time courses. Overall, our results provide some morphological and biochemical bases for explaining the effectiveness of fresh muscle-vein combined nerve guides and throw an interesting light on the possible role of alpha(2a-2b) NRG1 through the erbB2/erbB3 heterodimer receptor for nerve regeneration inside non-nervous conduits.

Animals↗

PubMed2002

In vitro control of human bone marrow stromal cells for bone tissue engineering.

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.

Alkaline Phosphatase↗

PubMed2002

Reverse micellar extraction for downstream processing of proteins/enzymes.

New developments in the area of downstream processing are, hopefully, to fulfill the promises of modern biotechnology. The traditional separation processes such as chromatography or electrophoresis can become prohibitively expensive unless the product is of high value. Hence, there is a need to develop efficient and cost-effective downstream processing methods. Reverse micellar extraction is one such potential and a promising liquid-liquid extraction technique, which has received immense attention for isolation and purification of proteins/enzymes in the recent times. This technique is easy to scale-up and offers continuous operation. This review, besides briefly considering important physico-chemical and biological aspects, highlights the engineering aspects including mass transfer, mathematical modeling, and technology development. It also discusses recent developments in reverse micellar extraction such as affinity based separations, enzymatic reactions in reverse micelles coupled with membrane processes, reverse micellar extraction in hollow fibers, etc. Special emphasis has been given to some recent applications of this technique.

Antigen-Antibody Reactions↗

PubMed1997

Masking of retroviral envelope functions by oligomerizing polypeptide adaptors.

We have constructed chimeric retroviral envelopes displaying N-terminal polypeptides that are known to form homotrimeric associations. The amphotropic receptor (RAM-1) binding domain from the trimeric surface (SU) glycoprotein of 4070A murine leukemia virus (MLV)-inhibited ecotropic receptor (Rec-1) mediated infection by the SU glycoprotein of Moloney MLV when grafted to its N-terminus. The block to Rec-1-mediated infection was reversed when the RAM-1 binding domain was cleaved from the vector particles using an engineered factor Xa protease-sensitive cleavage signal between the envelope glycoprotein and its N-terminal extension. Trimeric leucine zipper peptides and the trimeric C-terminal domain of CD40 ligand were shown to inhibit RAM-1-mediated infection of NIH3T3 cells by the 4070A envelope when fused to its N-terminus, whereas monomeric helical peptides and the monomeric epidermal growth factor domain did not. The block to RAM-1-mediated infection was reversed when the trimeric polypeptides were cleaved from the vector particles by addition of factor Xa protease. Envelope binding assays using cleaved and uncleaved chimeric 4070A envelopes revealed that binding to RAM-1 receptors on mammalian cells was hindered by trimeric, but not by monomeric, N-terminal polypeptides. These results have important implications for the design of protease-activatable vectors for targeted gene delivery.

3T3 Cells↗

PubMed2006

Combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering.

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.

Aggrecans↗

PubMed2006

In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.

Our purpose was to characterize human dental pulp cells isolated by various methods and to examine the behavior of cells grown under various conditions for the purpose of pulp/dentin tissue engineering and regeneration. We compared the growth of human pulp cells isolated by either enzyme digestion or the outgrowth method. Expression of dentin sialophosphoprotein, Cbfa1, and two types of collagen (I and III) in these cells was examined by Western blot or reverse transcription/polymerase chain reaction. Growth of pulp cells on dentin and in collagen gel was also characterized. We found that different isolation methods give rise to different populations or lineages of pulp cells during in vitro passage based on their collagen gene expression patterns. Cells isolated by enzymedigestion had a higher proliferation rate than those isolated by outgrowth. Pulp cells did not proliferate or grew minimally on chemically and mechanically treated dentin surface and appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule as revealed by scanning electron microscopy. The contraction of the collagen matrix caused by pulp cells was dramatic: down to 34% on day 14. Our data indicate that (1) the choice of the pulp cell isolation method may affect the distribution of the obtained cell populations, (2) a treated dentin surface might still promote odontoblast differentiation, and (3) a collagen matrix may not be a suitable scaffold for pulp tissue regeneration because of the marked contraction caused by pulp cells in the matrix. The present study thus provides important information and a basis for further investigations pre-requisite to establishing pulp tissue engineering/regeneration protocols.

Base Sequence↗

PubMed2002

Understanding the human condition: experimental strategies in mammalian genetics.

Mice have become the mammalian model of choice for the application of genetics in biomedical research due to the evolutionary conservation of physiological systems and their attendant pathologies among all mammals as well as the exceptional power of genetic research technologies in the species. Beginning from aberrant phenotypes, a large number of mouse mutants and natural polymorphisms have been cloned, providing much information about the molecular basis of physiological processes. Additionally, the variable expression of these mutations in different inbred strain backgrounds has demonstrated the importance of modifier genes, which are also susceptible to cloning. Research efforts are keeping pace with these developments. In the area of gene discovery, large, government-funded mutagenesis programs now exist, and as a matter of great practical importance, recent evidence suggests that the same genes may be involved in the natural polymorphisms affecting disease in mice and humans. In parallel, dramatic advances are also being made in our ability to measure physiological processes in mice, and the advent of expression profiling promises revolutionary advances in understanding phenotype at the molecular level. Gene-driven approaches have relied on engineering the mouse genome, including adding, subtracting, and replacing genes and, most recently, the ability to control gene activity reversibly. Together, these multiple advances in our technical abilities have created extraordinary opportunities for future discovery.

Animals↗

PubMed2003

In vitro chondrogenesis of bone marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel.

Mesenchymal stem cells (MSCs) from skeletally mature goats were encapsulated in a photopolymerizing poly(ethylene glycol)-based hydrogel and cultured with or without transforming growth factor beta1 (TGF) to study the potential for chondrogenesis in a hydrogel scaffold system amenable to minimally invasive implantation. Chondrogenic differentiation was evaluated by histological, biochemical, and RNA analyses for the expression of cartilage extracellular matrix components. The two control groups studied were MSCs cultured in monolayer and MSCs encapsulated in the hydrogel and cultured for 6 weeks in chondrogenic medium without TGF-beta1 (6wk-TGF). The three experimental time points for encapsulated cells studied were 0 days (0d), 3 weeks, and 6 weeks in chondrogenic medium with TGF-beta1 at 10 ng/ml (3wk+TGF and 6wk+TGF). MSCs proliferated in the hydrogels with TGF-beta1. Glycosaminoglycan (GAG) and total collagen content of the hydrogels increased to 3.5% dry weight and 5.0% dry weight, respectively, in 6wk+TGF constructs. Immunohistochemistry revealed the presence of aggrecan, link protein, and type II collagen. Upregulation of aggrecan and type II collagen gene expression compared with monolayer MSCs was demonstrated. Type I collagen gene expression decreased from 3 to 6 weeks in the presence of TGF-beta1. 6wk-TGF hydrogels produced no GAG and only moderate amounts of collagen. However, immunohistochemistry and RT-PCR demonstrated a small amount of spontaneous differentiation in this control group. This study demonstrates the ability to encapsulate MSCs to form cartilage-like tissue in vitro in a photopolymerizing hydrogel. This system may be useful for minimally invasive implantation, MSC differentiation, and engineering of composite tissue structures with multiple cellular phenotypes.

Animals↗

PubMed2004

Gas-inducible transgene expression in mammalian cells and mice.

We describe the design and detailed characterization of a gas-inducible transgene control system functional in different mammalian cells, mice and prototype biopharmaceutical manufacturing. The acetaldehyde-inducible AlcR-P(alcA) transactivator-promoter interaction of the Aspergillus nidulans ethanol-catabolizing regulon was engineered for gas-adjustable transgene expression in mammalian cells. Fungal AlcR retained its transactivation characteristics in a variety of mammalian cell lines and reversibly adjusted transgene transcription from chimeric mammalian promoters (P(AIR)) containing P(alcA)-derived operators in a gaseous acetaldehyde-dependent manner. Mice implanted with microencapsulated cells engineered for acetaldehyde-inducible regulation (AIR) of the human glycoprotein secreted placental alkaline phosphatase showed adjustable serum phosphatase levels after exposure to different gaseous acetaldehyde concentrations. AIR-controlled interferon-beta production in transgenic CHO-K1-derived serum-free suspension cultures could be modulated by fine-tuning inflow and outflow of acetaldehyde-containing gas during standard bioreactor operation. AIR technology could serve as a tool for therapeutic transgene dosing as well as biopharmaceutical manufacturing.

Acetaldehyde↗

PubMed2005

Effects of transforming growth factor beta1 (TGFbeta-1) and dentin non-collagenous proteins (DNCP) on human embryonic ectomesenchymal cells in a three-dimensional culture system.

Cranial neural crest-derived ectomesenchymal cells represent a population of pluripotent stem cells giving rise to many of the various oro-facial and dental tissues. The factors determining the terminal fate of these cells are still unclear. The potentiality of human embryonic ectomesenchymal cells from the first branchial arch have been investigated when isolated and grown in a three-dimensional (3D)-collagen gel culture system in the presence of dentin matrix-derived non-collagenous proteins (DNCP) and TGFbeta-1. Functional differentiation of cells showing some characteristics of odontoblast-like cells could be observed when the cells were cultured with DNCP+TGFbeta-1 or DNCP, however, only cytological differentiation was observed during culture with TGFbeta-1 alone. The characteristics of these cells was assessed by morphological appearance, expression of the odontoblast phenotype marker dentin sialophosphoprotein (DSPP), increased alkaline phosphatase levels and formation of mineralised nodules in vitro. The results indicate that these embryonic cells from the first branchial arch are capable of responding to the inductive stimulus of DNCP or DNCP+TGFbeta-1 when isolated and grown in the 3D collagen gel culture system. The capacity of the isolated cells to differentiate into mineralizing cells showing some characteristics of odontoblast-like cells under these growth conditions highlights the potential of such approaches for tissue engineering strategies for hard-tissue regeneration after injury.

Alkaline Phosphatase↗

PubMed2002

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation.

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.

Animals↗

PubMed1998

Protein-protein interactions as a means of purification

Hetero-association-based separations are characterized by great specificity and large protein-protein interaction energies and are well suited to application early in separation trains. Research efforts involving reverse micellar extraction and affinity chromatography processes, particularly with respect to affinity ligand engineering and processing, are improving selectivity and decreasing process, but not molecular, complexity. Self-association-based separations are less specific because the underlying interaction energies are smaller; they are prone to interference from contaminants. Efforts in precipitation processes are improving our understanding of protein solubility behavior. In spite of recent progress, the full ultrapurification potentials of bulk crystallization and self-interaction chromatography processes remain unrealized.

Journal Article↗

PubMed2002

Design, production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins.

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.

Avidin↗

PubMed1996

Insulin-secreting non-islet cells are resistant to autoimmune destruction.

Transgenic nonobese diabetic mice were created in which insulin expression was targeted to proopiomelanocortin-expressing pituitary cells. Proopiomelanocortin-expressing intermediate lobe pituitary cells efficiently secrete fully processed, mature insulin via a regulated secretory pathway, similar to islet beta cells. However, in contrast to the insulin-producing islet beta cells, the insulin-producing intermediate lobe pituitaries are not targeted or destroyed by cells of the immune system. Transplantation of the transgenic intermediate lobe tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic symptoms. The absence of autoimmunity in intermediate lobe pituitary cells engineered to secrete bona fide insulin raises the potential of these cell types for beta-cell replacement therapy for the treatment of insulin-dependent diabetes mellitus.

Animals↗

PubMed2006

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Animals↗

PubMed1998

Conformational correction mechanisms aiding antigen recognition by a humanized antibody.

The crystal structure of the complex between hen egg lysozyme and the Fv fragment of a humanized antilysozyme antibody was determined to 2.7-A resolution. The structure of the antigen combining site in the complex is nearly identical to that of the complexed form of the parent mouse antibody, D1.3. In contrast, the combining sites of the unliganded mouse and humanized antilysozymes show moderate conformational differences. This disparity suggests that a conformational readjustment process linked to antigen binding reverses adverse conformations in the complementarity determining regions that had been introduced by engineering these segments next to human framework regions in the humanized antibody.

Animals↗

PubMed2005

Dual promoter-controlled oncolytic adenovirus CG5757 has strong tumor selectivity and significant antitumor efficacy in preclinical models.

PURPOSE: Transcriptionally controlled oncolytic adenovirus CG5757 is engineered with two tumor-specific promoters from E2F-1 and human telomerase reverse transcriptase genes. This virus has broad anticancer spectrum and higher specificity. The objective of the current study is to show its antitumor selectivity and therapeutic potential. EXPERIMENTAL DESIGN: The antitumor specificity of E2F-1 and human telomerase reverse transcriptase promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and cytotoxicity assays followed by a newly developed ex vivo tumor culture assay. Subsequently, in vivo antitumor efficacy and toxicology studies were carried out to assess the therapeutic potential of this oncolytic agent. RESULTS: In a broad panel of cells, E2F-1 and human telomerase reverse transcriptase promoters were activated in a tumor-selective manner. Under the control of these promoters, expression of E1a and E1b genes appears only in tumor cells. This specificity is extended to viral replication and hence the cytotoxicity in a broad range of cancer cells. Furthermore, CG5757 only replicates in cancer tissues but not in normal tissues that are derived from clinical biopsies. The safety profile was further confirmed in in vivo toxicology studies, and strong efficacy was documented in several tumor xenograft models after CG5757 was given via different routes and regimens. CONCLUSIONS: CG5757 has strong antitumor selectivity and potency. It has low toxicity and has great potential as a therapeutic agent for different types of cancers.

Adenoviridae↗