Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “reverse engineering”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources
PubMed1999

In vivo studies of gene expression via transient transgenesis using lipid-DNA delivery.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.

Animals↗
PubMed2006

Modulation instability in nonlinear negative-index material.

We investigate modulation instability (MI) in negative-index material (NIM) with a Kerr nonlinear polarization based on a derived (3+1)-dimensional nonlinear Schrödinger equation for ultrashort pulse propagation. By a standard linear stability analysis, we obtain the expression for instability gain, which unifies the temporal, spatial, and spatiotemporal MI. It is shown that negative refraction not only brings some new features to MI, but also makes MI possible in ordinary material in which it is otherwise impossible. For example, spatial MI can occur in the defocusing regime, while it only occurs in the focusing regime in ordinary material. Spatiotemporal MI can appear in NIM in the case of anomalous dispersion and defocusing nonlinearity, while it cannot appear in ordinary material in the same case. We believe that the difference between the MI in NIM and in ordinary material is due to the fact that negative refraction reverses the sign of the diffraction term, with the signs of dispersion and nonlinearity unchanged. The most notable property of MI in NIM is that it can be manipulated by engineering the self-steepening effect by choosing the size of split-ring resonator circuit elements. To sum up the MI in ordinary material and in NIM, MI may occur for all the combinations of dispersion and nonlinearity.

Journal Article↗
PubMed2025

Disassembly activates Retron-Septu for antiphage defense.

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Cryoelectron Microscopy↗
PubMed2004

HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells.

A T-cell subset, defined as CD4(+)CD25(hi) (regulatory T-cells [Treg cells]), was recently shown to suppress T-cell activation. We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4(+) T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4(+) and higher levels of activated T-cells have greatly reduced levels of FoxP3(+)CD4(+)CD25(hi) T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset.

CD4-Positive T-Lymphocytes↗
PubMed2003

The cytotoxic T-lymphocyte response against a poorly immunogenic mammary adenocarcinoma is focused on a single immunodominant class I epitope derived from the gp70 Env product of an endogenous retrovirus.

The TS/A mouse mammary adenocarcinoma is a poorly immunogenic tumor widely used in preclinical models of cancer immunotherapy. CTLs have often been indicated as important in TS/A tumor destruction, but their generation in this model has been rarely studied, nor have their precise target(s) been identified. We hypothesized that the gp70 Env product of an endogenous murine leukemia virus could be a target antigen for TS/A-specific CTLs and investigated this possibility in four different TS/A cell lines engineered with the genes that encode IFN-alpha, IFN-gamma, interleukin-4, and B7.1, respectively. All tumor cell lines expressed gp70, albeit at different levels, as demonstrated by reverse transcription-PCR analysis. Transfected tumor cells exhibited a delayed growth in vivo, and partial tumor regression. Spleen cells from mice that displayed tumor regression had high percentages of CD8(+) T cells that were specifically stained with L(d) tetramers loaded with gp70(423-431), the antigenic epitope of gp70 protein. Mixed leukocyte-peptide and mixed leukocyte-tumor cultures, set up by stimulating splenocytes with the immunogenic peptide and with transfected TS/A tumor cells, respectively, resulted in similar large increases in tetramer-reactive CD8(+) T cells and showed high lytic activity specific for gp70(423-431). Finally, in a Cold Target Inhibition assay, lytic activity of a mixed leukocyte-tumor culture was inhibited in an overlapping fashion by both the TS/A line used for restimulation and 293L(d) cells loaded with gp70(423-431) peptide, but not by 293L(d) cells pulsed with an irrelevant H-2 L(d) epitope, thus demonstrating that all or most of the cytotoxic activity was directed exclusively against this antigenic epitope.

Adenocarcinoma↗
PubMed2004

Gastric banding for clinically severe obesity: results with the Swedish band.

Parallel with the rise of the obesity pandemic, bariatric surgery is quickly becoming one of the most frequently performed GI procedures. In selected, well-informed patients, restrictive surgery offers a good alternative to more complex malabsorption-inducing procedures. Laparoscopic gastric banding is a reversible, technically straightforward procedure. Both early and late complications seen with the original models are less common with the Swedish adjustable gastric band SAGB (Ethicon Endosurgery, Johnson & Johnson, Dilbeek, Belgium) engineered as a low-pressure device. This chapter is a review of our experience with the SAGB and provides an overview of current controversies regarding its place in management of severe obesity.

Adult↗
PubMed2005

Venous ulcers: pathophysiology and treatment options.

Venous ulcers affect approximately 1% of the world's population, increasing healthcare expenditures and decreasing quality of life. Several hypotheses may help explain their origin. Incompetent veins or valves or impaired muscle function may lead to abnormal calf muscle pump function that can elevate ambulatory venous pressure (venous hypertension). This hypertension subsequently results in local venous dilatation and pooling, concomitantly trapping leukocytes that may release proteolytic enzymes that destroy tissues. Venous pooling also induces interendothelial pore widening and deposition of fibrin and other macromolecules that "trap" growth factors within them, rendering them unavailable for wound repair. Compression therapy, the mainstay treatment, reduces edema, reverses venous hypertension, and improves calf muscle pump function. Several treatment options can be employed as adjuvants to compression--eg, systemic therapy with pentoxifylline or aspirin, autologous grafts, tissue-engineered skin, growth factor therapy, and/or vein surgery. The epidemiology, pathophysiology, diagnosis, and management options regarding venous ulcers are reviewed.

Anti-Inflammatory Agents↗
PubMed2026

Landscape of retron diversity across the SPIRE microbial metagenome resource reveals candidate novel type XI-like lineages.

Retrons are bacterial genetic elements encoding a specialized reverse transcriptase (RT) that synthesizes multicopy single-stranded DNA and are increasingly recognized as components of bacterial anti-phage defense systems. However, their diversity and ecological distribution across large-scale genomic resources remain poorly characterized. Here, we surveyed retron RTs across the SPIRE representative metagenome collection, a non-redundant, species-level data set spanning diverse microbial habitats. Using a curated panel of type-specific hidden Markov models, we identified retrons representing all canonical types together with additional divergent lineages. Retron distribution showed strong taxonomic and ecological structuring, with some groups restricted to specific bacterial phyla, whereas others were broadly distributed across environmental categories. Systematic novelty assessment identified two candidate type XI-like lineages, TXI_C2like and TXI_noncan_h, characterized by protease-independent architectures and distinct accessory modules associated with WYL- and DnaB_C-containing proteins, respectively. De novo covariance-based analyses further identified candidate msr/msd-like non-coding RNA structures in both lineages, supporting conservation of the canonical RT-ncRNA organizational framework despite extensive sequence divergence. Together, these findings expand the known diversity of retron systems and identify type XI-like retrons as a dynamic and previously underexplored evolutionary group.IMPORTANCERetrons are bacterial genetic elements that are increasingly exploited as programmable tools for genome editing, molecular recording, and biosensing in addition to their natural role in anti-phage defense. Despite this growing biotechnological interest, the true diversity of retrons across the bacterial world has remained largely unmapped. By mining a resource of over 100,000 processed microbial metagenomes, we uncovered thousands of retron sequences spanning known types as well as previously unrecognized lineages and found that their distribution is strongly shaped by both bacterial taxonomy and ecological niche. Among these, we identified two candidate new lineages related to type XI retrons that lack the protease domain typical of this group but instead carry distinct accessory proteins, expanding the known architectural diversity of these systems. These findings broaden the catalog of retron diversity available for functional characterization and biotechnological engineering and provide a framework for prioritizing candidate lineages for future experimental validation.

effectors↗
PubMed2002

Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide.

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1-derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G(0) to G(1) phases of the cell cycle. Compared to unmodified HIV-1-based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

Antigens, CD↗
PubMed1978

Reversible respiratory disease in beryllium workers.

A medical and environmental survey was carried out in a beryllium extraction and processing plant in 1971, and a follow-up study was done in 1974. Peak air concentrations of beryllium of as much as 50 times the accepted peak limit value were found in the plant in 1971. Radiographic changes of interstitial disease were found in 31 workers, hypoxemia was found in 20, and in 11 workers a combination of hypoxemia and radiographic changes of interstitial disease was seen. Follow-up in 1974 showed that air concentrations had all decreased significantly, mainly because of improvements in engineering and ventilation in the plant. Improvement in hypoxemia and decreased alveolar-arterial O2 tension difference at rest ([A-a]DO2) were noted in 13 of the 20 men who had hypoxemia in 1971 and were available for follow-up. In addition, radiographic abnormalities of interstitial disease were reversed in some workers. Our results suggest that improvement in gas exchange and radiographic findings of interstitial disease occurs when peak air concentrations of beryllium are decreased. Our approach in combining environmental and medical screening in workers exposed to beryllium enabled us to identify persons with early changes, to detect reversible abnormalities, and to correlate air concentrations with the medical status of workers.

Adult↗
PubMed1995

Control of protein-ligand recognition using a stimuli-responsive polymer.

Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

Acrylic Resins↗
PubMed2002

Reversible stretching of a monomeric unit in a dimeric bovine carbonic anhydrase B with the atomic force microscope.

We have previously shown that a full stretching of native carbonic anhydrase B (CAB) using the atomic force microscope could not be achieved, presumably due to the presence of a 'knot' in the C-terminal region of the protein. When we used an engineered dimer of CAB, where the N-terminal monomeric unit (unit I) was expected to be 'knotless', we successfully recorded extension of the protein up to 110 nm which was long enough to account for the full extension of unit I monomer. In this paper we report that, by limiting the maximum length of extension to 90 nm extensions (corresponding to about 80 nm extension of the dimer and 70 nm of unit I), retractions of the polypeptide chain can be repeated cyclically without breaking the covalent crosslinking system. The force-extension curves obtained from the forward and reverse cycles of such experiments were almost perfectly superimposable with each other and with the corresponding part of the curves obtained from full extension experiments suggesting that the structure of unit I in the dimer was reversibly stretched and contracted. During the stretching of unit I of the dimer in either type of the experiments mentioned above, we occasionally observed a force peak having the force of about 0.5-0.7 nN when extension length reached 40-50 nm. We interpreted the appearance of such force peaks as an indication of formation of a tightly folded domain structure in unit I of CAB dimer.

Animals↗
PubMed1995

Expression of factor VII by muscle cells in vitro and in vivo following direct gene transfer: modelling gene therapy for haemophilia.

Direct injection of plasmid DNA into skeletal muscle has been proposed as a method of effecting somatic gene therapy. This article describes the construction and testing of a plasmid derived expression cassette believed to confer skeletal muscle specific expression. Expression constructs were designed containing the full-length cDNAs for both coagulation factor VIII and factor VII. The engineered genes were flanked by two muscle specific regulatory elements from different myosin isoforms and by an artificial polyadenylation signal sequence. In vitro transfection of C2-myoblasts led to expression of the factor VIII gene, shown by reverse transcription and polymerase chain reaction, upon differentiation of the myoblasts. The expression of the FVII construct was tested in a C2 cell culture system and also when injected directly into mouse muscle. It was found that in cell culture the level of factor VII antigen outside the cell, ie in the cell culture medium was two- to three-fold higher than inside the cell, ie in the cell lysate. This level of expression was found to continue for the duration of cell culture maintenance and a fully functional protein was produced. In vivo transfection experiments in mice showed a substantial increase in factor VII antigen compared with the background level 4-5 days after injection. An anti-human factor VII antibody was detected 7-10 days after injection. We conclude that muscle cells in vitro secrete and efficiently carry out post-translational modifications of the engineered gene product and in vivo secrete the gene product resulting in elevation of systemic levels. The data provide the basis for the use of muscle cells as an in vivo expression system for coagulation proteins in the treatment of inherited haemostatic and thrombotic disorders.

Animals↗
PubMed2000

[Extension of life-span of normal human fibroblasts by reconstitution of telomerase activity].

Most of normal human somatic cells can divide only a finite number of times and inevitably become senescent. Telomerase is an enzyme that imparts replicative immortality by maintaining the length of the telomeres when expressed in reproductive and cancer cells. Cells that are mortal do not express the telomerase. Recently it was reported that the life-span of the normal human cells could be successfully extended by introduction of telomerase into these cells. We have found, in the previous work, that fibroblasts exhibited an osteogentic potential, and therefore, can be considered as a type of "seed cells" in tissue engineering for bone repairing and reconstruction. But this potential was impaired by the limitation in life-span and proliferative capacity of the normal fibroblasts. In the present work, plasmid pGRN145 bearing a cDNA insert of human telomerase reverse transcriptase (hTERT) was introduced into the fibroblasts with osteogenic potential by electroporation. The stable hTERT+ fibroblast clones was established and cultured for long-term in a medium containing hygromycin-B. The exogenous hTERT mRNA expression and telomerase activity were detected. The hTERT+ fibroblasts showed shorter population doubling time and no beta-galactosidase stain, which indicated a stronger proliferative capacity and fewer signs of cell senescence, compared to their hTERT- counterpart. These evidenced that the life-span of hTERT+ fibroblasts was extended. The assays for DNA euploidy by flow cytometry and chromosome karyotype by cytogenetic technique showed no signs of heteroploidy, providing the data for cell carcinogenesis and utilization safety. The results of the present study suggested that the introduction of hTERT could make the life-span of normal fibroblast extended without causing their malignant transformation, and such type of "longevous" fibroblasts might be clinically useful in tissue engineering for bone repairing and reconstruction.

Cells, Cultured↗
PubMed2006

CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore.

Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES-. We report here that the permeant, pseudohalide anion [Au(CN)2]- can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2]- produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]- and activity was not restored by washing [Au(CN)2]- from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]- -blocked channels to excess [CN]-. The results are consistent with the hypothesis that [Au(CN)2]- can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of -1, blocks the anion conduction pathway.

Amino Acid Substitution↗
PubMed2006

[Expressions of Cbfal and osterix in osteoblasts on human acellular amniotic membrane].

OBJECTIVE: To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane (HAAM). METHODS: To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and 10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The real-time PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simply-cultured osteoblasts were used as the control group (n=20). RESULTS: The expression of Cbfal was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day (P < 0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results (P < 0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference (P > 0.05). CONCLUSION: Cbfal and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.

Amnion↗
PubMed1995

An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch.

BACKGROUND: Studies of the mechanisms by which certain water-soluble proteins can assemble into lipid bilayers are relevant to several areas of biology, including the biosynthesis of membrane and secreted proteins, virus membrane fusion and the action of immune proteins such as complement and perforin. The alpha-hemolysin (alpha HL) protein, an exotoxin secreted by Staphylococcus aureus that forms heptameric pores in lipid bilayers, is a useful model for studying membrane protein assembly. In addition, modified alpha HL might be useful as a component of biosensors or in drug delivery. We have therefore used protein engineering to produce variants of alpha HL that contain molecular triggers and switches with which pore-forming activity can be modulated at will. Previously, we showed that the conductance of pores formed by the mutant hemolysin alpha HL-H5, which contains a Zn(II)-binding pentahistidine sequence, is blocked by Zn(II) from either side of the lipid bilayer, suggesting that residues from the pentahistidine sequence line the lumen of the transmembrane channel. RESULTS: Here we show that Zn(II) can arrest the assembly of alpha HL-H5 before pore formation by preventing an impermeable oligomeric prepore from proceeding to the fully assembled state. The prepore is a heptamer. Limited proteolysis shows that, unlike the functional pore, the prepore contains sites near the amino terminus of the polypeptide chain that are exposed to the aqueous phase. Upon removal of the bound Zn(II) with EDTA, pore formation is completed and the sites near the amino terminus become occluded. Conversion of the prepore to the active pore is the rate-determining step in assembly and cannot be reversed by the subsequent addition of excess Zn(II). CONCLUSIONS: The introduction of a simple Zn(II)-binding motif into a pore-forming protein has allowed the isolation of a defined intermediate in assembly. Genetically-engineered switches for trapping and releasing intermediates that are actuated by metal coordination or other chemistries might be generally useful for analyzing the assembly of membrane proteins and other supramolecular structures.

Bacterial Toxins↗
PubMed2002

Culture of primary human gingival fibroblasts on biodegradable membranes.

Repair and regeneration of periodontal tissues by tissue engineering is dependent on the use of biodegradable polymer scaffolds which serve as a carrier for cells or bioactive substances. There is a need to understand how a specific biomaterial may influence gene expression. The aim of this investigation was to develop and to optimize an in vitro technique for the adherance and proliferation of primary human gingivaL cells on implantable and biodegradable matrices. Square pieces of Bio-Gide matrix (BG) and slices of Ethisorb tamponade (ET) were coated with poly-L-lactide. The stability of coated and uncoated scaffolds was investigated by incubation in standard culture medium. Various concentrations of the cells were seeded onto coated and uncoated polymer matrices in tissue culture dishes without shaking ("static seeding") or continuous shaking ("agitated seeding"). Cultures were grown for 4 week and were then evaluated by light and scanning electron microscopy. After a culture period of 10 d, BG-carriers showed a delicate consistency which made histological processing difficult. Cells were grown only sparsely in coated and non-coated BG-scaffolds. Contrary. ET-specimens were stable during a 4 week culture period. After "static seeding" a significantly higher number of cells resulted in comparison to those in "agitated" cultures. The cells were evenly distributed throughout the ET-carriers and produced extracellular matrix compounds as well. Furthermore, the examination with RT-PCR (reverse transcription-polymerase chain reaction) revealed that the cells synthesized and secreted type I collagen, and expressed genes implicated in transducing bone morphogenetic protein (BMP) signals. Messenger RNAs for BMP-2, -4, -7, the BMP type I receptors Act R-1 (alk 2, activin-like kinase receptor), BMPR-IA (alk 3), -IB (alk 6), and the type II receptor BMPR-II were detected. These data reveal that static seeding favors the adherence and proliferation of primary gingival cells on polyglactin matrices. This system may serve as a valuable tool for periodontal tissue engineering.

Activin Receptors, Type I↗