Showing papers on "Alkaline phosphatase published in 2005"
TL;DR: A new stem-cell line was derived from human embryos under completely cell and serum free conditions, eliminating exposure of human embryonic stem cells and their progeny to animal and human feeder layers, and thus the risk of contamination with pathogenic agents capable of transmitting diseases to patients.
454 citations
TL;DR: Osteoblast-lineage cells circulate in physiologically significant numbers, correlate with markers of bone formation, and are markedly higher during pubertal growth; therefore, they may represent a previously unrecognized circulatory component to the process of bone Formation.
Abstract: Background Although current evidence suggests that only a minuscule number of osteoblast-lineage cells are present in peripheral blood, we hypothesized that such cells circulate but that their concentration has been vastly underestimated owing to the use of assays that required adherence to plastic. We further reasoned that the concentration of these cells is elevated during times of increased bone formation, such as during pubertal growth. Methods We used flow cytometry with antibodies to bone-specific proteins to identify circulating osteoblast-lineage cells in 11 adolescent males and 11 adult males (mean [±SD] age, 14.5±0.7 vs. 37.7±7.6 years). Gene expression and in vitro and in vivo bone-forming assays were used to establish the osteoblastic lineage of sorted cells. Results Cells positive for osteocalcin and cells positive for bone-specific alkaline phosphatase were detected in the peripheral blood of adult subjects (1 to 2 percent of mononuclear cells). There were more than five times as many cells ...
397 citations
TL;DR: It is concluded that LLLT promotes proliferation and maturation of human osteoblasts in vitro and may have clinical implications.
Abstract: Objectives: The aim of the present study was to investigate the effect of low-level laser irradiation on proliferation and differentiation of a human osteoblast cell line. Background data: It was previously found that lowlevel laser therapy (LLLT) enhances bone repair in experimental models. Materials and methods: Cultured osteoblast cells were irradiated using He-Ne laser irradiation (632 nm; 10 mW power output). On the second and third day after seeding the osteoblasts were exposed to laser irradiation. The effect of irradiation on osteoblast proliferation was quantified by cell count and colorimetric MTT (dimethylthiazol tetrazolium bromide) assay 24 and 48 h after second irradiation. Results: Asignificant 31–58% increase in cell survival (MTT assay) and higher cell count in the once-irradiated as compared to nonirradiated cells was monitored. Differentiation and maturation of the cells was followed by osteogenic markers: alkaline phosphatase (ALP), osteopontin (OP), and bone sialoprotein (BSP). A two-...
333 citations
TL;DR: In addition to enhanced osteolysis, MM cells also suppress bone formation at least in part through an inhibition of the canonical Wnt pathway by secreting sFRP-2, suggesting a predominant role for MM cell-derived sFRp-2 in the impairment of bone formation by MM.
325 citations
TL;DR: The present study demonstrates the expression of molecular markers that are distinctive for the osteoblastic phenotype in MBA‐15 marrow stroma cells and have crucial role in cell–matrix interaction, in establishing the cellular osteogenic phenotype and their survival.
Abstract: Marrow stroma cells (MSC) play a major role in osteogenesis. The potential of the MSC to differentiate to bone-forming cells relies upon molecular regulation. This study analyzed MBA-15 cells for the expression of genes and proteins that are key regulators of osteoblast differentiation. These cells express Cbfa1 and c-fos transcription factors (TF) of osteoprogenitor proliferating cells. RT-PCR and immunohistochemistry were used to demonstrate the message and protein expression of extracellular matrix proteins that are a prerequisite for matrix formation and mineralization, including alkaline phosphatase (ALP), osteocalcin, osteopontin, biglycan, and bone sialoprotein (BSP). The activity of ALP was correlated at various cell densities with co-expression of osteocalcin or osteopontin. Adhering cells must attach to the appropriate matrix to enable survival and differentiation. Using attachment assays, we demonstrated that MBA-15 cells adhered to collagenous matrix and the effect on survival measured by changes in intracellular calcium (Ca) levels. The cells' adhesion to matrix is mediated via cell surface molecules. We quantified the expression of cells surface molecules that are important players in mediating cell-matrix interaction. Flow cytometry analysis (FACS) was used to determine the expression of CD-31 (36%), and lower levels were identified for CD-62E and CD11b. In summary, the present study demonstrates the expression of molecular markers that are distinctive for the osteoblastic phenotype in MBA-15 marrow stroma cells and have crucial role in cell-matrix interaction, in establishing the cellular osteogenic phenotype and their survival.
242 citations
TL;DR: In this article, the effects of acidosis on osteoblast function were investigated by using mineralized bone nodule-forming primary Osteoblast cultures, harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone.
Abstract: The negative effect of acidosis on the skeleton has been known for almost a century. Bone mineral serves an important pathophysiologic role as a reserve of hydroxyl ions to buffer systemic protons if the kidneys and lungs are unable to maintain acid-base balance within narrow physiologic limits. Extracellular hydrogen ions are now thought to be the primary activation signal for osteoclastic bone resorption, and osteoclasts are very sensitive to small changes in pH within the pathophysiologic range. Herein, we investigated the effects of acidosis on osteoblast function by using mineralized bone nodule-forming primary osteoblast cultures. Osteoblasts harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone. pH was manipulated by addition of 5 to 30 mmol/L HCl and monitored by blood gas analyzer. Abundant, matrix-containing mineralized nodules formed in osteoblast cultures at pH 7.4, but acidification progressively reduced mineralization of bone nodules, with complete abolition at pH 6.9. Osteoblast proliferation and collagen synthesis, assessed by 3H-thymidine and 3H-proline incorporation, respectively, were unaffected by pH in the range 7.4 to 6.9; no effect of acidification on collagen ultrastructure and organization was evident. The apoptosis rate of osteoblasts, assessed by the enrichment of nucleosomes in cell lysates, was also unaffected by pH within this range. However, osteoblast alkaline phosphatase activity, which peaked strongly near pH 7.4, was reduced eight-fold at pH 6.9. Reducing pH to 6.9 also downregulated messenger ribonucleic acid (mRNA) for alkaline phosphatase, but upregulated mRNA for matrix Gla protein, an inhibitor of mineralization. The same pH reduction is associated with two-and four-fold increases in Ca2+ and PO4(3-) solubility for hydroxyapatite, respectively. Our results show that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response. Thus, in uncorrected acidosis, the deposition of alkaline mineral in bone by osteoblasts is reduced, and osteoclast resorptive activity is increased in order to maximize the availability of hydroxyl ions in solution to buffer protons.
233 citations
TL;DR: MS‐based proteomics can reveal novel markers for MSCs that can be used for their isolation and for monitoring OB differentiation, and no concordance was detected between the changes in levels of gene and protein expression during OB differentiation.
Abstract: One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely useful for analyzing complex protein expression patterns and, when applied quantitatively, can be used to resolve subtle differences between samples. Thus, we used MS to characterize changes in expression of membrane protein markers before and after short-term induction of osteoblast (OB) differentiation in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and cytochemistry. Quantitative real-time reverse transcription-polymerase chain reaction confirmed the presence of mRNA of these membrane proteins. However, with the exception of ALP, no concordance was detected between the changes in levels of gene and protein expression during OB differentiation. In conclusion, MS-based proteomics can reveal novel markers for MSCs that can be used for their isolation and for monitoring OB differentiation.
209 citations
TL;DR: Results showed that in response to LLLT, HOB cultured on titanium implant material had a tendency towards increased cellular attachment, proliferation, differentiation and production of TGF-beta(1), indicating that in vitro L LLT can modulate the activity of cells and tissues surrounding implant material.
195 citations
TL;DR: It is suggested that PRP has a favorable effect on human osteoblast-like cells, and acts both to enhance bone regeneration and as an activator in wound healing.
193 citations
TL;DR: It is demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation.
Abstract: Green tea has been reported to possess antioxidant, antitumorigenic, and antibacterial qualities that regulate the endocrine system. Previous epidemiological studies found that the bone mineral density (BMD) of postmenopausal women with a habit of tea drinking was higher than that of women without habitual tea consumption. However, the effects of green tea catechins on osteogenic function have rarely been investigated. In this study, we tested (-)-epigallocatechin-3-gallate (EGCG), one of the green tea catechins, on cell proliferation, the mRNA expressions of relevant osteogenic markers, alkaline phosphatase (ALP) activity and mineralization. In a murine bone marrow mesenchymal stem cell line, D1, the mRNA expressions of core binding factors a1 (Cbfa1/Runx2), osterix, osteocalcin, ALP increased after 48 h of EGCG treatment. ALP activity was also significantly augmented upon EGCG treatment for 4 days, 7 days and 14 days. Furthermore, mineralizations assayed by Alizarin Red S and von Kossa stain were enhanced after EGCG treatment for 2-4 weeks in D1 cell cultures. However, a 24-h treatment of EGCG inhibited thymidine incorporation of D1 cells. These results demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation. This finding suggests that the stimulatory effects of EGCG on osteogenesis of mesenchymal stem cells may be one of the mechanisms that allow tea drinkers to possess higher BMD.
176 citations
TL;DR: Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels.
Abstract: Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester- poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.
TL;DR: It was found that the protectability of the enzymes against harsh pH values is dependent on the nature of the surfactant.
Abstract: By entrapment in (surfactant modified) silica sol−gel matrixes, alkaline phosphatase (AlP) naturally with optimum activity at pH 9.5 was kept functioning at extreme acidic environments as low as pH 0.9, and acid phosphatase (AcP) naturally with optimum activity at pH 4.5 was kept functioning at extreme alkaline environments, up to pH 13.0. Propositions are offered as to the origin of the ability of the matrixes to provide such highly efficient protection and as to the origin of the synergetic enhancing effect when both the silica and the surfactants are used as a combined entrapping environment. It was found that the protectability of the enzymes against harsh pH values is dependent on the nature of the surfactant.
TL;DR: Vitamin D depletion was significantly more prevalent in the African-American patients than in the white patients, and studies evaluating the effects of gastric bypass on vitamin D metabolism must consider preoperative vitamin D nutritional status.
TL;DR: Observations indicate that glucocorticoids regulate the commitment of progenitors derived from dental pulp cells to form odontoblast-like cells, while reducing the proportion of SMA-positive cells.
Abstract: Regenerative dental pulp strategies require the identification of precursors able to differentiate into odontoblast-like cells that secrete reparative dentin after injury. Pericytes have the ability to give rise to osteoblasts, chondrocytes, and adipocytes, a feature that has led to the suggestion that odontoblast-like cells could derive from these perivascular cells. In order to gain new insights into this hypothesis, we investigated the effects of dexamethasone (Dex), a synthetic glucocorticoid employed to induce osteogenic differentiation in vitro, in a previously reported model of human dental pulp cultures containing pericytes as identified by their expression of smooth muscle actin (SMA) and their specific ultrastructural morphology. Our data indicated that Dex (10–8 M) significantly inhibited cell proliferation and markedly reduced the proportion of SMA-positive cells. Conversely, Dex strongly stimulated alkaline phosphatase (ALP) activity and induced the expression of the transcript encoding the major odontoblastic marker, dentin sialophosphoprotein. Nevertheless, parathyroid hormone/parathyroid hormone-related peptide receptor, core-binding factor a1/osf2, osteonectin, and lipoprotein lipase mRNA levels were not modified by Dex treatment. Dex also increased the proportion of cells expressing STRO-1, a marker of multipotential mesenchymal progenitor cells. These observations indicate that glucocorticoids regulate the commitment of progenitors derived from dental pulp cells to form odontoblast-like cells, while reducing the proportion of SMA-positive cells. These results provide new perspectives in deciphering the cellular and molecular mechanisms leading to reparative dentinogenesis.
TL;DR: In the present study, the biochemical manifestations of liver toxicity caused by co-administration of anti-TB drugs, rifampicin, isoniazid and pyrazinamide, in a sub-chronic mode (12 weeks), were investigated and Silymarin reversed these abnormal alterations.
TL;DR: It is suggested that low and repeated doses of MC-LR from cyanobacterial cells induce toxicity in tilapia fish although no adverse effects were detected.
TL;DR: Gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation, and this activity is associated with dramatic enhancement of bone formation in intact calvarial cultures.
Abstract: Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca2+/calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin Aα into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin Aα-/- mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.
TL;DR: It is suggested that large yellow croaker larvae could be fed successfully with a compound diet from early stages, at least from day 23, showing maturation of the intestinal digestive process.
TL;DR: Results do not support induction of ALT or AP in the liver as the cause of elevated serum activities of these enzymes due to PB.
Abstract: Phenobarbital (PB) therapy is frequently associated with elevated serum alanine aminotransferase (ALT) and alkaline phosphatase (AP) activities in dogs without clinical signs of liver disease. The goal of this study was to determine if increased serum ALT and AP activities in clinically healthy PB-treated epileptic dogs are due to hepatic enzyme induction or to subclinical liver injury. Liver biopsies were obtained from 12 PB-treated dogs without clinical signs of liver disease but with elevated serum ALT and/or AP activities or both. Liver biopsies were obtained from eight healthy control dogs not receiving PB. Biopsies were evaluated histopathologically (all dogs) and liver homogenates were assayed for ALT (all dogs) and AP (six treated dogs, all controls) activities. As a positive control, liver cytochrome P4502B, an enzyme known to be induced by PB, was measured by benzyloxyresorufin-O-dealkylase activity and immunoblotting (five treated dogs, all controls). Serum AP isoenzyme analyses were performed. Results showed that ALT and AP activities in liver homogenates were not increased in treated dogs compared with controls, whereas the positive control for induction, CYP2B, was dramatically increased in treated dogs. Histopathological examination of liver biopsies revealed more severe and frequent abnormalities in treated dogs compared to controls, but similar types of abnormalities were found in both groups. Serum AP isoenzyme analyses in treated dogs demonstrated increased corticosteroid-induced and liver isoenzyme activities compared to controls. Results do not support induction of ALT or AP in the liver as the cause of elevated serum activities of these enzymes due to PB.
TL;DR: ZnOA-coated titanium substrates represent a new class of materials for human repair that provide, mechanical stability, as well as chemical and biochemical signals to promote new bone growth.
TL;DR: Preliminary results suggest that NCB was not likely to produce severe toxicological effects on organ weights, haematological and biochemical indices when given at normal therapeutic doses.
Abstract: Graded doses of Nature Cure Bitters (NCB) were administered daily (100, 200 and 400 mg/kg p.o) to rats for 28 days and the effects on body weight, organ weight, clinical signs, gross pathology, haematology, histology and serum biochemical parameters were evaluated. The relative weights of the heart, liver and testes of treated rats were unaffected in contrast to a significant increase in the relative weights of the lungs, kidneys and spleen. The packed cell volume and haemoglobin concentrations were significantly reduced whereas total leucocyte counts and glucose levels were remarkably increased. A significant decrease in alkaline phosphatase occurred in all the groups but alanine aminotransferase and albumin levels were significantly elevated. NCB elicited hypo-cholesterolaemic effects in addition to lowering urea, uric acid, BUN and total protein concentrations. Histological findings did not reveal any treatment-related effects. The calculated therapeutic index was >37.5. These preliminary results suggest that NCB was not likely to produce severe toxicological effects on organ weights, haematological and biochemical indices when given at normal therapeutic doses.
Key words: Nature Cure Bitters, organ weight; pathology, haematology; serum biochemistry.
TL;DR: The data overall suggest that no significant effect can be ascribed to the ionic products of 58S bioactive gel-glass dissolution tested here and their ability to stimulate osteoblastic marker gene expression.
Abstract: Bioactive glasses dissolve upon immersion in culture medium, and release their constitutive ions into solution. There has been some evidence suggesting that these ionic-dissolution products influence osteoblast-specific processes. Here, the effect of 58S sol–gel-derived bioactive glass (60% SiO2, 36% CaO, 4% P2O5, in molar percentage) on primary osteoblasts derived from human fetal long bone explant cultures is investigated, and it is hypothesized that critical concentrations of sol–gel-dissolution products (consisting of a combination of simple inorganic ions) can enhance osteoblast phenotype in vitro by affecting the expression of a number of genes associated with the differentiation and extracellular matrix deposition processes. Cells were exposed to a range of 58S dosages continuously for a period of 4–14 days in monolayer cultures. Quantitative real-time RT-PCR analysis of a panel of osteoblast-specific markers showed a varied gene expression pattern in response to the material. The highest concentration of Ca and Si tested (96 and 50 ppm, respectively) promoted upregulation of gene expression for most markers (including alkaline phosphatase, osteocalcin, and osteopontin) at the latest time point, compared to non-58S-treated control, although this observation was not statistically significant. The same 58S concentration produced higher ALP activity levels and increased proliferation throughout the culture period, compared to lower dosages tested; however, the results generated were again not statistically significant. The data overall suggest that no significant effect can be ascribed to the ionic products of 58S bioactive gel-glass dissolution tested here and their ability to stimulate osteoblastic marker gene expression. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2005
TL;DR: It was found that the "odontogenic" properties of the isolated cells were supported by three-dimensional calcium phosphate and titanium scaffolds equally well and showed the ability to differentiate into odontoblast-like cells and produced calcified nodules, which had components similar to dentin.
Abstract: The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats. Immunochemistry by stem cell marker STRO-1 proved the existence of stem cells or progenitors in the isolated cell population. The dissociated cells were then cultured both on smooth surfaces and on three-dimensional (3-D) scaffold materials in medium supplemented with beta-glycerophosphate, dexamethasone, and L-ascorbic acid. Cultures were analyzed by light and scanning electron microscopy and, on proliferation, alkaline phosphatase activity and calcium content were determined and the polymerase chain reaction was performed for dentin sialophosphoprotein, osteocalcin, and collagen type I. These cells showed the ability to differentiate into odontoblast-like cells and produced calcified nodules, which had components similar to dentin. In addition, we found that the "odontogenic" properties of the isolated cells were supported by three-dimensional calcium phosphate and titanium scaffolds equally well.
TL;DR: It was found that mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium or a specific CaR activator, NPS-R467, which suggests that the CaR may be involved in osteoblast differentiation.
Abstract: We have previously shown that the extracellular calcium-sensing receptor (CaR) is expressed in various bone marrow-derived cell lines and plays an important role in stimulating their proliferation and chemotaxis. It has also been reported that the CaR modulates matrix production and mineralization in chondrogenic cells. However, it remains unclear whether the CaR plays any role in regulating osteoblast differentiation. In this study, we found that mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium (2.8 and 3.8 mM) or a specific CaR activator, NPS-R467 (1 and 3 μM). Next, we stably transfected MC3T3-E1 cells with either a CaR antisense vector (AS clone) or a vector containing the inactivating R185Q variant of the CaR (DN clone) that has previously been shown to exert a dominant negative action. Alkaline phosphatase activities were decreased compared with controls in both the AS and DN clones. However, the levels of type I procollagen and osteopontin mRNA in the AS clone, as detected by Northern blotting, were almost the same as in the controls. On the other hand, the expression of osteocalcin, which is expressed at a later stage of osteoblastic differentiation, was significantly reduced in both the AS and DN clones. Mineralization was also decreased in both clones. In conclusion, this study showed that the abolition of CaR function results in diminishing alkaline phosphatase activity, osteocalcin expression, and mineralization in mouse osteoblastic cells. This suggests that the CaR may be involved in osteoblastic differentiation.
TL;DR: It is suggested that VEGF induces initial differentiation of osteoblasts but requires other factors, present in C4-2B, to induce mineralization, as well as PTK787, a VEGFR tyrosine kinase inhibitor, which contributes to prostate cancer-induced osteoblastic activity in vivo.
Abstract: Prostate cancer frequently metastasizes to bone resulting in the formation of osteoblastic metastases through unknown mechanisms. Vascular endothelial growth factor (VEGF) has been shown recently to promote osteoblast activity. Accordingly, we tested if VEGF contributes to the ability of prostate cancer to induce osteoblast activity. PC-3, LNCaP, and C4-2B prostate cancer cell lines expressed both VEGF-165 and VEGF-189 mRNA isoforms and VEGF protein. Prostate cancer cells expressed the mRNA for VEGF receptor (VEGFR) neuropilin-1 but not the VEGFRs Flt-1 or KDR. In contrast, mouse pre-osteoblastic cells (MC3T3-E1) expressed Flt-1 and neuropilin-1 mRNA but not KDR. PTK787, a VEGFR tyrosine kinase inhibitor, inhibited the proliferation of human microvascular endothelial cells but not prostate cancer proliferation in vitro. C4-2B conditioned medium induced osteoblast differentiation as measured by production of alkaline phosphatase and osteocalcin and mineralization of MC3T3-E1. PTK787 blocked the C4-2B conditioned medium-induced osteoblastic activity. VEGF directly induced alkaline phosphatase and osteocalcin but not mineralization of MC3T3-E1. These results suggest that VEGF induces initial differentiation of osteoblasts but requires other factors, present in C4-2B, to induce mineralization. To determine if VEGF influences the ability of prostate cancer to develop osteoblastic lesions, we injected C4-2B cells into the tibia of mice and, after the tumors grew for 6 weeks, administered PTK787 for 4 weeks. PTK787 decreased both intratibial tumor burden and C4-2B-induced osteoblastic activity as measured by bone mineral density and serum osteocalcin. These results show that VEGF contributes to prostate cancer-induced osteoblastic activity in vivo.
TL;DR: Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.
Abstract: Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.
TL;DR: It is shown that IGF-I up-regulates Type I collagen, Runx2, and alkaline phosphatase (Alp) gene expression in hMSCs, genes indicative of early osteogenic differentiation, and transduction up-regulated markers of osteoblastic differentiation and in conjunction with recombinant BMP-2-induced matrix mineralization independently of Dex.
Abstract: Human adult mesenchymal stem cells (hMSCs) differentiate into an osteogenic lineage if the appropriate differentiative cues, such as dexamethasone or bone morphogenetic protein 2 (BMP-2), are present. This study was undertaken to determine the role of insulin-like growth factor I (IGFI) in the regulation of early osteoblast differentiation in hMSC. Previous studies have shown that IGF-I, regulates bone formation and remodeling by participating in the differentiation of mature cells of osteoblast lineage. We hypothesized that IGF-I exerted its effects early, but the effects were too subtle to be detected. Therefore, engineered hMSCs to produce IGF-I via adenoviral transfection and used quantitative real-time PCR (qPCR) to assess marker gene expression. Here we show that IGF-I up-regulates Type I collagen, Runx2, and alkaline phosphatase (Alp) gene expression in hMSCs, genes indicative of early osteogenic differentiation. We also observed mineral deposition in the absence of dexamethasone (Dex) in hMSC cult...
TL;DR: Fondaparinux is a new antithrombotic drug that specifically inhibits factor Xa that is associated with a risk of heparin‐induced osteoporosis and its effects on human osteoblasts were analysed in vitro.
Abstract: Background:
The prolonged administration of heparin for prevention and treatment of venous thromboembolism has been associated with a risk of heparin-induced osteoporosis. Fondaparinux is a new antithrombotic drug that specifically inhibits factor Xa. Because of the known interactions of other antithrombotic agents with bone remodelling, the effects of fondaparinux on human osteoblasts were analysed in vitro.
Methods:
Primary human osteoblast cell cultures were incubated with either the low molecular weight heparin dalteparin at concentrations of 30, 300 and 900 µg/ml or with fondaparinux at concentrations of 25, 50, 100, 150, 200 and 250 µg/ml. Cellular proliferation rate and protein synthesis were measured. Expression of genes encoding osteocalcin, collagen type I and alkaline phosphatase was examined by reverse transcriptase–polymerase chain reaction.
Results:
Incubation with dalteparin led to a significant, dose-dependent inhibition of osteoblast proliferation, inhibition of protein synthesis, and inhibited expression of phenotype markers (osteocalcin and alkaline phosphatase genes) after 3 and 7 days. No inhibitory effects were observed in the fondaparinux-treated cells.
Conclusion:
Fondaparinux did not inhibit osteoblast proliferation in vitro and may reduce the risk of heparin-induced osteoporosis associated with long-term heparin administration. Copyright © 2004 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.
Journal Article•
TL;DR: The calcification of isolated ICs was seen to be dependent on ALP activity and Levamisol inhibited the beta-glycerophosphate-induced calcification process, as well as the expression of osteoblastic differentiation markers.
Abstract: Background and aim of the study The calcification of heart valves is associated with valve degeneration and failure, but the mechanisms involved are poorly understood The presence of lamellar bone has been demonstrated in calcified aortic valves Since osseous calcification is closely associated with alkaline phosphatase (ALP) activity, it was hypothesized that ALP activity might be implicated in the calcification of isolated leaflet interstitial cells (ICs) Methods Human valve leaflet ICs were isolated from transplant-explanted hearts at the time of transplantation (n = 12) Results Isolated leaflet ICs expressed the fibroblast-specific antigen (100% of cells) and smooth muscle (SM) alpha-actin (70-80% of cells), but osteoblastic markers were not expressed Cultured ICs did not calcify spontaneously, however when the growth medium was supplemented with beta-glycerophosphate (an organic phosphate) it induced the formation of calcified nodules that expressed osteonectin and ALP, but not SM alpha-actin Beta-glycerophosphate-induced calcification of ICs showed a time-dependent effect on the calcium content of treated cells over a 14-day period ALP activity was considerably increased in beta-glycerophosphate-treated ICs, and this correlated with the calcium content (r = 05: p = 001) Levamisol (an ALP inhibitor) inhibited the beta-glycerophosphate-induced calcification process, as well as the expression of osteoblastic differentiation markers Conclusion Isolated and cultured leaflet ICs did not calcify spontaneously, though organic phosphate induced the formation of calcified nodules that expressed osteoblastic markers The calcification of isolated ICs was seen to be dependent on ALP activity
TL;DR: ALP appears to be a highly sensitive and fairly specific marker in the diagnosis of OSA.
Abstract: Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone neoplasia. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, and plasma cell myeloma. The purpose of this study is to determine the sensitivity and specificity of alkaline phosphatase (ALP) staining to differentiate OSA from other tumors that express vimentin by immunocytochemistry or immunohistochemistry. ALP is a hydrolytic enzyme present in multiple tissues including liver, kidney, intestine, placenta, and bone. Hypothetically, neoplasms actively producing bone should be specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 8-10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3- indolyl phosphate toluidine salt-phosphatase substrate. A positive reaction stains the membrane of the cells gray to black. Samples were counterstained with a Romanowsky's stain to determine whether the sample was of representative cellularity. A total of 61 vimentin-positive neoplasms have been evaluated and confirmed histopathologically. Tumors that expressed vimentin and were positive for ALP included 33 OSAs, one multi- lobular tumor of bone, one amelanotic melanoma, and one chondrosarcoma. Tumors that expressed vimentin and were negative for ALP included chondrosarcomas (three of four), multiple fibrosarcomas, and multiple synovial cell sarcomas. The sensitivity is 100%, and the specificity is 89%. In conclusion, ALP appears to be a highly sensitive and fairly specific marker in the diagnosis of OSA.