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)

Hyaluronan (HA) is a component of cartilage matrix with known effects on chondrocytes. We tested the effects of adding HA to 3-dimensional (3-D) collagen. sponges on chondrocyte function in vitro. Bovine articular chondrocytes isolated by collagenase digestion were injected into either collagen or HA/collagen scaffolds comprising different amounts of HA (2, 5, 10, and 14% w/w). Expression of aggrecan and type II collagen genes was measured by gene-specific quantitative competitive reverse transcriptase-polymerase chain reactions, and the extracellular matrix was estimated by histomorphometrical analyses. After 7-day culture, the chondrocytes in 2% (w/w) HA sponges expressed fourfold more mRNA transcripts for type II collagen (p = 0.002) and twofold more mRNA transcripts for aggrecan (p = 0.022) than in control collagen sponges. Furthermore, there was 45% more extracellular matrix in 2% (w/w) HA sponges and 43% less matrix in the 10% (w/w) HA sponges compared with plain collagen sponges (p > 0.05). In sum, a small amount of HA in 3-D collagen scaffolds enhanced chondrogenesis, but a greater amount was inhibitory. This 3-D system represents a novel tool to identify mechanisms by which extracellular matrix molecules influence chondrocyte function. Further, these results show the potential for modifying scaffolds to improve production of engineered cartilage for in vivo applications.
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PMID:Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds. 1142 90

The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type 11 collagen matrices with and without CS. Type II collagen matrices were prepared using purified, pepsin-treated, type II collagen. Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.
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PMID:Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering. 1210 90

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

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with alpha(1)-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.
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PMID:Trypsin-2 degrades human type II collagen and is expressed and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue. 1619 46

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

A novel biodegradable graft copolymer chondroitin sulfate-grafted poly(L-lactide) (CS-PLLA) was synthesized. The graft copolymer was blended with PLLA to form biomimetic porous scaffolds. Natural CS was introduced into the polyester matrix to promote the proliferation of cells. Three-dimensional spongelike scaffolds were fabricated by a combination of salt leaching and solvent casting methods. The morphology of the scaffolds was observed with scanning electron microscopy with an average pore size between 50 and 250 microm, and its porosity was high (>85%). Compression analysis indicated that the mechanical properties of the scaffold were adequate to support the proliferation of cells. The hydrophilicity increased with an increase in the copolymer content in the blend, as determined by measuring the contact angle. Hematoxylin and eosin, Masson, and Safranin-O staining showed that cells formed a chondro tissue gradually. Histological results revealed that abundant cartilaginous matrixes surrounded spherical chondrocytes in the center of the explants. Chondrocytes cultured in this extracellular-matrix-like scaffold maintained a round morphology phenotype, characterized by a significant quantity of extracellular matrixes of sulfated glycosaminoglycans and collagens. Additionally, phenotypic gene expression (reverse transcriptase-polymerase chain reaction) indicated that chondrocytes expressed transcripts that encoded type II collagen and aggrecan and generated sulfated glycosaminoglycans.
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PMID:Biomimetic porous scaffolds made from poly(L-lactide)-g-chondroitin sulfate blend with poly(L-lactide) for cartilage tissue engineering. 1682 88

Tissue engineering of articular cartilage usually requires the isolation and culture of chondrocytes. Previous studies have suggested that enzymatic isolation may alter the metabolic activity and growth rate of chondrocytes. This study examined the effects of 4 common isolation protocols on chondrocyte gene expression, morphology, and total cell yield immediately following the digest (t = 0) and after 2 culture periods (24 h and 1 week). Cartilage explants were digested using 1 of 4 protocols: (1) 6-h collagenase digest, (2) 22-h collagenase digest, (3) 45-min trypsin digest followed by a 3-h collagenase digest, or (4) 1.5-h pronase digest followed by a 3-h collagenase digest. Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, type II collagen, aggrecan, superficial zone protein, matrix metalloproteinase- 1, and tissue inhibitor of metalloproteinase-1 were measured at t = 0 h, 24 h, and 1 week using quantitative reverse transcriptase-polymerase chain reaction. In this study, cell yield was greatest for the 22-h collagenase and pronase-collagenase digests. However, the data indicate that a 6-h collagenase digest has the fewest gene expression changes compared to native cells. For tissue engineering, data from this study suggest that when cell yield is critical, a 22-h collagenase digest is preferable, but when obtaining cells closest to native chondrocytes is more desired, the 6-h collagenase digest is more beneficial.
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PMID:The effects of isolation on chondrocyte gene expression. 1699 90

The purpose of this study was to examine the role of the synovium in the transitional zone between the articular cartilage and the synovial membrane in cartilage repair and the relationship between the origin of the repaired cartilage and the grafted synovium. We used 8-week-old Sprague Dawley (SD) rats and green fluorescent protein (GFP) transgenic rats. In study 1, a full-thickness cartilage defect was created at the medial condyle of the femur, and the synovium 5 x 5 mm extending up to the cartilage defect was resected in the left knee (cartilage defect without synovium group) but not resected in the right knee (cartilage defect with intact synovium group). In study 2, after the creation of a full-thickness cartilage defect and resection of the synovium, the synovium of the GFP rats was transplanted into the unilateral knee (cartilage defect with transpl.synovium group). At 2, 4, 6, and 8 weeks after surgery, we evaluated the repaired tissue in cartilage defects histologically and immunohistochemically, and the expression of aggrecan and type II collagen in the repaired tissue was also investigated using reverse transcriptase-polymerase chain reactions (RT-PCR). At 6 and 8 weeks after surgery, the defect was filled with cartilage-like tissue in cartilage defect with intact synovium group and cartilage defect with transpl.synovium group, but not in cartilage defect without synovium group. GFP positive cells were observed in the repaired tissue and the expression of aggrecan and type II collagen was found in cartilage defect with transpl.synovium group. We concluded that the synovium in the transitional zone between the articular cartilage and the grafted synovial membrane invades the cartilage defects where the cells could be detected as GFP-positive cells. Those cells may take part in the repair and may induce chondrogenesis.
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PMID:The role of the synovium in repairing cartilage defects. 1727 25


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