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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biopsies removed from 57 patients considered for cartilage transplantation were grown at CTI Ltd. (47 biopsies) and at Tel Aviv University (10 biopsies). Tissue processing took place in dedicated laboratories. Explant cultures allowed cell number expansion. Fifty-four out of 57 biopsies grew cells. Fanning out of the cells began after 5-15 days in culture. Two passages later, cell numbers in the 10(7) range were achieved. Cells from all cultures expressed mRNA of aggrecan and link protein but not of alkaline phosphatase. Histochemical stains such as alcian blue pH 1 were negative in sparse monolayer cultures, but positive in pellet cultures. Immunohistochemistry demonstrated expression of collagen type I in monolayer cultures, switching to collagen type II in micromass cultures. Fibroblast growth factor receptor 3, a recently described characteristic receptor of precartilaginous cells, was expressed in monolayers and disappeared in micromass cultures. In conclusion, explants of articular chondrocytes cultured in vitro consistently yield monolayer cultures. The cells appear to revert to dedifferentiated chondrocytes, expressing a mesenchymal stem cell protein profile. Simultaneously, these cells regained their capacity to proliferate. Cultures held as micromass allowed reexpression of the differentiated phenotype traits.
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PMID:Characteristics of cartilage biopsies used for autologous chondrocytes transplantation. 1133 35

We investigated the effects of transplantation of osteoblastic cells with a bone morphogenetic protein (BMP)/carrier complex on bone repair by in vitro and in vivo experiments. Poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS) was used as a carrier for cell transplantation. In the in vitro experiments, three cell types, C3H10T1/2 cells, MC3T3-E1 cells, and primary osteoblastic cells, isolated from newborn rat calvariae (ROB cells), were cultured for 2 weeks on PGS alone or PGS containing BMP-2 (PGS/BMP). C3H10T1/2 cells cultured on PGS/BMP expressed several markers related to differentiation of both osteoblasts and chondrocytes, such as alkaline phosphatase (ALP) activity and mRNAs for osteocalcin and aggrecan, whereas the cells cultured on PGS alone expressed no such markers. MC3T3-E1 cells cultured on PGS/BMP exhibited a more ALP-positive cells than those cultured on PGS alone. PGS/BMP promoted ROB cell differentiation into both osteoblasts and chondrocytes. In the in vivo experiments, we transplanted ROB cells, which had been cultured on PGS alone or PGS/BMP in vitro for 2 weeks, into bone defects created in rat calvariae. Transplantation of ROB cells cultured on PGS alone generated little new bone. Transplantation of ROB cells cultured on PGS, which absorbed a low dose (10 ng) of rhBMP-2,; induced significantly higher bone mineral content than PGS/BMP alone, although application of a high dose (1 microg) of rhBMP-2 induced no difference in bone mineral content between transplantation of PGS/BMP with or without ROB cells. These results show that transplantation of osteoblastic cells after induction of osteoblast maturation in vitro by cultivation on PGS/BMP is a potent technique for cell therapy of bone repair.
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PMID:The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)/carrier complex on bone repair. 1150 79

The localized expression of a number of extracellular matrix genes was evaluated over time in a novel rat rotator cuff injury model. The supraspinatus tendons of rats were severed at the bony insertion and repaired surgically. The healing response was evaluated at 1, 2, 4, and 8 weeks post-injury using histologic and in situ hybridization techniques. Expression patterns of collagens (I, II, III, IX, X, XII), proteoglycans (decorin, aggrecan, versican, biglycan, fibromodulin), and other extracellular matrix proteins (elastin, osteocalcin, alkaline phosphatase) were evaluated at the healing tendon to bone insertion site. Histologic results indicate a poor healing response to the injury, with only partial recreation of the insertion site by 8 weeks. In situ hybridization results indicate a specific pattern of genes expressed in each zone of the insertion site (i.e., tendon, fibrocartilage, mineralized cartilage, bone). Overall, expression of collagen types I and XII, aggrecan, and biglycan was increased, while expression of collagen type X and decorin was decreased. Expression of collagen type I, collagen type XII, and biglycan decreased over time, but remained above normal at 8 weeks. Results indicate that the rat supraspinatus tendon is ineffective in recreating the original insertion site, even at 8 weeks post-injury, in the absence of biological or biomechanical enhancements.
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PMID:The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study. 1203 18

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.
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PMID:CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates proliferation and differentiation, but not hypertrophy of cultured articular chondrocytes. 1211 36

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-beta1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated proteoglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, beta-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor gamma2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.
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PMID:Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. 1238 74

Stem cell-based therapies for degenerative disorders and injuries are promising in the new era. Multipotential mesenchymal stem cells (MSCs) from bone marrow (BM) are on the leading edge because they are easy to expand in culture while maintaining their multilineage potential. In vitro assessment of the chondrogenic and osteogenic potentials of cultured MSCs has been established, and the BM used in those experiments was exclusively from healthy donors via iliac crest aspiration. It is unknown whether human marrow obtained from femurs also contains these multipotential MSCs. We collected marrow from proximal femurs of two patients undergoing total hip replacement surgery for femoral head osteonecrosis and isolated and culture expanded MSCs to about 20 population doublings. These cells were homogeneously positive for beta1-integrin. When pelleted into aggregates and cultured in a medium containing transforming growth factor-beta3 for 14 days, the cells began to express mRNA for aggrecan and collagen type II and to deposit immunoreactive collagen type II and sulfated proteoglycans in the matrix, hallmarks of chondrogenic differentiation. These MSCs could also be differentiated into osteocytic lineage in vitro, as shown by increased expression of alkaline phosphatase activity and deposition of mineral content onto culture plates. These results indicate that femoral BM obtained during hip surgeries also contained multipotential MSCs. These data imply that direct replacement therapy using MSCs from in situ marrow may be possible in the future and that an MSC bank may be established by using marrow from this approach, bypassing the necessity for iliac marrow aspiration from healthy donors.
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PMID:Multipotential mesenchymal stem cells from femoral bone marrow near the site of osteonecrosis. 1263 15

The characteristics of the nucleus pulposus cells from adult rabbits maintained in in vitro cultures were described in another study. Herein, the authors provide a parallel profile of adult rabbit nucleus pulposus in situ, therefore allowing direct comparisons between in vitro and in situ investigations. Nucleus pulposus specimens from adult rabbits were evaluated using biochemical and immunohistochemical morphologic techniques. The nucleus pulposus from adult rabbits contained cell clusters embedded in proteoglycan-collagen matrix. The cells exhibited a well-defined Golgi system, an extensive endoplasmic reticulum, and a complex vesicular system filled with beaded structures (proteoglycans). Neither necrotic nor apoptotic cells were evident. There was a lack of mitochondria. The extensive extracellular matrix contained amorphous, beaded, and fibrillar components. The fibrillar banding was indicative of Type VI collagen. The nucleus pulposus of adult rabbits expressed aggrecan, collagen Type I and Type II, and CD44, but not collagen Type X and displayed low alkaline phosphatase activity.
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PMID:Intervertebral disc tissue engineering I: characterization of the nucleus pulposus. 1278 89

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

Osteogenic protein-1 (OP-1, also called BMP-7), a member of the BMP family and the TGF-beta superfamily, induces formation of new bone and cartilage, but also regulates a wide array of processes. In the present study, the expression of several characteristic biochemical markers of ligaments, such as Six1, Scleraxis, aggrecan, and type I collagen in primary cultures of adult rat medial collateral ligament (MCL) cells was determined. The effects of OP-1 on cell proliferation and on gene expression were subsequently examined. OP-1 stimulated cell proliferation, alkaline phosphatase (AP) activity, and the steady-state mRNA levels of the transcription factor Runx2/Cbfa1 in a dose- and time-dependent manner. The mRNA levels of type I collagen only increased slightly, but the activity of the cloned collagen promoter increased by 2-fold in transiently transfected MCL cells. OP-1 also stimulated aggrecan mRNA expression. The mRNA levels of Six1 and Scleraxis were not detectably altered by OP-1. In control cultures, the steady-state mRNA levels of ActR-I, BMPR-IA, BMPR-IB, and BMPR-II increased as a function of time in culture. The mRNA levels of BMP-1 and -4 increased significantly after 12 days, but those of BMP-2 and -6 did not change. The GDF-1, -3, -5, -6, and -8 mRNA levels in the control cultures also increased as a function of time. OP-1 treatment stimulated mRNA expression of BMPR-IA and BMPR-II, but had little effect on ActR-I and BMPR-IB mRNA expression. OP-1 lowered the BMP-1, -2, and -6 mRNA levels without changing the BMP-4 mRNA level. OP-1 treatment also reduced the mRNA levels of GDFs detected. In summary, the present study demonstrated that OP-1 stimulated cell proliferation and mRNA expression of several biochemical markers in this ligament cell culture model and established the spatial and temporal appearance of several members of the TGF-beta superfamily.
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PMID:Effects of osteogenic protein-1 (OP-1, BMP-7) on gene expression in cultured medial collateral ligament cells. 1458 33

Recent studies suggest that human adipose tissue contains pluripotent stem cells similar to bone marrow-derived stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. In the present study, we extend this approach to characterize adipose tissue-derived stromal cells, sometimes called processed lipoaspirate (PLA) cells. Adipose-derived stromal cells (ASCs) were isolated from inguinal fat pads of GFP transgenic mice after extensive washing with phosphate-buffered saline and treatment with collagenase. After primary culture in a control medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum) and expansion to two passages, the cells were incubated in either an osteogenic medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum+dexamethasone+ascorbate-2-phosphate+beta-glycerophosphate) or a chondrogenic medium (Dulbecco's modified Eagle's medium+1% fetal bovine serum+insulin+ascorbate-2-phosphate+transforming growth factor-beta1) for 2-4 weeks to induce osteogenesis and chondrogenesis, respectively. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining, while chondrogenic differentiation was assessed by Alcian blue staining. Expression of osteocyte specific osteopontin, osteocalcin, and alkaline phosphatase, and chondrocyte specific aggrecan and type II/X collagen was confirmed by RT-PCR. ASCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes, except osteocalcin, was also detected. Incubation with chondrogenic medium induced Alcian blue positive cells and expression of aggrecan and type II/X collagen genes. No osteochondrogenic differentiation was observed in cells incubated in the control medium. ASCs from GFP transgenic mice have both osteogenic and chondrogenic potential in vitro. Since this cell population can be easily identified through fluorescence microscopy, it may be an ideal source of ASCs for further experiments on stem cell biology and tissue engineering.
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PMID:Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice. 1470 23


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