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ALP Activity (alp + activity)
Selected AbstractsEfficiency of combined methotrexate/chloroquine therapy in adjuvant-induced arthritisFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 4 2005M.A.R.C.P. Silva Abstract The present study evaluates the effects of methotrexate (MTX) and chloroquine (CQ), and of combined MTX + CQ treatment, on the inflammatory response and on plasma and liver phosphatase and transaminase activities, employing an adjuvant-induced arthritis model in rats. Arthritis was induced by the intradermal injection of a suspension of Mycobacterium tuberculosis in mineral oil into the plantar surface of the hind paws. Development of the inflammatory response was assessed over a 21-day period. Animal groups received either: (i) MTX, administered i.p., weekly, in 0.15, 1.5, 3, 6 or 12 mg/kg doses; (ii) CQ, given intragastrically, in daily 25 or 50 mg/kg doses; or (iii) MTX + CQ, administered in two combinations (MTX1.5 mg/kg + CQ50 mg/kg, or MTX6 mg/kg + CQ50 mg/kg). At the end of the experimental period, the animals were anesthetized and killed, blood and liver samples were collected and prepared for measurement of acid and alkaline phosphatase (AP, ALP), and aspartate (AST) and alanine aminotransferase (ALT) activities. MTX at 6 and 12 mg/kg reduced the inflammatory response while CQ had no effect. MTX6 mg/kg + CQ50 mg/kg reduced the inflammatory response similar to MTX12 mg/kg, without affecting the bone marrow. Plasma AP and liver ALP activities were very elevated in the arthritic rats. While MTX treatment partially reduced both plasma AP and liver ALP activities at all doses used in the arthritic rats, CQ treatment reduced plasma AP, but increased liver AP activity. MTX + CQ treatment decreased plasma AP and liver ALP activities in the arthritic rats to control values. Plasma and liver AST activities were unaltered in the arthritic rats, and were unaffected by treatment. However, plasma and liver ALT activities were significantly reduced in the arthritic rats. While MTX or CQ treatment did not alter plasma transaminase activity in the arthritic rats, after MTX + CQ treatment, plasma ALT activity returned to normal values. In conclusion, the present data suggest that MTX + CQ treatment provides more effective anti-inflammatory protection against adjuvant-induced arthritis than does MTX alone, reverting the alterations in enzyme activities induced by this inflammatory disease in rats. [source] Expression of Osterix in mechanical stress-induced osteogenic differentiation of periodontal ligament cells in vitroEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2008Yanhong Zhao Osterix (Osx) is an osteoblast-specific transcription factor required for the differentiation of pre-osteoblasts into functional osteoblasts. This study sought to examine the changes of Osx expression in periodontal ligament cells (PDLC) subjected to mechanical force, and to investigate whether Osx is involved in the mechanical stress-induced differentiation of PDLC. Human PDLC were exposed to centrifugal force for 1,12 h. Real-time polymerase chain reaction (PCR), western blot, and immunofluorescence assays were used to examine the mRNA and protein expression of Osx and its subcellular localization. Furthermore, PDLC were transfected with the expression vector pcDNA3.1 flag-Osx and subjected to mechanical force for 6 h. The changes in alkaline phosphatase (ALP) activity and in the expression of core-binding factor alpha1 (Cbfa1), ALP, osteopontin, bone sialoprotein, osteocalcin, and collagen I were measured. After the application of mechanical force, Osx was upregulated in a time-dependent manner at both mRNA and protein levels, and Osx protein was translocated from the cytosol into the cell nuclei. Overexpression of Osx did not affect the expression of Cbfa1, but it significantly enhanced the ALP activity and the mRNA expression of all the aforementioned osteogenic marker genes, all of which increased further under mechanical stress. These results suggest that Osx might play an important role in the mechanical stress-induced osteogenic differentiation of PDLC and therefore be involved in alveolar bone remodeling during orthodontic therapy. [source] Prostaglandin F2, stimulates MEK-ERK signalling but decreases the expression of alkaline phosphatase in dental pulp cellsINTERNATIONAL ENDODONTIC JOURNAL, Issue 6 2010M. C. Chang Chang MC, Chen YJ, Lee MY, Lin LD, Wang TM, Chan CP, Tsai YL, Wang CY, Lin BR, Jeng JH. Prostaglandin F2, stimulates MEK-ERK signalling but decreases the expression of alkaline phosphatase in dental pulp cells. International Endodontic Journal, 43, 461,468, 2010. Abstract Aim, To study prostaglandin F2, (PGF2,) receptor expression and downstream signalling in cultured human dental pulp cells and the effect of PGF2, on the alkaline phosphatase (ALP) activity of dental pulp cells. Methodology, Human dental pulp cells were cultured and exposed to PGF2,. The expression of PGF2, (FP) receptors was analysed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The activation of extracellular regulated kinase (ERK) and cAMP responsive element binding protein/activating transcription factor-1 (CREB/ATF-1) signalling was determined by Western blotting. The expression of ALP in pulp cells after exposure to PGF2, was evaluated by ALP staining and PCR. Results, Dental pulp cells expressed FP receptor mRNA and protein. Exposure to PGF2, revealed little cytotoxicity to pulp cells. PGF2, induced both ERK and CREB/ATF-1 phosphorylation in pulp cells. Exposure to PGF2, (>1 ,mol L,1) further decreased the ALP activity and mRNA expression. However, U0126 (an inhibitor of MEK1) showed little preventive effect on the decline of ALP activity in dental pulp cells by PGF2,. Conclusion, PGF2, may potentially activate FP receptors leading to ERK/CREB-ATF-1 activation during its production in inflamed dental pulp. PGF2, attenuated the ALP activity of pulp cells possibly via pathways not solely by MEK/ERK activation. PGF2, is a contributing factor of pulpal inflammation by regulating the activities of pulp cells. [source] Effect of antisense oligonucleotide against mouse dentine matrix protein 1 on mineralization ability and calcium ions metabolism in odontoblast-like cell line MDPC-23INTERNATIONAL ENDODONTIC JOURNAL, Issue 7 2006J. L. Pang Abstract Aim, To study the mineralization ability and the dynamic changes of intracellular and extracellular concentrations of calcium ions in the odontoblast-like cell line MDPC-23 affected by antisense oligonucleotide (AS-ODN) against mouse dentine matrix protein 1 (DMP1). Methodology, The expression of DMP1 in MDPC-23 cells was detected by an immunohistochemical method and its blocking outcome by the Western blot method. The alkaline phosphatase (ALP) activity, size and number of mineralized nodules, and the intracellular free ([Ca2+]if), total ([Ca2+]it) and the extracellular ([Ca2+]e) calcium ion concentrations in MDPC-23 cells in the experimental group affected with AS-ODN were compared with those in the control group (paired-samples t -test). Results, Dentine matrix protein 1 was stably expressed in a stable way in MDPC-23 cells; the expression was only just detectable at 12 h and became negative after 24 h affected by AS-ODN. Compared with the control groups, ALP activity of MDPC-23 cells in the AS-ODN group was decreased (P < 0.05), and both the number and size of mineralized nodules were smaller than those in the control group. [Ca2+]if in the AS-ODN group increased and then decreased after 24 h. [Ca2+]it dropped substantially to the lowest point at 24 h (P < 0.01). [Ca2+]e increased before treatment for 24 h and then dropped, however, it was still higher than that of the control group. Conclusions, Antisense oligonucleotide against DMP1 could decrease mineralization ability and affect the intracellular and extracellular concentrations of calcium ions in MDPC-23 cells. This would indicate that DMP1 regulates the metabolism and transportation of calcium ions in odontoblasts, and thus boosts dentine mineralization. [source] Chronic ethanol intake inhibits in vitro osteogenesis induced by osteoblasts differentiated from stem cellsJOURNAL OF APPLIED TOXICOLOGY, Issue 2 2008Maria L. Rosa Abstract The study investigated whether chronic ethanol (ETH) intake and subsequent ETH exposure of cell cultures affects osteoblast differentiation by evaluating key parameters of in vitro osteogenesis. Rats were treated with 5,20% (0.85,3.43 mm) ETH, increasing by 5% per week for a period of 4 weeks (habituation), after which the 20% level was maintained for 15 days (chronic intake). Bone-marrow stem cells from control (CONT) or ETH-treated rats were cultured in osteogenic medium which was either supplemented (ETH) or not supplemented (CONT) with 1.3 mm ethanol. Thus, four groups relating to rat treatment/culture supplementation were evaluated: (1) CONT/CONT, (2) ETH/CONT, (3) CONT/ETH and (4) ETH/ETH. Cell morphology, proliferation and viability, total protein content, alkaline phosphatase (ALP) activity and bone-like nodule formation were evaluated. Chronic ethanol intake significantly reduced both food and liquid consumption and body weight gain. No difference was seen in cell morphology among treatments. Cell number was affected at 7 and 10 days as follows: CONT/CONT = CONT/ETH < ETH/CONT = ETH/ETH. Doubling time between 3 and 10 days was greater in groups of CONT animals: ETH/ETH = ETH/CONT < CONT/ETH = CONT/CONT. Cell viability and ALP activity were not affected by either animal treatment or culture exposure to ethanol. At day 21, the total protein content was affected as follows: ETH/ETH = CONT/ETH < ETH/CONT = CONT/CONT. Bone-like nodule formation was affected as follows: ETH/ETH < CONT/ETH < ETH/CONT < CONT/CONT. These results show that chronic ethanol intake, followed by the exposure of osteoblasts to ethanol, inhibited the differentiation of osteoblasts, as indicated by an increased proliferation rate and reduced bone-like nodule formation. Copyright © 2007 John Wiley & Sons, Ltd. [source] Diosmetin Induces Human Osteoblastic Differentiation Through the Protein Kinase C/p38 and Extracellular Signal-Regulated Kinase 1/2 Pathway,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008Ya-Ling Hsu Abstract Introduction: The survival of osteoblasts is one of the determinants of the development of osteoporosis. This study is the first to investigate the osteoblastic differentiation induced by diosmetin, a flavonoid derivative, in osteoblastic cell lines MG-63, hFOB, and MC3T3-E1 and bone marrow stroma cell line M2-10B4. Materials and Methods: Osteoblastic differentiation was determined by assaying alkaline phosphatase (ALP) activity and mineralization degree and measuring various osteoblast-related markers using ELISA. Expression and phosphorylation of Runt-related transcription factor 2 (Runx2), protein kinase C, (PKC,), extracellular signal-regulated kinase (ERK), p38, and c- jun -N-terminal kinase (JNK) was assessed by immunoblot. Rac1 activity was determined by immunoprecipitation, and Runx2 activity was assessed by EMSA. Genetic inhibition was performed by small hairpin RNA plasmids or small interfering RNA (siRNA) transfection. Results: Diosmetin exhibited an effect on osteoblastic maturation and differentiation by means of ALP activity, osteocalcin, osteopontin, and type I collagen production, as well as Runx2 upregulation. Induction of differentiation by diosmetin was associated with increased PKC, phosphorylation and the activations of Rac1 and p38 and ERK1/2 kinases. Blocking PKC, by siRNA inhibition significantly decreased osteoblastic differentiation by inhibiting Rac1 activation and subsequently attenuating the phosphorylation of p38 and ERK1/2. In addition, blocking p38 and ERK1/2 by siRNA transfection also suppressed diosmetin-induced cell differentiation. Conclusions: In this study, we show that diosmetin induced osteoblastic differentiation through the PKC,-Rac1-MEK3/6-p38 and PKC,-Rac1-MEK1/2- ERK1/2-Runx2 pathways and that it is a promising agent for treating osteoporosis. [source] Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005Daichi Chikazu Abstract We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5,-AACCACA-3,) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2,/, mice. In vivo, BMP-2 (10 ,g/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (,CT), was decreased by 78% in COX-2,/, mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo. [source] Ghrelin Directly Regulates Bone Formation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2005Nobuhiro Fukushima Abstract To clarify the role of ghrelin in bone metabolism, we examined the effect of ghrelin in vitro and in vivo. Ghrelin and its receptor, GHS-R1a, were identified in osteoblasts, and ghrelin promoted both proliferation and differentiation. Furthermore, ghrelin increased BMD in rats. Our results show that ghrelin directly affects bone formation. Introduction: Ghrelin is a gut peptide involved in growth hormone (GH) secretion and energy homeostasis. Recently, it has been reported that the adipocyte-derived hormone leptin, which also regulates energy homeostasis and opposes ghrelin's actions in energy homeostasis, plays a significant role in bone metabolism. This evidence implies that ghrelin may modulate bone metabolism; however, it has not been clarified. To study the role of ghrelin in skeletal integrity, we examined its effects on bone metabolism both in vitro and in vivo. Materials and Methods: We measured the expression of ghrelin and growth hormone secretagogue receptor 1a (GHS-R1a) in rat osteoblasts using RT-PCR and immunohistochemistry (IHC). The effect of ghrelin on primary osteoblast-like cell proliferation was examined by recording changes in cell number and the level of DNA synthesis. Osteoblast differentiation markers (Runx2, collagen ,1 type I [COLI], alkaline phosphatase [ALP], osteocalcin [OCN]) were analyzed using quantitative RT-PCR. We also examined calcium accumulation and ALP activity in osteoblast-like cells induced by ghrelin. Finally, to address the in vivo effects of ghrelin on bone metabolism, we examined the BMD of Sprague-Dawley (SD) rats and genetically GH-deficient, spontaneous dwarf rats (SDR). Results: Ghrelin and GHS-R1a were identified in osteoblast-like cells. Ghrelin significantly increased osteoblast-like cell numbers and DNA synthesis in a dose-dependent manner. The proliferative effects of ghrelin were suppressed by [D-Lys3]-GHRP-6, an antagonist of GHS-R1a, in a dose-dependent manner. Furthermore, ghrelin increased the expression of osteoblast differentiation markers, ALP activity, and calcium accumulation in the matrix. Finally, ghrelin definitely increased BMD of both SD rats and SDRs. Conclusions: These observations show that ghrelin directly stimulates bone formation. [source] Effects of Secreted Frizzled-Related Protein 3 on Osteoblasts In Vitro,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2004Yoon-Sok Chung Abstract To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnt's. Introduction: Secreted frizzled-related proteins (sFRPs) are a truncated form of frizzled receptor, missing both the transmembrane and cytosolic domains. Because previous studies have shown that sFRPs bind and act as decoy receptors for Wnt proteins that promote osteoblast differentiation, we postulated that sFRP3 acts as an inhibitor of osteoblast differentiation. Materials and Methods: We examined the effects of mouse recombinant sFRP3 and/or Wnt-3A on cell proliferation and differentiation using MC3T3-E1 mouse osteoblasts and primary cultures of mouse bone marrow stromal cells. We evaluated the effects of sFRP3 on ,-catenin levels using Western immunoblot analyses. Results: We found that sFRP3 suppressed osteoblast cell number in a dose-dependent manner that was the result of a decrease in proliferation and not because of an increase in apoptosis. Surprisingly, sFRP3 increased osteoblast differentiation, which could not be explained based on sFRP3 acting as a decoy receptor for stimulatory Wnt's. Furthermore, sFRP3 did not inhibit Wnt3A-induced increase in alkaline phosphatase (ALP) activity. Wnt3A, but not sFRP3 treatment, increased cellular ,-catenin levels, and sFRP3 failed to block Wnt3A-induced increase in cellular ,-catenin levels. Treatment with endostatin, an agent known to degrade ,-catenin, did not inhibit sFRP3-induced increase in ALP activity. sFRP1, like sFRP3, inhibited proliferation and stimulated ALP activity in MC3T3-E1 mouse osteoblasts. Conclusions: Based on our findings, we conclude that sFRP3 decreased osteoblast proliferation and unexpectedly increased parameters of osteoblast differentiation. Based on our findings, we propose that sFRP3 may stimulate differentiation through a ,-catenin-independent pathway in addition to its previously known function as a decoy receptor for Wnt's. [source] The Balance Between Concurrent Activation of ERs and PPARs Determines Daidzein-Induced Osteogenesis and Adipogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2004ZhiChao Dang PhD Abstract The soy phytoestrogen daidzein has biphasic dose responses, but the underlying mechanisms are not yet clear. Transcriptional and biochemical data show that PPARs, in addition to ERs, are molecular targets of daidzein, which divergently regulates osteogenesis and adipogenesis. Dose responses are the result of a balance among PPARs and between ERs and PPARs. Introduction: Soy phytoestrogens have been used for the purposes of treatment and prevention of osteoporosis. Biphasic dose responses of daidzein, one of the main soy phytoestrogens, have long been recognized, but the underlying molecular mechanisms of action are not yet clear. Materials and Methods: Mouse bone marrow cells and mouse osteoprogenitor KS483 cells that concurrently differentiate into osteoblasts and adipocytes were cultured. Biochemical measurement of alkaline phosphatase (ALP) activity, RT-PCR, and gene reporter assays were used in this study. Results: Daidzein, one of the major soy phytoestrogens, had biphasic effects on osteogenesis and adipogenesis. Daidzein stimulated osteogenesis (ALP activity and nodule formation) and decreased adipogenesis (the number of adipocytes) at concentrations below 20 ,M, whereas it inhibited osteogenesis and stimulated adipogenesis at concentrations higher than 30 ,M. When estrogen receptors (ERs) were blocked by ICI182,780, daidzein-induced effects were not biphasic. A decrease in osteogenesis and an increase in adipogenesis were observed at the concentrations higher than 20 and 10 ,M, respectively. In addition to ERs, daidzein transactivated not only peroxisome proliferator-activate receptor , (PPAR,), but also PPAR, and PPAR, at micromolar concentrations. Activation of PPAR, had no direct effects on osteogenesis and adipogenesis. In contrast, activation of PPAR, stimulated osteogenesis but had no effects on adipogenesis, whereas PPAR, inhibited osteogenesis and stimulated adipogenesis. Transfection experiments show that an activation of PPAR, or PPAR, by daidzein downregulated its estrogenic transcriptional activity, whereas activation of PPAR, upregulated its estrogenic transcriptional activity. Activation of ER, or ER, by daidzein downregulated PPAR, transcriptional activity but had no influence on PPAR, or PPAR, transcriptional activity. Conclusions: Daidzein at micromolar concentrations concurrently activates different amounts of ERs and PPARs, and the balance of the divergent actions of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis. [source] Smad3 Promotes Alkaline Phosphatase Activity and Mineralization of Osteoblastic MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002Hideaki Sowa Abstract Transforming growth factor (TGF) , is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-, signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-, on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-, inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L -azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs. [source] BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/,-catenin signallingJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Ni Tang Abstract Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-,/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/,-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. ,-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, ,-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and ,-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between ,-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs. [source] Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Olivia Fromigué Abstract Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites. J. Cell. Biochem. 104: 620,628, 2008. © 2007 Wiley-Liss, Inc. [source] Evidence that both 1,,25-dihydroxyvitamin D3 and 24-hydroxylated D3 enhance human osteoblast differentiation and mineralizationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2006M. van Driel Abstract Vitamin D plays a major role in the regulation of mineral homeostasis and affects bone metabolism. So far, detailed knowledge on the vitamin D endocrine system in human bone cells is limited. Here we investigated the direct effects of 1,,25-(OH)2D3 on osteoblast differentiation and mineralization. Also, we studied the impact of 24-hydroxylation, generally considered as the first step in the degradation pathway of vitamin D, as well as the role of the nuclear and presumed membrane vitamin D receptor (VDR). For this we used a human osteoblast cell line (SV-HFO) that has the potency to differentiate during culture forming a mineralized extracellular matrix in a 3-week period. Transcriptional analyses demonstrated that both 1,,25-(OH)2D3 and the 24-hydroxylated metabolites 24R,25-(OH)2D3 and 1,,24R,25-(OH)3D3 induced gene transcription. All metabolites dose-dependently increased alkaline phosphatase (ALP) activity and osteocalcin (OC) production (protein and RNA), and directly enhanced mineralization. 1,,24R,25-(OH)3D3 stimulated ALP activity and OC production most potently, while for mineralization it was equipotent to 1,,25-(OH)2D3. The nuclear VDR antagonist ZK159222 almost completely blocked the effects of all metabolites. Interestingly, 1,,25-(OH)2D3, an inhibitor of membrane effects of 1,,25-(OH)2D3 in the intestine, induced gene transcription and increased ALP activity, OC expression and mineralization. In conclusion, not only 1,,25-(OH)2D3, but also the presumed 24-hydroxylated "degradation" products stimulate differentiation of human osteoblasts. 1,,25-(OH)2D3 as well as the 24-hydroxylated metabolites directly enhance mineralization, with the nuclear VDR playing a central role. The intestinal antagonist 1,,25-(OH)2D3 acts in bone as an agonist and directly stimulates mineralization in a nuclear VDR-dependent way. J. Cell. Biochem. 99: 922,935, 2006. © 2006 Wiley-Liss, Inc. [source] Role of TNF alpha and PLF in bone remodeling in a rat model of repetitive reaching and grasping,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2010Shobha Rani We have previously developed a voluntary rat model of highly repetitive reaching that provides an opportunity to study effects of non-weight bearing muscular loads on bone and mechanisms of naturally occurring inflammation on upper limb tissues in vivo. In this study, we investigated the relationship between inflammatory cytokines and matricellular proteins (Periostin-like-factor, PLF, and connective tissue growth factor, CTGF) using our model. We also examined the relationship between inflammatory cytokines, PLF and bone formation processes. Rats underwent initial training for 5 weeks, and then performed a high repetition high force (HRHF) task (12,reaches/min, 60% maximum grip force, 2,h/day, 3 days/week) for 6 weeks. We then examined the effect of training or task performance with or without treatment with a rat specific TNF, antibody on inflammatory cytokines, osteocalcin (a bone formation marker), PLF, CTGF, and behavioral indicators of pain or discomfort. The HRHF task decreased grip strength and induced forepaw mechanical hypersensitivity in both trained control and 6-week HRHF animals. Two weeks of anti-TNF, treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNF, treatment attenuated task-induced increases in inflammatory cytokines (TNF,, IL-1,, and MIP2 in serum; TNF, in forelimb bone and muscles) and serum osteocalcin in 6-week HRHF animals. PLF levels in forelimb bones and flexor digitorum muscles increased significantly in 6-week HRHF animals, increases attenuated by anti-TNF, treatment. CTGF levels were unaffected by task performance or anti-TNF, treatment in 6-week HRHF muscles. In primary osteoblast cultures, TNF,, MIP2 and MIP3a treatment increased PLF levels in a dose dependent manner. Also in primary osteoblast cultures, increased PLF promoted proliferation and differentiation, the latter assessed by measuring Runx2, alkaline phosphatase (ALP) and osteocalcin mRNA levels; ALP activity; as well as calcium deposition and mineralization. Increased PLF also promoted cell adhesion in MC3T3-E1 osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNF,, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro. J. Cell. Physiol. 225: 152,167, 2010. © 2010 Wiley-Liss, Inc. [source] Oversulfated chondroitin sulfate-E binds to BMP-4 and enhances osteoblast differentiationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2008Tatsuya Miyazaki Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N -acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization. J. Cell. Physiol. 217: 769,777, 2008. © 2008 Wiley-Liss, Inc. [source] Osterix is a key target for mechanical signals in human thoracic ligament flavum cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Dongwei Fan Mechanical stress is considered to be an important factor in the progression of thoracic ossification of the ligament flavum (TOLF). To elucidate the mechanism underlying mechanical stress-induced TOLF, we investigated the effect of stretching on cultured flavum ligament cells derived from TOLF and non-TOLF patients. We found that the mRNA expression of alkaline phosphatase (ALP), osteocalcin, Runx2, and osterix, but not that of Dlx5 and Msx2, was significantly increased by stretching in TOLF cells. In addition, the effect seems to be finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, a p38 specific inhibitor, SB203580, significantly inhibited stretching-induced osterix expression as well as ALP activity, whereas a specific inhibitor of ERK1/2, U0126, prevented stretching-induced Runx2 expression. We showed that overexpression of osterix resulted in a significant increase of ALP activity in TOLF cells, and osterix-specific RNAi completely abrogated the stretching-induced ALP activity, indicating that osterix plays a key role in stretching-stimulated osteogenic effect in TOLF cells. These results suggest that mechanical stress plays important roles in the progression of TOLF through induction of osteogenic differentiation of TOLF cells, and our findings support that osterix functions as a molecular link between mechanostressing and osteogenic differentiation. J. Cell. Physiol. 211: 577,584, 2007. © 2007 Wiley-Liss, Inc. [source] Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Takahito Yuasa The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 ,g) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1,5 ,g) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP. © 2002 Wiley-Liss, Inc. [source] Effect of elevated concentration of alkaline phosphatase on cardiac troponin I assaysJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2001Amitava Dasgupta Abstract Troponin I is the regulatory subunit of troponin complex associated with the actin thin filament within muscle cells. Cardiac troponin I (cTnI) is a good marker for diagnosis of myocardial damage. Several immunoassays are available for determination of cTnI in serum. The Stratus cTnI fluorometric enzyme immunoassay (Dade International) uses alkaline phosphatase (ALP) substrate. The microparticle enzyme immunoassay (MEIA) for cTnI (Abbott Laboratories) also uses ALP conjugate. On the other hand, the chemiluminescent assay (CLIA) for cTnI (Bayer Diagnostics) does not use ALP. ALP activity may frequently be elevated in serum of patients being evaluated for suspected myocardial infarction. Therefore, we studied the potential interference of ALP in cTnI assays. Serum pools were prepared from patients, and various concentrations of ALP solution were added to different aliquots. The cTnI concentrations were measured by the Stratus, MEIA, and CLIA assays. We observed no interference of ALP in the MEIA and CLIA assay for cTnI. On the other hand, we observed significant positive interference of ALP when cTnI concentrations were measured using the Stratus. J. Clin. Lab. Anal. 15:175,177, 2001. © 2001 Wiley-Liss, Inc. [source] Alkaline phosphatase isozyme activity in serum from patients with chronic periodontitisJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2003P. Gibert Background:, High alkaline phosphatase activity (ALP) is shown in the periodontal ligament due to the constant renewal of this tissue or pathological circumstances. We have previously shown that the activity level of this enzyme could be reflected at the serum level. Objectives:, Because the local production of ALP in the periodontal ligament is often of the bone-type enzyme, we studied the activity of this isozyme among the other isoforms in the serum of patients with chronic periodontitis in comparison with that of control subjects. Material and methods:, This study included 83 patients (59 with periodontal disease, 24 as control group) and we determined the total seric ALP activity and the percentage of the different isoforms (essentially bone, kidney and intestinal-types) by Ektachem analyser and gel agarose electrophoresis respectively. Conclusions:, By comparisons between the two groups, our results showed a relationship between loss of attachment in periodontal disease and a drop in bone ALP activity in serum. Moreover, these results suggested a gender based difference as well, with lower activity more frequent in women than in men. [source] Melatonin suppresses osteoclastic and osteoblastic activities in the scales of goldfishJOURNAL OF PINEAL RESEARCH, Issue 4 2002Nobuo Suzuki Abstract: The effects of melatonin on osteoclastic and osteoblastic cells were examined using a culture system of the goldfish scale. Tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) were used as markers of osteoclastic and osteoblastic cells, respectively. In Earle's minimum essential medium containing melatonin (10,9 to 10,5 m), activities of both enzymes in scales were significantly suppressed at 6 hr after incubation (TRACP: 10,8, 10,6, 10,5 m; ALP: 10,7 to 10,5 m), but at 18 hr only ALP activity was significantly lowered (10,8, 10,7 m). Estradiol-17ß (E2) enhanced both activities, which were significantly inhibited and brought down to the level of the controls when co-incubated with E2 and melatonin (TRACP at 6 hr: 10,9 to 10,5 m; ALP at 6 hr: 10,7 m; ALP at 18 hr: 10,8 m). Moreover, using reverse-transcription polymerase chain reaction, the mRNA expression of the estrogen receptor (ER) and insulin-like growth factor (IGF)-1, which are related to osteoblastic growth and differentiation, was decreased in the melatonin-treated scales. These results suggest that melatonin acts directly on the scale osteoclastic and osteoblastic cells where it suppresses the ALP activity via down-regulation of ER and IGF-1 mRNAs expression. This is the first report on the function of melatonin in osteoclasts and on the suppressive effect of melatonin in osteoblasts among vertebrates. [source] The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cellsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 5 2010Jun Liu Abstract Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, due to their multipotent nature. For instance, osteogenic hMSCs are applied in bone tissue engineering, but current differentiation protocols need further optimization before they can be clinically applied. Protein kinase C (PKC) family members have been implicated in bone metabolism, which prompted us to use a pharmaceutical approach to manipulate PKC signalling in hMSCs. Inhibition of PKC resulted in a dose-dependent inhibition of dexamethasone-induced osteogenic differentiation. Surprisingly, PKC activation using phorbol 12-myristate 13-acetate (PMA) also resulted in inhibition of osteogenesis, although we observed that inhibition was more pronounced at low than at high concentrations of PMA. Furthermore, we observed that inhibition of PKC, blocked alkaline phosphatase (ALP, an early marker of osteogenic differentiation) expression, whereas inhibition of the conventional PKC subfamily and PKCµ using Gö6976 resulted in an induction of ALP activity, collagen (I) expression and mineralization. In conclusion, inhibition of the conventional PKCs/PKCµ and activation of PKC, could further benefit osteogenic differentiation of hMSCs in vitro and in vivo, which is currently under investigation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Effect of low-level laser irradiation on odontoblast-like cellsLASER PHYSICS LETTERS, Issue 9 2008C.F. Oliveira Abstract Low-level laser therapy (LLLT), also referred to as therapeutic laser, has been recommended for a wide array of clinical procedures, among which the treatment of dentinal hypersensitivity. However, the mechanism that guides this process remains unknown. Therefore, the objective of this study was to evaluate in vitro the effects of LLL irradiation on cell metabolism (MTT assay), alkaline phosphatase (ALP) expression and total protein synthesis. The expression of genes that encode for collagen type-1 (Col-1) and fibronectin (FN) was analyzed by RT-PCR. For such purposes, odontoblast-like cell line (MDPC-23) was previously cultured in Petri dishes (15000 cells/cm2) and submitted to stress conditions during 12 h. Thereafter, 6 applications with a monochromatic near infrared radiation (GaAlAs) set at predetermined parameters were performed at 12-h intervals. Nonirradiated cells served as a control group. Neither the MTT values nor the total protein levels of the irradiated group differed significantly from those of the control group (Mann-Whitney test; p > 0.05). On the other hand, the irradiated cells showed a decrease in ALP activity (Mann-Whitney test; p < 0.05). RT-PCR results demonstrated a trend to a specific reduction in gene expression after cell irradiation, though not significant statistically (Mann-Whitney test; p > 0.05). It may be concluded that, under the tested conditions, the LLLT parameters used in the present study did not influence cell metabolism, but reduced slightly the expression of some specific proteins. (© 2008 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA) [source] Syringetin, a flavonoid derivative in grape and wine, induces human osteoblast differentiation through bone morphogenetic protein-2/extracellular signal-regulated kinase 1/2 pathwayMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 11 2009Ya-Ling Hsu Abstract Syringetin (3,5,7,4,-tetrahydroxy-3,,5,dimethoxyflavone), a flavonoid derivative, is present in grape and wine. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen ELISA, we have shown that syringetin exhibits a significant induction of differentiation in MC3T3-E1 mouse calvaria osteoblasts and human fetal osteoblastic 1.19 cell line human osteoblasts. ALP and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that syringetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by syringetin is associated with increased bone morphogenetic protein-2 (BMP-2) production. The BMP-2 antagonist noggin blocked syringetin-mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 production is required in syringetin-mediated osteoblast maturation and differentiation. Induction of differentiation by syringetin is associated with increased activation of SMAD1/5/8 and extracellular signal-regulated kinase 1/2 (ERK1/2). Cotreatment of ERK1/2 inhibitor 2,-amino-3,-methoxyflavone inhibited syringetin-mediated ALP upregulation and osteocalcin production. In conclusion, syringetin increased BMP-2 synthesis, and subsequently activated SMAD1/5/8 and ERK1/2, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass. [source] Stimulatory effect of undecylenic acid on mouse osteoblast differentiationPHYTOTHERAPY RESEARCH, Issue 4 2010Myung Hee Kim Abstract Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid (UA) on osteoblast differentiation in mouse osteoblastic MC3T3-E1 subclone 4 cells and primary mouse calvarial cells. Low concentrations of UA (up to 5,,M) exhibited no cytotoxicity and significantly increased the expression and activity of alkaline phosphatase (early differentiation marker of osteoblast) and calcium deposition with the induction of expression of the osteocalcin gene in both cells. Interestingly, at low concentration of UA, the induction of NF-,B p65 translocation into nucleus and the up-regulation of AP-1 and NFATc1 transcript levels were also observed, suggesting that the stimulatory effect of UA on osteoblast differentiation could be mediated through the activation of transcription factors. Additionally, although the patterns of UA-induced activation of MAP kinases (JNK and p38) were not completely consistent with the increase of both ALP activity and calcium deposition by UA, MAP kinases might be partially involved in the biological function of UA during the early and late stages of osteoblast differentiation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Involvement of the Klotho Protein in Dentin Formation and MineralizationTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2008Hironobu Suzuki Abstract Klotho -deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho -deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho -deficient mice,comparing the findings with those from their wild-type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho -deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho -deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho -deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization. Anat Rec, 2007. © 2008 Wiley-Liss, Inc. [source] Study of the Structure of Canine Mesenchymal Stem Cell Osteogenic CultureANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 5 2010M. B. Eslaminejad With 6 figures and 1 table Summary This study was designed to investigate the morphological features of osteogenic cultures that were established from canine marrow derived-mesenchymal stem cells (MSCs). Tripotent canine MSCs were plated in osteogenic conditions for 3 weeks, at the end of which the cultures were observed by light and transmission electron microscopy. Alkaline phosphatase (ALP) activity of the culture was determined during the differentiation period. To assess whether endochondral or intramembranous ossification was involved in MSC bone differentiation, the cultures were explored for cartilage-related gene expression. Multiple nodule-like cell aggregates appeared to form in the osteogenic cultures. These nodules were covered by a periosteum-like layer and osteocyte-like cells of varying morphology were located in lacuna-like cavities within the nodule mass. Furthermore, the bone nodules possessed an abundant matrix in which clearly striated collagen I fibres were arranged in perpendicular bundles. Matrix vesicles involving in matrix mineralization were evident in the nodules. This was in accordance with increased ALP activity in the culture. No expression of cartilage-related genes was observed, which suggested that osteogenesis might occur by intramembranous ossification. In conclusion, canine MSCs could be an appropriate model for studying in vitro bone development. [source] Effect of weaning on behavior and serum parameters in dairy goat kidsANIMAL SCIENCE JOURNAL, Issue 4 2008Cengiz ATASOGLU ABSTRACT This study aimed at investigating the effects of weaning kids abruptly at an average of 55 ± 13 days of age on intake, behavioral and serum parameters and, lasted for a total of six weeks; two weeks pre-weaning and four weeks post-weaning. Sixteen single kids with equal gender were used. Kids were only allowed to stay with their mothers for suckling (45 min/period) both in the morning and in the evening period during pre-weaning. Grower concentrate and hay were offered ad libitum. The duration of the study was divided into three periods for the sampling of behavioral and serum parameters; (i) pre-weaning period lasting for two weeks (P-BW) (ii) early post-weaning period lasting for one week (P-AW1) and (iii) late post-weaning period lasting for three weeks (P-AW2). Daily weight gain of kids gradually decreased as the observation period progressed (P = 0.001). However concentrate feed intake increased from 0.154 kg/day in P-BW to 0.479 kg/day in P-AW1 and 0.499 kg/day in P-AW2. Water intake, rumination and standing behaviors decreased in P-AW2 (P < 0.001), whereas activity towards concentrate feed (CF) (P < 0.001) and roughage (P = 0.012) increased as compared to P-BW and P-AW1. Abnormal oral activity was not affected by the periods (P = 0.906). CF was significantly higher in females (P = 0.003), whereas males displayed higher lying behavior (P = 0.007). Glucose, total protein, urea, cholesterol, HDL-cholesterol, LDL-cholesterol concentrations (P = 0.001) and ALP activity (P = 0.003) were significantly affected by the periods. The results of the present study suggest that behavioral and serum parameters across the periods describe changes in the nutritional conditions as a result of the transition from milk to solid feed in association with weaning. [source] Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineeringBIOELECTROMAGNETICS, Issue 7 2007Ming-Tzu Tsai Abstract Bone tissue engineering is an interdisciplinary field involving both engineers and cell biologists, whose main purpose is to repair bone anatomical defects and maintain its functions. A novel system that integrates pulsed electromagnetic fields (PEMFs) and bioreactors was applied to bone tissue engineering for regulating osteoblast proliferation and differentiation in'vitro. Osteoblasts were acquired from the calvaria of newborn Wistar rats and isolated after sequential digestion. Poly(DL -lactic-co-glycolic acid) (PLGA) scaffolds were made by the solvent merging/particulate leaching method. Osteoblasts were seeded into porous PLGA scaffolds with 85% porosity and cultured in bioreactors for the 18-day culture period. Cells were exposed to PEMF pulsed stimulation with average (rms) amplitudes of either 0.13, 0.24, or 0.32 mT amplitude. The resulting induced electric field waveform consisted of single, narrow 300 µs quasi-rectangular pulses with a repetition rate of 7.5'Hz. The results showed that PEMF stimulation for 2 and 8 h at .13 mT increased the cell number on days 6 and 12, followed by a decrease on day 18 using 8 h stimulation. However, ALP activity was decreased and then increased on days 12 and 18, respectively. On the other hand, PEMF-treated groups (irrespective of the stimulation time) at 0.32 mT inhibited cell proliferation but enhanced ALP activity during the culture period. These findings suggested that PEMF stimulation with specific parameters had an effect on regulating the osteoblast proliferation and differentiation. This novel integrated system may have potential in bone tissue engineering. Bioelectromagnetics 28:519,528, 2007. © 2007 Wiley-Liss, Inc. [source] Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3-E1 osteoblast-like cellsCELL BIOCHEMISTRY AND FUNCTION, Issue 1 2009Magdalena Coetzee Abstract Osteoblasts in culture can differentiate into mature mineralizing osteoblasts when stimulated with osteogenic agents. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) may benefit bone health. The aim of this study was to investigate whether arachidonic acid (AA) and docosahexaenoic acid (DHA) affect osteogenesis in osteoblasts and the transdifferentiation into adipocytes. Results from this study show that long-term exposure to AA inhibited alkaline phosphatase (ALP) activity in these cells, which might be prostaglandin E2 (PGE2)-mediated. DHA exposure also inhibited ALP activity which was evident after both short- and long-term exposures. The mechanism whereby DHA inhibits ALP activity is not clear and needs to be investigated. Although long-term exposure to PUFAs inhibited ALP activity, the mineralizing properties of these cells were not compromised. Furthermore, PUFA exposure did not induce adipocyte-like features in these cells as evidenced by the lack of cytoplasmic triacylglycerol accummulation. More research is required to elucidate the cellular mechanisms of action of PUFAs on bone. Copyright © 2008 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||||||||