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)

Owing to their plasticity and high proliferation capacity in vitro, mesenchymal stem cells (MSC) isolated from human bone marrow are promising candidates for use in tissue engineering approaches for the repair or replacement of mesenchymal tissues such as bone, cartilage or tendon. In keeping with the tissue engineering concept, these cells are cultivated on three-dimensional (3D) scaffolds to replace 3D tissue defects. Among the scaffolds tested for tissue engineering of bone, those containing phosphorus and calcium, as natural bone does, are the most promising candidates for this purpose. In this study, MSC from five patients were isolated from bone marrow. After in vitro expansion, cells were cultivated and differentiated towards the osteogenic lineage on mineralized collagen sponges and alpha-tricalcium phosphate (alpha-TCP). To analyze how appropriate these scaffolds would be for tissue engineering purposes, we established an in vitro characterization system to describe seeding efficiency, cell distribution and proliferation behavior on each scaffold. Real-time reverse transcriptase polymerase chain reaction quantification of important genes involved in osteogenic differentiation [e.g. bone sialoprotein (BSP), bone morphogenic protein 2, alkaline phosphatase and osteocalcin] was used to monitor the differentiation process of cells seeded on mineralized collagen and alpha-TCP. Using this in vitro characterization, we were able to demonstrate effective 3D growth of MSC on both scaffolds investigated. Improved osteogenic differentiation was observed on the scaffolds as compared to control monolayers. Of the two matrices, mineralized collagen was superior to alpha-TCP with regard to seeding efficacy (98 vs. 67%, p = 0.0003), increase in osteogenic marker genes (BSP expression on day 24, Pcollagen vs. TCP = 0.046) and 3D cell alignment (cell infiltration up to 500 vs. 200 microm). In conclusion, our data suggest that mineralized collagen is a promising candidate for use as a scaffold in tissue engineering of bone.
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PMID:Evaluation of mineralized collagen and alpha-tricalcium phosphate as scaffolds for tissue engineering of bone using human mesenchymal stem cells. 1529 81

MEPE, 56.6 kDa protein isolated from tumors associated with hypophosphatemic osteomalacia, increases renal phosphate excretion and is expressed in normal human bone cells. AC-100, a central 23-amino acid fragment of MEPE, contains motifs that are important in regulating cellular activities in the bone microenvironment. Thus, we assessed in vitro effects of AC-100 on multipotential normal human marrow stromal (hMS) cells that have the capacity to differentiate into mature osteoblasts. Proliferation was quantified by [H3]thymidine uptake and cell counting and differentiation by the levels of mRNA for the alpha2-chain of type I procollagen (COL1A2), alkaline phosphatase (AP), and osteocalcin (OC) measured using real time reverse transcriptase PCR (RT-PCR) and by the formation of mineralized nodules. AC-100 increased proliferation by 257 +/- 89% (P < 0.005), increased gene expression of COL1A2 by 339 +/- 85% (P < 0.005), AP by 1,437 +/- 40% (P < 0.001), and OC by 1,962 +/- 337% (P < 0.001). In addition, it increased mineralized nodule formation by 81 +/- 14% (P < 0.001) in a dose- and time-dependent fashion. In equimolar dosages, the parent compound, MEPE, had the full activity of the AC-100 fragment. AC-100 elicited a comparable response to both IGF-I and BMP-2 with respect to proliferation and differentiation of hMS cells. Using gene expression microarray analysis, we demonstrated that AC-100 increased (by approximately 3-fold) the mRNA for cyclooxgenase-2 (COX-2), an inducible enzyme required for prostaglandin synthesis. Moreover, NS-398, a specific inhibitor of COX-2 action completely blocked AC-100-induced increases in proliferation and differentiation. Thus, AC-100 has potent anabolic activity on osteoblast precursor cells in vitro and these effects require the induction of COX-2.
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PMID:A fragment of the hypophosphatemic factor, MEPE, requires inducible cyclooxygenase-2 to exert potent anabolic effects on normal human marrow osteoblast precursors. 1544 21

It is important to elucidate whether the leptin receptor, especially the long signal-transducing form of the leptin receptor (OB-Rb) is expressed in human osteoblasts. We detected the expression of human OB-Rb in cultured commercially available human osteoblasts (NHOst cells) using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). After confirming the expression of OB-Rb, we investigated the effect of leptin on NHOst cells. Leptin enhanced cell proliferation of the cells shown by the MTT assay. Furthermore, leptin changed the copy numbers of Bax and Bcl-2 mRNAs in the cultured cells as shown by real-time quantitative RT-PCR, although the effect was not consistent. Leptin did not change the production of osteocalcin and osteopontin by the cells. Leptin did not change the expression of OB-Rb mRNA in the cells. In conclusion, OB-Rb mRNA is expressed in cultured commercially available human osteoblasts. Leptin may have some effects on bone metabolism by directly modulating cell proliferation and apoptosis of osteoblasts in humans.
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PMID:The leptin receptor in human osteoblasts and the direct effect of leptin on bone metabolism. 1562 71

The biological mechanisms underlying bone-titanium integration and biomechanical properties of the integrated bone are poorly understood. This study assesses intrinsic biomechanical properties of mineralized tissue cultured on titanium having different surface topographies. The osteoblastic phenotypes associated with mineral deposition and collagen synthesis underlying the biomechanical modulation are also reported. Rat bone marrow-derived osteoblastic cells were cultured either on the machined titanium disc or acid-etched titanium disc. Nano-indentation study of day 28 culture revealed that the mineralized tissue on the acid-etched surface shows 3-3.5 times greater hardness than that on the machined surface (p < 0.01). Elastic modulus of the mineralized tissue was also 2.5-3 times greater on the acid-etched surface than on the machined surface (p < 0.01). After 28 days of culture, mineralized nodule area was significantly lower on the acid-etched surface than on the machined surface (p = 0.0105), while total calcium deposition did not differ between the two surfaces, indicating denser mineral deposition on the acid-etched surface. Osteopontin and osteocalcin gene expressions assayed by the reverse transcriptase-polymerase chain reaction were upregulated in the acid-etched titanium culture. Collagen synthesis measured by Sirius red stain-based colorimetry was 1.5-10 times higher on the acid-etched surface than on the machined surface in the initial culture period of day 1 to day 14 (p < 0.0001). The amount of collagen synthesis corresponded with the enhanced gene expression of prolyl 4-hydroxylase, a key enzyme for post-translational modification of collagen chains. Scanning electron microscopic images revealed that tissue cultured on the acid-etched titanium exhibited plate-like, compact surface morphology, while the tissue on the machined titanium appeared porous and was covered by fibrous and punctate structures. We conclude that culturing osteoblasts on rougher titanium surfaces enhances hardness and elastic modulus of the mineralized tissue, associated with condensed mineralization, accelerated collagen synthesis, and upregulated expression of selected bone-related genes.
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PMID:Enhanced intrinsic biomechanical properties of osteoblastic mineralized tissue on roughened titanium surface. 1565 12

Fluorine was administered to a system of hydroxyapatite (HA)/polycaprolactone (PCL) ceramic-polymer bioactive composites for applications as hard tissue regeneratives. The HA was fluoridated at different levels (5%, 25%, 50% and 75%) in order to produce the fluor-hydroxyapatite (FHA)/PCL composites. The osteoblastic cellular responses to the composites were examined in terms of the cell attachment, proliferation and differentiation as well as the expression of bone-associated genes. The amount of fluorine released from the composites was controlled by changing the degree of fluoridation, and the cellular responses were strongly influenced by the level of fluoridation. The MG63 cells on the FHA-PCL attached and proliferated at a similar level to those on HA-PCL. However, the fluoridation of HA increased significantly the alkaline phosphatase (ALP) activity and osteocalcin (OC) production by the cells on the composites, which was measured by an enzymatic assay. Moreover, the gene expression level of ALP and OC in the cells was up regulated on the FHA-PCL, which was confirmed semi-quantitatively by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. These findings on the fluorine-administered biological composites (FHA-PCL) suggested that fluorine plays a significant role in stimulating the bone derived cellular activity, and the FHA-PCL composites have high potential for use as hard tissue regeneratives.
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PMID:Effect of fluoridation of hydroxyapatite in hydroxyapatite-polycaprolactone composites on osteoblast activity. 1570 68

CdSe/ZnS quantum dots (QDs) have recently been used as cell tracers for long term imaging of live cells. A number of studies indicate that introduction of quantum dots to cells have no apparent deleterious effects on the morphology or growth of cells. In the present study, the human bone marrow mesenchymal stem cells (hBMSCs) were used as a model to examine the effects of QDs on the growth and osteogenic differentiation of the cells. The CdSe/ZnS QDs were delivered into hBMSCs by liposome-mediated transfection with high efficiency; analysis by transmission electron microscopy revealed that the internalized QDs could be located in the endosome-like vesicles. Uptake of QDs into hBMSCs did not affect the proliferation and cell cycle distribution of the cells. When induced to differentiate along the osteogenic lineage, the QD-containing-hBMSCs were shown to have mineral deposition on the extracellular matrix. However, the cells displayed lower alkaline phosphatase activity as compared to those without QDs. Analysis by reverse transcriptase polymerase chain reaction further demonstrated that the expression of two osteogenic markers, osteopontin and osteocalcin, was significantly inhibited. Together our results show that the presence of QDs in hBMSCs prevents the full response of the cells to induced osteogenic differentiation.
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PMID:The inhibition of osteogenesis with human bone marrow mesenchymal stem cells by CdSe/ZnS quantum dot labels. 1618 13

Tissue differentiation is based on the expression of transcription factors, receptors for cytokines, and nuclear receptors that regulate a specific phenotype. The purpose of this study was to select cells from various skeletal tissues in order to analyse differential gene expression of cells in the native environment in vivo. It is a difficult task to obtain cells from skeletal tissues, such as cartilage, periost, bone and muscle, that are structured together and do not exist as individual organs. We used laser capture microdissection which permits the selection and isolation of individual cells from tissue sections. The RNA isolated from these tissues was used for reverse transcriptase-polymerase chain reactions for molecular analysis. We analysed the expression of transcription factors (cFOS, cbfa1, MyoD), receptors for cytokines, nuclear receptors, alkaline phosphatase and the structural proteins osteocalcin and collagen II. The results obtained demonstrate differential patterns of gene expression according to the tissue arrangement in their native in vivo environment, with reliable interpretation of the functions of the analysed genes in the context of intact skeletal tissue physiology.
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PMID:Gene expression in skeletal tissues: application of laser capture microdissection. 1626 58

Human osteoblast cell line (MG63) cells were treated with long wave (45 kHz, intensity 30 mW/cm(2)) continuous ultrasound (US) for 5 min and incubated for various time periods following the treatment. The reverse transcriptase polymerase chain reaction (RT-PCR) technique was used for observing the genetic expression and real-time PCR for quantitative analysis of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) along with alkaline phosphatase (ALP), an early bone marker, and osteocalcin (OCN) a late marker. ELISA was performed to estimate the amount of the cytokine released into the culture media. The osteoblasts responded to US by significantly upregulating both the OPG mRNA and protein levels. There was no RANKL mRNA expression observed in both the US and control groups and the protein levels were also very low in both groups. There was also no TNF-alpha expression and the TNF-alpha protein levels were insignificant. ALP and OCN mRNA were significantly upregulated in the US group. To our knowledge, this is the first study that shows the effect of US on OPG, RANKL and TNF-alpha expression. US appears to upregulate OPG and may downregulate RANKL production. From these findings, we conclude that therapeutic ultrasound may increase bone regeneration by altering the OPG/RANKL ratio in the bone microenvironment.
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PMID:Long wave ultrasound may enhance bone regeneration by altering OPG/RANKL ratio in human osteoblast-like cells. 1656 38

This study analyzed human mesenchymal stem cell (hMSC) behavior in a fibrin sealant. hMSC morphology, proliferation, and osteogenic differentiation were analyzed after up to 28 days of incubation in eight different formulations of fibrin gels (Tisseel) prepared with various concentrations of fibrinogen complex (FC) and thrombin. Cell morphology and distribution within the gels were observed by fluorescence microscopy after cell staining with calcein dye. Cell proliferation was assessed by measuring the fluorescence intensity of the cell suspension stained with calcein dye after dissolution of the gels. A standard alkaline phosphatase (ALP) assay, von Kossa staining, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze hMSC osteogenic differentiation. Cell behavior varied depending on the gel formulation. Proliferation was higher in the formulations containing a low FC concentration, but ALP activity was higher in the formulations containing a high FC concentration. Variations in thrombin concentration had a lesser effect. Small nodules of mineralization were observed at days 21 and 28 in a formulation containing a high FC concentration, in addition to a marked increase in bone sialoprotein (BSP) gene expression level as well as a lower increase in ALP and osteopontin (OPN) levels. However, there was no significant increase in osteocalcin (OCN) expression, a late marker of osteogenic differentiation, up to day 28. In conclusion, this study demonstrated that hMSC morphology, proliferation, and osteogenic differentiation in fibrin gels depended on the FC/thrombin ratio. hMSCs appeared to undergo osteogenic differentiation when seeded in Tisseel fibrin sealant containing a high FC concentration, but they did not fully differentiate into mature osteoblasts.
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PMID:Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. 1696 77

The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.
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PMID:Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum. 1697 24


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