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At least 577 records · Page 32Linked to original sources
PubMed2006

Do human osteoblasts grow into open-porous titanium?

A titanium foam for spine fusion and other applications was tested by cell culture. Its high porosity and surface roughness should enable bone cells to grow through it, resulting in a better fixation of the vertebral body. The foam was tested by in vitro experiments with human osteoblasts under static culture conditions and in a perfused system. By means of cell number, viability, scanning electron microscopy and histological staining, cell proliferation could be observed. The expression of osteogenic genes like collagen-I, alkaline phosphatase and osteocalcin was proven by reverse transcription polymerase chain reaction (RT-PCR) as well as in the case of alkaline phosphatase with biochemical methods. The conducted experiments showed that human osteoblasts could grow through the interconnected porosity of the metal foam and that they expressed an osteoblast like phenotype. The results suggest that in vivo osteoblasts are likely to form a trabecular bone bridge through this titanium foam. Consequently, with this osteoconductive material, there may be a reduced need for autologous bone in spinal fusion procedures.

Alkaline Phosphatase↗
PubMed2003

Establishment of an immortalized human hepatic stellate cell line to develop antifibrotic therapies.

Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-beta receptor (PDGF-betaR), alpha-smooth muscle actin (alpha-SMA), and type I collagen (alpha1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-gamma or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (alpha1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-gamma (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (alpha1), PDGF-betaR, and alpha-SMA expression and suppressed TGF-beta1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.

Actins↗
PubMed2006

[Synthetic RGD-containing peptide K16GRGDSPC affected the adhesion, proliferation and osteogenic differentiation of rabbit bone marrow stromal cells on PLGA-[ASP-PEG] scaffold materials].

OBJECTIVE: To explore the effects of synthetic RGD-containing peptide K16GRGDSPC covalent bonding with PLGA-[ASP-PEG] scaffold materials on the adhesion, proliferation and osteogenic differentiation of rabbit bone marrow stromal cells (BMSCs). METHODS: The peptide was synthesized by solid-phase synthesis method and characterized by mass spectrometry and high pressure liquid chromatography. PLGA-[ASP-PEG] scaffold materials were modified with the peptide by cross linker Sulfo-LC-SPDP and detected by XPS. The BMSCs obtained from rabbit were cultured on PLGA-[ASP-PEG] modified with the peptide and those cultured on unmodified PLGA-[ASP-PEG] were also observed as control group. The adhesion and proliferation behaviors of the cells were analyzed by conventional precipitation method, micropipette aspiration technique, MTT assay and Coomassie Brilliant Blue dyes. The osteogenic differentiation of the cells was showed by the activity of alkaline phosphatase (ALP) assayed by ALP Assay Kit and the mRNA levels of ALP, osteocalcin (OCN), osteopontin (OPN) and collagen I assessed by real-time PCR (RT-PCR). Immunofluorescence stain was also used to detect the expression of core binding factor a1 (Cfba1) which was an osteogenic maker as well. RESULTS: The peptide was successfully manufactured and linked to the surface of the PLGA-[ASP-PEG] by the cross-linker. The abilities of adhesion and proliferation and the expressions of osteogenic makers of the cells were significantly higher than those of control group (P < 0.05). CONCLUSION: RGD-containing peptide K16GRGDSPC could promote the adhesion, proliferation and osteogenic differentiation of BMSCs on the biomimetic bone-matrix materials.

Alkaline Phosphatase↗
PubMed2005

Wound chronicity and fibroblast senescence--implications for treatment.

A proportion of chronic wounds fail to heal in response to standard therapy. For venous leg ulcers, a correlation exists between longer duration before treatment initiation and poor healing response to compression therapy. Differences identified between the healing wound microenvironment and that of the non healing chronic wound suggests that many potential mechanisms exist to impair healing. One contributory mechanism may be inhibition of fibroblast proliferation and induction of a stress-induced premature senescence phenotype by the continuing inflammation found in chronic wounds. Senescent fibroblasts exhibit an extracellular matrix degradative phenotype that contributes to wound chronicity. Accumulation of greater than 15% senescent fibroblasts has been described as a threshold beyond which wounds become hard to heal. The ratio of senescent : non senescent cells is therefore critical to determining response to treatment, and adjunctive therapies that modulate this ratio in favour of non senescent cells are likely to enhance therapeutic healing rates. A number of tissue-engineered dermal replacements contain non senescent fibroblasts and can donate cells to the wound environment additional to releasing growth factors and reversing the antiproliferative activity of chronic wound exudate. Recognition of the role of fibroblast senescence in wound chronicity may allow for identification of those wounds that will respond positively to these products.

Cellular Senescence↗
PubMed2005

Expression pattern of the chromosome 21 transcription factor Ets2 in cell-seeded three-dimensional bone constructs.

The ability to generate new bone for reconstructive surgery use is a major clinical need. Tissue engineering with osteoprogenitor cells isolated from the patient's periosteum and seeded into bioresorbable scaffolds offers a promising approach to the generation of skeletal tissue. To our knowledge, there is no description about the expression of Ets2 in tissue engineered "bone neotissue." The aim of our study was to manufacture cell-seeded three-dimensional bone constructs with human periosteal cells on poly (lactic-co-glycolic acid) polymer fleeces to describe the expression pattern of Ets2 and its target genes osteocalcin and osteopontin; expression analysis of type I collagen, core-binding factor-1, alkaline phosphatase, and osteonectin; the ability of matrix mineralization and ALP enzymatic activity showed the osteogenic character of the constructs. A significant correlation between the expression of Ets2 and osteopontin mRNA (r = -0.70; p < 0.05) could be shown. A 1.35-fold increase of Ets2 expression from days 1 to 9 was detected, followed by a slight decrease from days 11 to 15. Until the end of the culture period, the expression of Ets2 reached a comparable high level as detected on day 9. In contrast, the expression level of osteopontin mRNA reached a maximum at day 7, followed by a progressive 3.04-fold decrease until day 21. This study shows for the first time that Ets2 gene and its transcriptional target genes are expressed in tissue-engineered bone constructs. These findings have the potential to provide much-needed information about the role and function of Ets2 in human osteogenesis processes and creation of "bone neotissue."

Alkaline Phosphatase↗
PubMed2000

A ligand-reversible dimerization system for controlling protein-protein interactions.

Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (F(M)) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a "reverse dimerization" regulatory system involving F(M) fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated F(M) domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.

Dimerization↗
PubMed2007

Isolation and chondroinduction of a dermis-isolated, aggrecan-sensitive subpopulation with high chondrogenic potential.

OBJECTIVE: To develop a process that yields tissue-engineered articular cartilage constructs from skin-derived cells. METHODS: Dermis-isolated, aggrecan-sensitive (DIAS) cells were isolated using a modified rapid adherence process. The chondrogenic potential was measured by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Filamentous actin (F-actin) and vinculin organization was detected using fluorescence microscopy. RESULTS: The rapid adherence process led to a selection of DIAS cells, <10% of the entire population. DIAS cells displayed greater chondroinduction potential, as evidenced by the formation of large numbers of chondrocytic nodules on aggrecan-coated surfaces. In addition, these cells showed higher gene expression and protein production in terms of chondrocytic markers when compared with unpurified dermis cells. Similar patterns of F-actin and vinculin organization were observed between DIAS cells and chondrocytes. Three-dimensional constructs from chondroinduced DIAS cells produced greater amounts of cartilage matrix than constructs from the rest of the dermis populations. CONCLUSION: These findings show a series of steps that work together to form tissue-engineered articular cartilage constructs using DIAS cells. Since skin presents a minimally invasive, relatively abundant cell source for tissue engineering, this study offers evidence of an efficient and stable technique to form cartilage constructs for future cartilage regeneration with autologous cells from skin.

Actin Cytoskeleton↗
PubMed2003

Establishing the protein MIA (melanoma inhibitory activity) as a marker for chondrocyte differentiation.

Melanoma inhibitory activity (MIA), also referred to as cartilage derived retinoic acid-sensitive protein (CD-RAP), is detected physiologically in cartilage tissue and pathologically in malignant melanomas. To measure MIA/CD-RAP quantitatively we developed a sensitive ELISA system. Recently, we described diagnostic applications of the MIA-ELISA in patients with cartilage diseases. The study described herein was performed to determine whether there is any relation between MIA/CD-RAP levels and the degree of chondrocyte differentiation in tissue culture and to analyse whether MIA/CD-RAP may serve as a useful marker to control chondrocyte differentiation in in vitro tissue engineering. Our data provide evidence that measuring MIA in tissue culture supernatant by a quantitative ELISA can be used as a marker for differentiated chondrocytes.

Biomarkers↗
PubMed2003

Site-specific selfish genes as tools for the control and genetic engineering of natural populations.

Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.

Animals↗
PubMed2001

A new RT-PCR method for the identification of reoviruses in seawater samples.

The frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. In order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (RT-PCR) was set up to detect reoviruses. Two primers were engineered to amplify a 538 base pair fragment of the Sigma 2 gene. Reovirus strains obtained from ATCC (Jones, Lang, Dearing, Abney, NC-TEV, SV59 and SV12) were used as references. Twenty-four samples of 101 were collected from two beaches of the Adriatic sea and 12 from the neighbourhood of Fano Harbour Channel. The presence of environmental reoviruses was tested on both concentrated seawater samples and lysates of BGM cells infected with the concentrated seawater samples. The new method was used in parallel with the detection of a 3:3:4 electrophoretic pattern of reovirus RNA in polyacrylamide gel electrophoresis (PAGE). Enterovirus and bacteria were also screened in compliance with EEC directives. No enteroviruses were isolated, and it was not attributable to reovirus interference. All the reovirus found by PAGE (8/72) were confirmed by RT-PCR, while several genomes (14/72) were detected only by RT-PCR. Presumptive methods of virus identification, that is CPE on BGM cells and haemagglutination test, were not able to detect them. The specificity of RT-PCR products was checked by direct nucleotide sequence analyses of the amplicons. The phylogenetic analyses showed heterogeneous taxa including human and animal reoviruses, with strong evidence that they were spreading consistently from the Harbour-Channel. This novel approach for reovirus detection will be very useful as a trace route of faecal pollution; more importantly, it could be very useful in contributing to the creation of a databank of circulating enteric viruses.

Base Sequence↗
PubMed2006

Enrichment of cardiomyocytes derived from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cell-derived cardiomyocytes transplantation and tissue engineering together represent a promising approach for the treatment of myocardial infarction, despite the limited supply of cardiac myocytes. This study examines whether functional cardiomyocytes can be efficiently enriched from mouse embryonic stem (mES) cells. METHODS: mES cells were induced by ascorbic acid to differentiate into cardiomyocytes. Beating cells were observed after 1 week and increased in number with time while under differentiation conditions. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll gradient density centrifugation. RESULTS: The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac troponin T (cTnT). The enriched population contained 88.7% cardiomyocytes and showed expression of cardiomyocyte markers of troponin T and cardiac genes, including alpha-MHC, beta-MHC, ANF and Nkx2.5. However, Oct-4, a marker of early-stage ES cells, was not expressed in the mES cell-derived cardiac cell clusters. Moreover, the mES cell-derived and Percoll-enriched cardiomyocytes responded appropriately to cardioactive drugs, as did normal neonatal rat cardiomyocytes. CONCLUSIONS: mES cell-derived functional cardiomyocytes can be enriched by the method of discontinuous Percoll gradient centrifugation. The ability to differentiate and enrich for functional mouse cardiomyocytes makes it possible for further development of these cells as a model of myocardial repair through cell transplantation or tissue engineering.

Animals↗
PubMedDate not supplied

Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage.

A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro-Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography.

Amino Acid Sequence↗
PubMed2003

Mechanism of dimerization of bicoid mRNA: initiation and stabilization.

Dimerization of bcd mRNA was shown to be important for the formation of ribonucleoprotein particles and their localization in Drosophila embryo. The cis-element responsible for dimerization is localized in a stem-loop domain (domain III) containing two essential complementary 6-nucleotide sequences in a hairpin loop (LIIIb) and an interior loop (LIIIa). Such an RNA element can potentially generate single or double "hand-by-arm" interactions leading to open and closed complexes, respectively. The former retains the possibility of forming multimers, whereas the latter does not. We showed previously that dimerization proceeds through a two-step mechanism, which includes a transition from the reversible initiation complex into a very stable one. Here we have addressed the nature of the initial interactions and the mechanism of transition. We engineered a series of different RNA fragments with the capacity to form defined open dimers, multimers, or closed dimers. We compared their thermodynamic and kinetic behavior and mapped nucleotides involved in intermolecular interactions by enzymatic and chemical footprinting experiments and chemical modification interference. Our results indicate that the initiation step leads to a reversible open dimer, involving a more limited number of intermolecular base pairs than expected. The two loops play distinct roles in this process, and the structure of loop IIIb is more constrained than that of loop IIIa. Thus, loop IIIa appears to be the driving element of the recognition process. The initial open dimer is then converted into a stable closed dimer, possibly through a kinetically controlled mechanism.

Animals↗
PubMed2002

Mycobacterium tuberculosis genes induced during infection of human macrophages.

We identified Mycobacterium tuberculosis genes preferentially expressed during infection of human macrophages using a promoter trap adapted for this pathogen. inhA encodes an enoyl-acyl carrier protein reductase that is required for mycolic acid biosynthesis (A. Quemard et al., Biochemistry 34:8235-8241, 1995) and is a major target for isoniazid (INH) in mycobacterial species (A. Banerjee et al., Science 263:227-230, 1994). Since overexpression of inhA confers INH resistance in Mycobacterium smegmatis (Banerjee et al., Science 263:227-230, 1994), we designed a promoter trap based on this gene. A library of clones, containing small fragments of M. tuberculosis DNA cloned upstream of inhA in a plasmid vector, was electroporated into M. tuberculosis, and the resulting culture was used to infect the human monocytic THP-1 cell line. Selection was made for clones surviving INH treatment during infection but retaining INH sensitivity on plates. The DNA upstream of inhA was sequenced in each clone to identify the promoter driving inhA expression. Thirteen genes identified by this method were analyzed by quantitative reverse transcription-PCR (R. Manganelli et al., Mol. Microbiol. 31:715-724, 1999), and eight of them were found to be differentially expressed from cultures grown in macrophages compared with broth-grown cultures. Several of these genes are presumed to be involved in fatty acid metabolism; one potentially codes for a unique DNA binding protein, one codes for a possible potassium channel protein, and the others code for proteins of unknown function. Genes which are induced during infection are likely to be significant for survival and growth of the pathogen; our results lend support to the view that fatty acid metabolism is essential for the virulence of M. tuberculosis.

Bacterial Proteins↗
PubMed2005

[The effect of bone-related growth factors on the proliferation and differentiation of marrow mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the effect of dexamethasone, recombinant human fibroblast growth factor (rhFGF) and recombinant human bone morphogenetic protein 2 (rhBMP-2) on the proliferation and differentiation of marrow stromal stem cells (MSCs) for their further application in tissue engineering. METHODS: MSCs were isolated and cultured in vitro, and then exposed to different dose of dexamethasone (10(-8) mol/l, 10(-7) mol/L, 10(-6) mol/L), rhFGF (50 ng/ml, 200 ng/ml, 500 ng/ml) and rhBMP-2 (50 ng/ml, 500 ng/ml, 1000 ng/ml) respectively. The total protein and alkaline phosphatase (ALP) activity of each group was measured on 4th and 7th day. RESULTS: Exposure of MSCs with 10(-6) mol/L dexamethasone inhibited protein synthesis without obvious effects on ALP expression. The application of rhFGF significantly promoted cell proliferation but inhibited ALP activity. In comparison, ALP expression was significantly enhanced by treatment of rhBMP-2 at concentration of 500 ng/ml, 1000 ng/ml. CONCLUSION: The exposure of dexamethasone as well as rhBMP-2 to MSCs with an appropriate concentration promotes osteogenic expression without reverse effects on cell proliferation, which indicates the great potential value in cell-based strategy of bone tissue engineering.

Alkaline Phosphatase↗
PubMed1992

Inhibition of bovine leukaemia virus replication by the antisense RNA in cell line CC81.

A model system has been developed for quantitative evaluation of bovine leukaemia virus (BLV) replication in a permanent cell line CC81. Transfection of the BLV DNA into these cells evoked typical signs of retroviral infection: formation of syncytia, manifestation of reverse transcriptase activity and appearance of characteristic budding retroviral particles. To inhibit BLV replication, a recombinant plasmid pAGR with an antisense RNA gene targeted at the R-U5 region (147th-342th nt) of the viral genome has been engineered. Cotransfection of CC81 cells with infectious BLV DNA and pAGR led to effective inhibition of BLV replication by the antisense RNA, evidenced by a drop in the number of syncytia and reverse transcriptase activity. Maximal inhibition of BLV replication (95-97%) was observed at a weight ratio of input viral and plasmid DNAs equal to 1:10.

Animals↗
PubMed1990

Behavioural Effect of Engineered Cells that Synthesize l-dopa or Dopamine after Grafting into the Rat Neostriatum.

Cell lines in which tyrosine hydroxylase was introduced either by infection or transfection were used in grafting experiments in a rat model of Parkinson's disease obtained by unilateral lesion of the substantia nigra. A neuroblastoma NS20 Y cell line which synthesizes only l-dopa and a neuroendocrine AtT-20 cell line which produces dopamine were obtained. They were grafted into denervated striata and their ability to compensate for the dopaminergic deficit was studied. Both modified cell types displayed a rapid partial reversal of apomorphine-induced turning behaviour. No effect was observed with the control unmodified cell lines. We discuss the usefulness of engineered cell lines to address the fundamental issues in grafting experiments and more particularly in the therapy of Parkinson's disease.

Journal Article↗
PubMed1993

An improved method for the detection of hepatitis C virus RNA in plasma utilizing heminested primers and internal control RNA.

The majority of transfusion-associated, non-A, non-B hepatitis cases are caused by hepatitis C virus (HCV), a positive-stranded RNA virus. Although high titers of HCV in clinical specimens have been reported, in some cases extremely low titers of virus are not uncommon. Therefore, an extremely sensitive and reliable assay is required to determine viremia and replication of HCV accurately. We report here the systematic investigation of factors influencing the detection of HCV RNA by a reverse transcription-polymerase chain reaction (RT-PCR) assay utilizing "drop in-drop out" heminested primers derived from the conserved 5' non-coding region of the viral genome. A genetically engineered 5' noncoding region has been constructed and used as an internal control. Addition of the control RNA to each test not only allowed semiquantitation of positive reactions but also validated the performance of reverse transcription and PCR for every specimen. The optimized heminested PCR (HN-PCR) protocol is capable of amplifying one molecule of cloned HCV DNA or 10 molecules of in vitro-transcribed HCV RNA to levels detectable in ethidium bromide-stained agarose gels. We evaluated the improved method for the detection of HCV RNA on a human plasma sample containing the pedigreed strain H of HCV with a chimpanzee infectious dose of 10(6)/ml. Utilizing the internal control RNA, we calculated 2 x 10(7) virions in 1 ml of the original human plasma. The HN-PCR achieves the sensitivity and specificity of the double-nested PCR (DN-PCR) in a simplified format that avoids the false-positive results associated with DN-PCR.

Base Sequence↗