Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Collagen is the primary structural component in connective tissue. The poor mechanical properties of most cell-seeded cartilage grafts used for cartilage repair can be attributed to the low level of collagen synthesized compared with native cartilage. In this study, the synthesis and assembly of collagen by chondrocytes in hydrogels were investigated, with particular attention paid to the role of cross-link formation in this process. Primary bovine chondrocytes were seeded in alginate and collagen synthesis was assessed in the presence and absence of beta-aminopropronitrile (BAPN), a potent inhibitor of the enzyme lysyl oxidase and collagen cross-link formation. Cultures on days 21, 35, and 49 were evaluated by stereology, biochemistry, and real-time reverse transcriptase-polymerase chain reaction. All measures of collagen synthesis (except hydroxyproline) significantly increased in the presence of 0.25 mM BAPN. By 35 days of culture, the average collagen fibril diameter was 62 +/- 10 nm in control cultures and 109 +/- 20 nm with BAPN supplementation. The collagen volume density increased from 5 +/- 3% in control cultures to 17 +/- 1% in the presence of BAPN. Likewise, the expression of cartilage-specific collagens (type II and XI) and aggrecan increased significantly as a result of BAPN culture. These findings demonstrate the prominent role of collagen cross-linking in collagen fibrillogenesis and suggest approaches by which collagen synthesis and assembly could be controlled in tissue-engineered constructs.
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PMID:Collagen fibrillogenesis by chondrocytes in alginate. 1254 43

The main objective of the current investigation was to regenerate cells of the nucleus pulposus without loss of phenotype. Nucleus pulposus cells were isolated from intervertebral discs from adult rabbits, grown in monolayer culture, and then maintained as a micromass pellet in tube culture. The specimens were evaluated by transmission and light microscopy, reverse transcriptase polymerase chain reaction, and immunohistochemistry. Nucleus pulposus cells proliferated in monolayer culture. When almost confluent, the cells were transferred to a tube and sedimented to form a pellet. The cells reverted to a rounded configuration and formed cell nests surrounded by extensive extracellular matrix, similar to that seen in vivo. These cells did not proliferate. Similar to that observed in situ, cells in pellet culture also expressed aggrecan, CD44, collagen Type II, and collagen Type I, but not collagen Type X, and had low alkaline phosphatase activity. The results of the investigation indicated that nucleus pulposus cells grown in monolayer culture might revert to their original characteristics when transferred to an environment that allows three-dimensional growth, such as upon implantation, a one-step approach. The results also indicated that the two-stage culture procedure might provide an expedient technique to regenerate nucleus pulposus tissue for disc repair.
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PMID:Intervertebral disc tissue engineering II: cultures of nucleus pulposus cells. 1278 90

We have evaluated a biomaterial to serve as a scaffold for the propagation and amplification of chondrocytes that promotes the original cellular phenotype of these cells. The goal of the present study was to investigate the use of thermally reversible polymer gels poly(NiPAAm-co-AAc), as a biocompatible supporting scaffold for the propagation of chondrocytic cells. The polymer gels at temperatures above its lower critical solution temperature whereas liquefying at temperatures below its lower critical solution temperature of 34.5 degrees C. Hence, the polymer, in its gelled form, has the ability to hold cells in situ, forming a matrix similar to the natural cellular environment or the extracellular matrix that comprises cartilage. We tested the hypothesis that the polymer gel promotes cell viability and function. Human osteoblast-like cells, nasal chondrocytes, and articular chondrocytes (1 x 10(5)/150 microL) were resuspended in enriched Dulbecco's minimal essential media and were plated onto control (without gel) and gel containing 24-well plates. The plates were reincubated at 37 degrees C, 5% CO(2) for the time point of interest. Additional media was added to the plates and exchanged as needed. After cell culture, cells were retrieved, enumerated, and cell viability was determined. Other aliquots of the cells were stained for morphological analysis whereas expression of chondrocyte markers including collagen type II and aggrecan were determined using reverse transcriptase-polymerase chain reaction. The polymer gel was not cytotoxic because the cell number retrieved from three-dimensional culture gel was found to be one to two times higher than that retrieved from monolayer culture. Chondrocytes propagated in the thermo-reversible polymers expressed enhanced or maintained expression of collagen type II and aggrecan. Collagen type I expression was decreased or unaltered. The N-isopropylacrylamide and acrylic acid copolymer gel has potential use as a cell culture substrate and as a cell delivery vehicle.
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PMID:Thermally reversible polymer gel for chondrocyte culture. 1462 18

The proteoglycans aggrecan, versican, neurocan, and brevican bind hyaluronan through their N-terminal G1 domains, and other extracellular matrix proteins through the C-type lectin repeat in their C-terminal G3 domains. Here we identify tenascin-C as a ligand for the lectins of all these proteoglycans and map the binding site on the tenascin molecule to fibronectin type III repeats, which corresponds to the proteoglycan lectin-binding site on tenascin-R. In the G3 domain, the C-type lectin is flanked by epidermal growth factor (EGF) repeats and a complement regulatory protein-like motif. In aggrecan, these are subject to alternative splicing. To investigate if these flanking modules affect the C-type lectin ligand interactions, we produced recombinant proteins corresponding to aggrecan G3 splice variants. The G3 variant proteins containing the C-type lectin showed different affinities for various ligands, including tenascin-C, tenascin-R, fibulin-1, and fibulin-2. The presence of an EGF motif enhanced the affinity of interaction, and in particular the splice variant containing both EGF motifs had significantly higher affinity for ligands, such as tenascin-R and fibulin-2. The mRNA for this splice variant was shown by reverse transcriptase-PCR to be expressed in human chondrocytes. Our findings suggest that alternative splicing in the aggrecan G3 domain may be a mechanism for modulating interactions and extracellular matrix assembly.
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PMID:Alternative splicing in the aggrecan G3 domain influences binding interactions with tenascin-C and other extracellular matrix proteins. 1472 76

The effects of cyclic, mechanical compression on human bone marrow-derived mesenchymal progenitor cells undergoing chondrogenic differentiation were examined in this study. Mesenchymal progenitor cells were injected into cylindrical biodegradable scaffolds (hyaluronan-gelatin composites), cultured in a defined, serum-free chondrogenic medium and subjected to cyclic, mechanical compression. Scaffolds were loaded for 4 hours daily in the first 7 days of culture. At 1, 7, 14 and 21 days of culture, scaffolds were harvested for reverse transcriptase Polymerase Chain Reaction (RT-PCR), histology, quantitative DNA, proteoglycan and collagen analysis. Scaffolds loaded for 7 days showed a significant upregulation especially of chondrogenic markers (type II collagen, aggrecan; p<0.0001). No significant difference could be found for DNA content between loaded samples and unloaded controls. At day 1 in culture no significant differences in proteoglycan- and collagen contents could be detected between unloaded and loaded samples. After 21 days the proteoglycan (p<0.001) and collagen contents (p<0.0001) were significantly higher in the loaded samples compared to unloaded controls. By histological analysis (toluidine blue) a higher amount of proteoglycan-rich, extracellular matrix production throughout the matrix could be detected for loaded samples compared to unloaded controls. This study indicates that cyclic, mechanical compression enhances the expression of chondrogenic markers in mesenchymal progenitor cells differentiated in vitro resulting in an increased cartilaginous matrix formation, and suggests that mechanical forces may play an important role in cartilage repair.
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PMID:Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds. 1529 66

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.
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PMID:Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures. 1557 69

Adult porcine nucleus pulposus cells were seeded onto gelatin, demineralized bone matrix (DBM), and polylactide scaffolds and cultured in vitro. Cellular behavior in response to the scaffolds was analyzed using biochemical assays, histology, and real-time quantitative reverse transcriptase-polymerase chain reaction. Scanning electron microscopy showed pronounced differences in surface texture of the scaffolds. Nucleus pulposus cells attached and assumed an elongated fibroblast-like morphology on the gelatin and DBM scaffolds. The cells cultured on the gelatin and DBM were metabolically active and expressed types I and II collagen and aggrecan. Detached cellular aggregates with a rounded morphology were noted in the culture tubes containing the polylactide scaffolds. Both surface chemistry and texture likely had a role in causing differences in cellular behavior in response to scaffold material. Promising results were observed using the gelatin and demineralized bone scaffolds, but the behavior of cells cultured on these scaffolds will need to be characterized further. This initial research will be used to direct future work involved in developing this composite injectable tissue engineering system.
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PMID:Evaluation of polymer scaffolds to be used in a composite injectable system for intervertebral disc tissue engineering. 1592 Jul 36

To assess effects of Celecoxib, selective cyclo-oxygenase (COX)-2 inhibitor, on matrix synthesis by chondrocytes under mechanical stress in vitro. Chondrocytes from 7-day-old rat articular cartilage were cultured. Cyclic mechanical stress (0.5 Hz, 7% elongation) was loaded using a Flexercell strain unit in the presence of Celecoxib, Indomethacin, and Ketoprofen. Concentrations of chondroitin sulfate (CS) in culture media were measured by high-performance liquid chromatography. Nitric oxide (NO) formation was detected by measuring NO2-accumulation in culture supernatants during the Griess reaction, using sodium nitrite as a standard. Levels of mRNA for aggrecan (AGC), type II collagen (CII), COX-2, and membrane-associated prostaglandin E synthase-1 (mPGES-1) were measured using real time reverse transcriptase-polymerase chain reaction. Under mechanical stress, levels of AGC and CII mRNA were decreased, while COX-2 mRNA levels were increased. Of the three drugs tested, only Celecoxib prevented the decrease of AGC mRNA and inhibited the release of CS. In addition, Celecoxib suppresses the level of mPGES-1 mRNA. These findings show that excessive mechanical stress decreases synthesis of matrix components in chondrocytes through a NO-regulated pathway, and suggest that Celecoxib has a protective effect on matrix metabolism.
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PMID:The influence of Celecoxib on matrix synthesis by chondrocytes under mechanical stress in vitro. 1627 90

Articular cartilage degeneration in osteoarthritis (OA) involves type II collagen degradation and chondrocyte differentiation (hypertrophy). Because these changes resemble growth plate remodeling, we hypothesized that collagen degradation may be inhibitable by growth factors known to suppress growth plate hypertrophy, namely transforming growth factor (TGF)-beta2, fibroblast growth factor (FGF)-2, and insulin. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with TGF-beta2, FGF-2, and insulin in combination (growth factors) or individually. In cultured explants from five OA patients, collagenase-mediated type II collagen cleavage was significantly down-regulated by combined growth factors as measured by enzyme-linked immunosorbent assay. Individually, FGF-2 and insulin failed to inhibit collagen cleavage in some OA explants whereas TGF-beta2 reduced collagen cleavage in these 5 explants and in 19 additional explants. Moreover, TGF-beta2 effectively suppressed cleavage at low concentrations. Together or individually these growth factors did not inhibit glycosaminoglycan (primarily aggrecan) degradation while TGF-beta2 occasionally did. Semiquantitative reverse transcriptase-polymerase chain reaction of articular cartilage from six OA patients revealed that TGF-beta2 suppressed expression of matrix metalloproteinase-13 and matrix metalloproteinase-9, early (PTHrP) and late (COL10A1) differentiation-related genes, and proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha). In contrast, TGF-beta2 up-regulated PGES-1 expression and prostaglandin E(2) release. These observations show that TGF-beta2 can suppress collagen resorption and chondrocyte differentiation in OA cartilage and that this may be mediated by prostaglandin E(2). Therefore TGF-beta2 could provide therapeutic control of type II collagen degeneration in OA.
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PMID:Transforming growth factor-beta2 suppresses collagen cleavage in cultured human osteoarthritic cartilage, reduces expression of genes associated with chondrocyte hypertrophy and degradation, and increases prostaglandin E(2) production. 1640 16

Growth/differentiation factors (GDFs) play a significant role in numerous skeletal tissues and processes. Previous work using the brachypod mouse has suggested that GDF-5 affects Achilles tendon composition, ultrastructure, and material behavior, as well as tendon repair. The aim of the present study was to examine the role of a related GDF family member, GDF-7 (BMP-12), in intact tendon by studying the Achilles tendon of genetically engineered knockout mice. Achilles tendons from 16-week-old GDF-7 -/- mice contained 14% less GAG/DNA than did wild type littermates (p = 0.0481), although collagen content was comparable to controls. Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) results show that GDF-5 was upregulated two-threefold in response to the absence of GDF-7 protein. GDF-6 was also upregulated in knockouts, but to a lesser extent (twofold, p = 0.0013). On an ultrastructural level, GDF-7 deficient Achilles tendons exhibited a shift towards smaller diameter fibrils which resulted in a small but significant reduction in mean fibril diameter (-8%, p = 0.05). GDF-7 deficiency did not noticeably affect the expression of fibrillar collagens (I, III, V) or tendon proteoglycans (decorin, fibromodulin, lumican, biglycan, versican, aggrecan). Differences in tendon composition and ultrastructure were not biologically significant enough to have a noticeable effect on the structural or material behavior of the tendons. These results demonstrate that GDF-7 deficiency has a subtle effect on the composition and ultrastructure of murine Achilles tendon. The small magnitude of the observed differences may be due to overcompensation by related GDF family members.
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PMID:Achilles tendon characterization in GDF-7 deficient mice. 1651 25


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