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)

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

Hydrostatic pressure is one of the most frequently used mechanical stimuli in chondrocyte experiments. A variety of hydrostatic pressure loading devices have been used in cartilage cell experiments. However, no gas-controlled system with other than a low pressure load was used up to this time. Hence we used a polyolefin bag from which gas penetration was confirmed. Chondrocytes were extracted from bovine normal knee joint cartilage. After 3 passages, dedifferentiated chondrocytes were applied to form a pellet. These pellets were cultured in chemically defined serum-free medium with ITS+Premix for 3 days. Then 5 MPa of cyclic hydrostatic pressure was applied at 0.5 Hz for 4 h per day for 4 days. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 5-fold increase in the levels of aggrecan mRNA due to cyclic hydrostatic pressure load (p<0.01). Type II collagen mRNA levels were also upregulated 4-fold by a cyclic hydrostatic pressure load (p<0.01). Type I collagen mRNA levels were similarly reduced in the cyclic hydrostatic pressure load group and in the control group. The partial oxygen pressure (PO2) and partial carbon dioxide pressure (PCO2) of the medium in the bag reached equilibrium in 24 h, and no significant change was observed for 3 days afterwards. PO2 and PCO2 were very well controlled. The loaded pellet showed better safranin O/fast green staining than did the control pellet. Metachromatic staining by Alcian blue staining was found to be stronger in the loaded than in the control pellets. The extracellular matrices excretion of loaded pellets was higher than that of control pellets. These results suggest that gas-controlled cyclic hydrostatic pressure enhanced the cartilaginous matrix formation of dedifferentiated cells differentiated in vitro.
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PMID:Redifferentiation of dedifferentiated bovine articular chondrocytes enhanced by cyclic hydrostatic pressure under a gas-controlled system. 1734 96

The purpose of this study was to evaluate the benefits of in vitro preconditioning of mesenchymal stem cells (MSCs) using low-intensity ultrasound (US) in the induction of chondrogenic differentiation of MSCs in vivo. After rabbit bone marrow-derived MSCs were seeded onto a polyglycolic acid (PGA) scaffold, the PGA-MSCs constructs were divided into 4 subgroups: untreated control, low-intensity US group, transforming growth factor-beta [TGF]-treated group and low-intensity US/TGF group. The chondrocyte-seeded PGA construct served as a positive control. For 1 week before implantation, the low-intensity US groups were subjected to ultrasound treatment for 20 min daily at an intensity of 200 mW/cm(2). The TGF groups were treated with 10 ng/mL TGF-beta1. The cells were then implanted into the nude mouse subcutaneously. Retrieved 1, 2, 4, and 6 weeks after implantation, each construct underwent gross examination, histology, biochemical assays, mechanical testing, and reverse transcriptase polymerase chain reaction (RT-PCR). Substantial size reduction and blood invasion were found much earlier in the groups that did not undergo low-intensity US than in those that did. Safranin O/Fast green staining revealed that the chondrogenic differentiation of MSCs was more widespread throughout the constructs in the low-intensity US groups. In the biochemical and mechanical analyses, the low-intensity US and low-intensity US/TGF groups were significantly better in forming hyaline cartilage-like tissue by 4 weeks than the non-low-intensity US groups. Presented by von Kossa staining, the development of osteogenic phenotypes was highly suppressed until 4 weeks in the low-intensity US groups, along with compressive strength comparable to the positive control. In the RT-PCR analysis before implantation, the messenger RNA levels of Sox-9, aggrecan, and tissue inhibitors of metalloproteinase-2 were higher in the low-intensity US groups, while those of type I and type X collagens and matrix metalloproteinase-13 were higher in the non-low-intensity US groups. Blood invasion into the constructs was also considerably hindered in the low-intensity US groups. These results strongly indicate that low-intensity US preconditioning in vitro could be an effective cue to upregulate chondrogenic differentiation of MSCs in vivo.
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PMID:Preconditioning of mesenchymal stem cells with low-intensity ultrasound for cartilage formation in vivo. 1751 69

The temporomandibular joint (TMJ) disc is maintained by a population of fibrochondrocytes. Although articular chondrocytes exhibit zonal differences and de-differentiate in monolayer culture, such variations are unknown for fibrochondrocytic populations. This study's objective was to define topographical cellular variations in the porcine TMJ disc and investigate changes in the disc's gene expression levels over multiple passages using quantitative reverse transcriptase polymerase chain reaction. For topographical characterization, samples were acquired from posterior, anterior, lateral, medial, and intermediate zone sections and subdivided into inferior and superior halves. For passage characterization, cells were plated and passaged for 35 days, with samples acquired at every passage. The medial region had the lowest expression of genes indicative of fibroblastic activity, but in general, topographical variations were limited. Passage effects were evident; gene expression levels of aggrecan, collagen type I, and collagen type II dropped 20%, 23%, and 73% per passage, respectively. In contrast, decorin and glyseraldehyde-3-phosphate dehydrogenase gene expression increased 33% and 27% per passage, respectively. These data indicate that TMJ disc cells undergo significant changes due to monolayer expansion, experiencing losses in major chondrocytic markers (aggrecan and collagen type II) and fibroblastic markers (collagen type I) and posing a serious impediment to studies in which cell passaging is required.
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PMID:Effect of passage and topography on gene expression of temporomandibular joint disc cells. 1751 84

Chondrocytes, the cellular component of the articular cartilage, have long been recognized as strain-sensitive cells, and have the ability to sense mechanical stimulation through surface receptors and intracellular signaling pathways. This strain-induced biological response of chondrocytes has been exploited to facilitate chondrocyte culture in in vitro systems; examples include the application of hydrostatic pressure, dynamic compression, hydrodynamic shear (i.e., rotating bioreactors), and low-intensity pulsed ultrasound (US). While the ability of US to influence chondrogenesis has been documented, the precise mechanisms of US-induced stimulation continue to be investigated. There remains a critical need to evaluate the impact of US on chondrocytes in 3D culture, which is a necessary microenvironment for maintaining the chondrocyte phenotype. In this study, a continuous US wave for predetermined time intervals was employed, as opposed to pulsed US used in previous studies, to stimulate chondrocytes seeded in 3D scaffolds. The chondrocytes (n = 6) were subjected to US stimulation as follows: 1.5 MHz for 161 seconds, 5.0 MHz for 51 seconds, and 8.5 MHz for 24 seconds, and the US signal was applied twice in a 24-hour period. Scaffolds that are not stimulated by US served as the control. Both the control and the US-stimulated groups were maintained in culture for 10 days, and at the conclusion of the culture period, chondrocytes were assayed for total DNA content, morphology, and cartilage-specific gene expression by reverse transcriptase polymerase chain reaction. Our results show that chondrocytes when stimulated with continuous US for predetermined time intervals possessed higher cellular viability (1.2 to 1.4 times) and higher levels of type II collagen and aggrecan mRNA expression when compared to controls.
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PMID:Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices. 1751 7

Adipose-derived adult stem cells (ADASCs) or bone marrow-derived mesenchymal stem cells (BMSCs) are considered as alternative cell sources for cell-based cartilage repair due to their ability to produce cartilage-specific matrix. This article addresses the differential expression pattern of extracellular matrix (ECM) molecules in BMSCs or ADASCs following chondrogenic differentiation. Human BMSCs or ADASCs were encapsulated in alginate and cultured in TGF-beta1-containing medium for 2 or 3 weeks. Chondrospecific mRNA expression was analyzed and alternative splicing of alpha(1)-procollagen type II mRNA was monitored via reverse transcriptase-polymerase chain reaction (RT-PCR). Corresponding ECM synthesis was demonstrated using immunohistochemistry. After chondroinduction, expression of collagen type II, type X, COMP and aggrecan mRNA was 3-15-fold higher than in ADASCs. The type IIA splicing form of alpha(1)-procollagen type II was expressed in both populations, and the type IIB splicing form was exclusively detected in BMSCs. In response to TGF-beta, collagen type II and X were secreted more strongly by BMSCs than by ADASCs. BMSCs express a more mature phenotype than ADASCs after chondroinduction. TGF-beta1 induces alternative splicing of the alpha(1)-procollagen type II transcript in BMSCs, but not in ADASCs. These findings may direct the development of a cell-specific culture environment either to prevent hypertrophy in BMSCs or to promote chondrogenic maturation in ADASCs.
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PMID:Differential expression pattern of extracellular matrix molecules during chondrogenesis of mesenchymal stem cells from bone marrow and adipose tissue. 1751 54

This study investigated the differential effects of ramped and steady applications of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in 3-dimensional culture in the absence of transforming growth factor-beta (TGF-beta). A custom hydrostatic pressure system was designed and manufactured. hMSCs were seeded in agarose and exposed to steady (7.5 MPa) or ramped (1 MPa to 7.5 MPa over a 14-day period) CHP for 4 h/d at f = 1 Hz for 14 days. Real-time reverse transcriptase polymerase chain reaction analysis was performed on days 0, 4, 9, and 14 to determine changes in messenger ribonucleic acid (mRNA) expression levels of Sox9, aggrecan, collagen I, and collagen II. Collagen II and aggrecan mRNA expression remained unchanged. Collagen I increased at day 4 in CHP specimens before decreasing to levels at or below same-day unloaded controls at days 9 and 14. On average, ramped and steady regimens of CHP increased Sox9, with the largest upregulation occurring at day 4 in response to steady pressure. These findings indicate that hydrostatic pressure may induce chondrogenesis in hMSC-seeded agarose constructs without TGF-beta, and that hMSCs are capable of withstanding high initial pressures that may initiate chondrogenesis faster than lower pressures.
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PMID:Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure. 1751 10

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after initial progeny in a monolayer with a serum-free medium. Two subgroups of nontreated and plasma-treated (using low-pressure plasma technique) scaffolds were investigated. The constructs were cultivated after seeding in six-well plates with serum-free medium for 7 days and implanted subcutaneously into nude mice for 6 and 12 weeks. Chondrogenic differentiations were investigated using immunhistology and reverse transcriptase-polymerase chain reaction. Cell adhesion only differed from 50% to 65% without a significant difference between the groups. During further cultivation for 7 days, the aggrecan synthesis of the seeded constructs was always higher in the PGA groups (p < 0.05). The mRNA gene expression for collagen type II was significantly higher in the PGA groups after 6 and 12 weeks (p < 0.05). A decrease in the expression of collagen type I was investigated in all groups. The expression for collagen type X and cartilage oligomeric matrix protein (COMP) increased in all groups over time. After cell proliferation in serum-free medium, the long-term chondrogenic differentiation in PGA scaffolds in vitro is cartilage specific and may be utilized in cartilage tissue engineering applications.
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PMID:Chondrogenic differentiation of human articular chondrocytes differs in biodegradable PGA/PLA scaffolds. 1769 68


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