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Sample records for cultured rat osteoblasts

  1. Enzymatic Cell Isolation and Explant Cultures of Rat Calvarial Osteoblast Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Osteoblast cells were isolated from the calvarial bones of newborn Wistar rats and cultured in vitro via both collagenase digestion method and explant technique, and a comparative study was carried out on the two culture methods. The biologic characteristics of tbs osteoblast cells were studied via cell number counting,morphology observation, alkaline phosphatase staining of the cells and alizarine- red staining of the calcified nodules. The results show that osteoblast cells can be cultured in vitro via collagenase digestion method and explant technique, and the obtained cells are of good biologic characteristics. In comparison with the explant techniqne,the operative procedure of the enzymatic digestion method is more complicated. The digestion time must be carefully controlled. However, with this method, one can obtain a lager number of cells in a short time. The operative procedure of the explant technique is simpler, but it usually takes longer time to obtain cells of desirable number.

  2. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

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    Ishaug-Riley, S. L.; Crane-Kruger, G. M.; Yaszemski, M. J.; Mikos, A. G.

    1998-01-01

    Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study

  3. Expression of precerebellins in cultured rat calvaria osteoblast-like cells.

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    Rucinski, Marcin; Zok, Agnieszka; Guidolin, Diego; De Caro, Raffaele; Malendowicz, Ludwik K

    2008-10-01

    Cerebellin (CER), originally isolated from rat cerebellum, is a hexadecapeptide derived from the larger precursor called precerebellin 1 (Cbln1). At present 4 propeptides designated as Cbln1, Cbln2, Cbln3 and Cbln4 are recognized. They belong to precerebellin subfamily of the C1q family proteins. Precerebellins act as transneuronal regulators of synapse development and synaptic plasticity in various brain regions. Initially CER was thought to be a cerebellum specific peptide, however subsequent studies revealed its presence in other brain regions as well as in extraneuronal tissues. We investigated whether precerebellins are expressed and involved in regulation of cultured rat calvarial osteoblast-like (ROB) cells. Classic RT-PCR revealed the presence of Cbln1 and Cbln3 mRNA in fragments of rat calvaria, in freshly isolated ROB cells and in ROB cells cultured for 7, 14 and 21 days. Cbln2 and Cbln4 mRNA, on the other hand, could not be demonstrated in ROB cells but was found to be present in the brain. In freshly isolated ROB cells expression of Cbln1 gene was very low and gradually increased in relation to the duration of culture. Expression of Cbln3, on the other hand, was very low in fragments of rat calvaria, and increased notably after digestion with collagenase-I. The highest expression of this precerebellin was observed at day 14 of culture while at days 7 and 21 levels of expressions were notably lower. Neither Cbln2 nor Cbln4 was found to be expressed in the ROB cells. Neither CER nor des-Ser1-CER (10(-10)-10(-6)M) affect osteocalcin production and proliferation rate of studied cells. The above findings suggest that CER, which theoretically would be derived from Cbln1, modulate neither differentiated (osteocalcin secretion) nor basic (proliferation) functions of cultured rat osteoblast-like cells. The obtained data raise an intriguing hypothesis that precerebellins may be involved in regulating of spatial organization of osteoblastic niches in the bone.

  4. [Experimental study on the effects of pharmaco-serum of diabetic rats fed with Chinese herbs qianggubao decoction ([Chinese characters: see text]) on osteoblast culture in vitro].

    Science.gov (United States)

    Su, You-xin; Zheng, Liang-pu; Chen, Zhi-neng; Yang, Lian-zi; Wang, He-ming

    2008-03-01

    To investigate the optimum phase and dose of pharmaco-serum of diabetic rats fed with Qianggubao decoction ([Chinese characters: see text]) on the differentiation and mineralization of osteoblast (OB). METHODS (OB) was isolated from the skull of 10 newly born SD rats aged 1 to 2 days by means of Trypsin-collagenase digestion. After the OB was identified, different kinds of pharmaco-serum of diabetic rats fed with inactive Qianggubao decoction ([Chinese characters: see text]) of different phase (rats were fed with medicine three days or five days after last fed with medicine one hour or three hours) and concentration (5%, 10%, 20%) were added to the OB and incubated. After 7 days and 18 days of culture,the effects of the differentiation and mineralization of osteoblast were detected. The secretion of ALP and formation of mineralized nodules of osteoblast in the different doses of pharmaco-serum groups were almost the same as that of normal control group, but were superior to that in the model control group. And the group with concentration of 20% pharmaco-serum was the best in the secretion of ALP and formation of mineralized nodules of osteoblast. As to the phases of pharmaco-serum, the best one on the differentiation and mineralization of osteoblast was the serums from diabetic rat-model fed with Qianggubao decoction ([Chinese characters: see text]) three days or five days, after one hour of last fed with medicine. The pharmaco-serum of diabetic rats fed with Qianggubao decoction ([Chinese characters: see text]) can promote the differentiation and mineralization of osteoblast. Allow for time and the cost of experiment,we presume that pharmaco-serum of diabetic rats fed with Qianggubao decoction ([Chinese characters: see text]) three days, after one hour of last fed, with concentration of 20% and not-inactivation is the optimum on the differentiation and mineralization of osteoblast.

  5. Three-dimensional Expansion: In Suspension Culture of SD Rat's Osteoblasts in a Rotating Wall Vessel Bioreactor

    Institute of Scientific and Technical Information of China (English)

    KE-DONG SONG; TIAN-QING LIU; XIANG-QIN LI; ZHAN-FENG CUI; XIANG-YU SUN; XUE-HU MA

    2007-01-01

    Objective To study large-scale expansion of SD (Sprague-Dawley) rat's osteoblasts in suspension culture in a rotating wall vessel bioreactor (RWVB). Methods The bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm,which could provide low shear on the microcarriers around 1 dyn/cm2. The cells were isolated via sequential digestions of neonatal (less than 3 days old) SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation. Results The results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones. Conclusions The developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional (3D)interactions among cells and is suitable for osteopath expansion in vitro.

  6. Antiosteoporotic Effects of Huangqi Sanxian Decoction in Cultured Rat Osteoblasts by Proteomic Characterization of the Target and Mechanism

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    Chong-Chong Guo

    2015-01-01

    Full Text Available Huangqi Sanxian decoction (HQSXD is routinely used for the treatment of osteoporosis in the Chinese traditional healthcare system. However, the targets and mechanism underlying the effect of HQSXD on osteoporosis have not been documented. In the present study, seropharmacology and proteomic approaches (two-dimensional gel electrophoresis combined with mass spectrometry were used to investigate the effects and possible target proteins of HQSXD on osteoblast. We found that HQSXD-treated rat serum significantly enhanced osteoblast proliferation, differentiation, and mineralization. In HQSXD-S-treated osteoblasts, there were increases in the expression of N-formyl peptide receptor 2 and heparan sulfate (glucosamine 3-O-sulfotransferase 3A1 and reduction in the expression of alpha-spectrin, prohibitin, and transcription elongation factor B (SIII, polypeptide 1. The identified proteins are associated with cell proliferation, differentiation, signal transcription, and cell growth. These findings might provide valuable insights into the mechanism of antiosteoporotic effect affected by HQSXD treatment in osteoblasts.

  7. Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid

    Science.gov (United States)

    Peter, S. J.; Liang, C. R.; Kim, D. J.; Widmer, M. S.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 x 10(-7) micromole/min/cell. Osteocalcin production reached 20 x 10(-6) ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model.

  8. [Experimental study on the effects of inactivated and un-inactivated pharmaco-serum of diabetic rats fed with Chinese herbs Qianggubao decoction on the proliferation of osteoblast cultured in vitro].

    Science.gov (United States)

    Chen, Zhi-Neng; Su, You-Xin; Yang, Lian-Zi; Zheng, Liang-Pu; Lin, Jiu-Mao; Wang, Pei-Qing

    2008-06-01

    To study the effect of inactivated and un-inactivated pharmaco-serum of diabetic rats fed with Chinese herbs Qianggubao decoction on the proliferation of osteoblast cells (OB)cultured in vitro. OB was isolated from the skull of newly born SD rats aged 1 to 2 days by means of Trypsin-collagenase digestion and identified by image analysis under inverted microscope, V-G collagen staining, ALP staining, calcification nod staining etc. After the OB was identified, in activated and un-inactivated pharmaco-serum of diabetic rats fed with Qianggubao decoction of ferent phase (rats were fed with medicine 3 days or 5 days after last fed with medicine 1 hour or 3 hours) and concentration (5%, 10%, 20%) were added to the OB and incubated. After determined times, the effects of the proliferation of osteoblasts were detected by MTT analysis. There was significant difference between un-inactivated pharmaco-serum and inactivated pharmaco-serum on the proliferation of osteoblasts, and un-inactivated serum had stronger effects to improve the proliferation of osteoblasts (P diabetic rats fed with Chinese herbs Qianggubao decoction can influence the proliferation of, and the un-inactivated pharmaco-serum has stronger effects.

  9. Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh.

    NARCIS (Netherlands)

    Dolder, J. van den; Bancroft, G.N.; Sikavitsas, V.I.; Spauwen, P.H.M.; Jansen, J.A.; Mikos, A.G.

    2003-01-01

    The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cell

  10. Culture and regulation of osteoblasts in multiple myeloma patients

    Institute of Scientific and Technical Information of China (English)

    高珊

    2014-01-01

    Objective To investigate the biological characteristics of osteoblasts cultured in vitro from bone marrow(BM)of multiple myeloma(MM)patients and to explore their generation and osteogenic potential.Effects of some factors such as bortezomib and MM patient serum on the osteoblasts were observed.Methods Twenty MM patients and 10 healthy donors as controls were enrolled in this study.Osteoblasts from MM patients’BM were cultured

  11. 新生大鼠下颌骨成骨细胞的分离、培养与鉴定%The isolation, culture and identification of osteoblasts from neonatal rat mandible

    Institute of Scientific and Technical Information of China (English)

    张巍; 史册; 倪世磊; 李琛; 乔春燕; 孙宏晨; 李德超

    2011-01-01

    Objective To explore an efficient, economical and convenient method for isolating neonatal rat mandibular osteoblasts. Methods Under sterile conditions,the mandibles were taken from neonatal rats which were bom within 24 hours. Tissue block adherent method was used. ALP staining and alizarin red staining were used for identification. The mandibular osteoblasts were compared to the osteoblasts of skull. Results The cultured cells exhibited the typical morphological characteristics of osteoblasts. ALP staining was positive and alizarin red staining showed the formation of mineralized nudules. These cells had the similar shape and function with the osteoblasts of skulL Conclusion Osteoblasts of neonatal rat mandible were successfully isolated. This laid the foundation for in vitro study of osteoblasts of mandible.%目的 探求高效、经济、方便的新生大鼠下颌骨成骨细胞的原代培养方法,为研究下颌骨成骨细胞的相关特性奠定基础.方法 无菌条件下取新生24h大鼠的下颌骨,采用组织块贴壁法培养成骨细胞,通过细胞形态学观察、碱性磷酸酶染色(alkaline phosphatase,ALP)、茜素红染色对细胞进行鉴定,并与颅骨分离的成骨细胞进行对比.结果 所分离培养的细胞通过倒置相差显微镜观察呈梭型,碱性磷酸酶染色阳性,能形成钙结节,与颅骨分离的成骨细胞有相似的形态和相同的功能.结论 采用组织块贴壁法成功分离出大量纯化的新生大鼠下颌骨成骨细胞,为体外研究下颌骨成骨细胞的特性奠定了实验基础.

  12. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

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    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

  13. 17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures

    Science.gov (United States)

    McCarthy, T. L.; Ji, C.; Shu, H.; Casinghino, S.; Crothers, K.; Rotwein, P.; Centrella, M.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind

  14. Culture of osteoblasts on bio-derived bones

    Institute of Scientific and Technical Information of China (English)

    LAN Xu; YANG Zhi-ming; GE Bao-feng; LIU Xue-mei

    2005-01-01

    Objective: To study the effect of bio-derived bones, as substitutes of autogenous bone grafts and demineralized cadaver bones, on the attachment, spreading and proliferation of isolated osteoblasts. Methods: Osteoblasts were isolated from the calvaria of a fetal rabbit through sequential collagenase digestion. In the attachment study, the osteoblasts labeled with 3H-leucine were incubated with the bio-derived bone materials in sterile microcentrifugale tubes for 15, 90 and 180 minutes, and 24 hours, respectively. The attached cells were collected and the radioactivity was measured with liquid scintillation spectrometry. In the proliferation study, the osteoblasts were cultured with the bio-derived bone materials for 24 hours and 3H-thymidine was added during the last 2 hours of the incubation. The attached cells were collected and the radioactivity was measured with liquid scintillation spectrometry. Osteoblasts were seeded on the bone graft materials for 60 or 120 minutes, 24 or 48 hours, and 3 or 7 days, then the co-culture was processed for scanning electron microscopy to observe the interaction of osteoblasts and the bio-derived bone materials. Results: Osteoblasts attached to the bio-derived bone materials in a time-dependent manner. There were significantly (P<0.05) more attached cells after 180 minutes than after 15 and 90 minutes of incubations (P<0.05). Osteoblasts were proliferated in a large amount on the surface and in the materials. Osteoblasts seeded onto 100 mg bio-derived bones resulted in significantly (P<0.05) more measurable proliferation than those seeded onto 10 mg bones. Osteoblasts appeared round as they attached to the materials, then flattened and spread over with time passing. Conclusions: Bio-derived bones can provide a good environment for the attachment and proliferation of osteoblasts.

  15. Modulation of Isoflavones on Bone-nodule Formation in Rat Calvaria Osteoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective To observe the effects of two main isoflavones, daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro. Methods Osteoblasts obtained from newborn Sprague-dawley rat calvarias were cultured for several generations. The second generation cells were cultured in Minimum Essential Medium supplemented with ascorbic acid and Na-beta-glycerophosphate for several days, in the presence of daidzein and genistein, with or without the estrogen receptor antagonist ICI 182780. Number of nodules was counted at the end of the incubation period (day 20) by staining with Alizarin Red S calcium stain. The release of osteocalcin, as a marker of osteoblast activity, was also determined on day 7 and day 12 during the incubation period. Results Compared with the control, the numbers of nodules were both increased by incubation with daidzein and genistein. 17a-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation than daidzein and genistein. The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein, as well as 17a-estradiol on day 7 and day 12 (day 12 were higher). The estrogen receptor antagonist ICI 182780 completely blocked the genistein- and 17a-estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts. However, the effects induced by daidzein could not be inhibited by ICI 182780. Conclusion These findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts. The effects, like those induced by 17a-estradiol, are mediated by the estrogen receptor dependent pathway. Daidzein also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts, but it is not, at least not merely, mediated by the estrogen receptor dependent pathway.

  16. A subset of osteoblasts expressing high endogenous levels of PPARgamma switches fate to adipocytes in the rat calvaria cell culture model.

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    Yuji Yoshiko

    Full Text Available BACKGROUND: Understanding fate choice and fate switching between the osteoblast lineage (ObL and adipocyte lineage (AdL is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPARgamma. METHODOLOGY/PRINCIPAL FINDINGS: Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL, a synthetic ligand for PPARgamma. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically when BRL was present. Profiling of ObL and AdL markers by qRT-PCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARgamma mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteo-adipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2, PPARgamma, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARgamma and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment. CONCLUSIONS/SIGNIFICANCE: We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARgamma expression can be switched to the AdL. The fact that some Ob

  17. Effect of La3+ on osteoblastic differentiation of rat bone marrow stromal cells

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In the present work, the effect of La3+ on osteoblastic differentiation of primary rat bone marrow stromal cells (MSCs) as well as the related mechanisms are studied. Differentiation is monitored by detection of alkaline phosphatase (ALP) activity, osteocalcin secretion, the mRNA levels of Type I collagen and osteocalcin, and matrix mineralization. The results show that La3+ inhibits osteoblastic differentiation of MSCs in the early and middle stages of culture, as demonstrated by the decrease of ALP activity, osteocalcin secretion, and down-regulation of the mRNA level of osteocalcin. However, La3+ does not affect the matrix mineralization in advanced MSCs, because it up-regulates the mRNA levels of Type I collagen, and promotes ALP activity and osteocalcin secretion in MSCs in the late stage of culture. In addition, Western blot analysis exhibits that La3+ induces the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Furthermore, MAPK kinase inhibitor PD98059 completely blocks the inhibitory effect of La3+ on ALP activity of MSCs in the middle stage of culture. These results suggest that La3+ affects MSCs osteoblastic differentiation depending on differentiation stages. La3+ inhibits osteoblastic differentiation of MSCs in the early and middle stages by a MAPK-dependent mechanism, but does not affect the matrix mineralization in advanced MSCs.

  18. Comparison on the Effects of Three Sex Hormones on the Fetal Rat Calvarial Osteoblasts

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    CHEN Lulu; ZENG Tianshu; XIA Wenfang; KE Li

    2000-01-01

    17β-estradiol(E2), progesterone (P) and testosterone (T) were investigated for their effects on the proliferation and differentiation of primary rat calvarial osteoblasts in vitro. Rat calvarial osteoblasts were cultured with l0-10 mol/L E2, 10-9-10-6 mol/L P and l0-10 mol/L T for 20days. Cell proliferation was determined by 3H-thymidine incorporation and cell counting. Cell differentiation was examined by measuring alkaline phosphatase (ALP) activity, osteocalcin gene expression and production, bone nodule formation in different periods of culture. Our results showed that no effect of three sex hormones was observed on cell proliferation, but, the responses of cell differentiation to the hormones were more or less different. Among these three hormones used in this study, P appeared to have multi-stimulating effect on cell differentiation. Effect of T seemed not so significant as that of P on cell differentiation. Although ALP activity and osteocalcin production were increased significantly by T, it had slight effect on osteocalcin mRNA and bone nodule formation. Besides, E2 did not demonstrate any effect on cell differentiation. It is concluded that the proliferation of rat calvarial osteoblasts was independent of the presence of sex hormones. However, the differentiation of these cells were stimulated in different levels and to different extent either by P or T. P appeared to be a multi-stimulator on differentiation of such cells.

  19. Curcumin increases rat mesenchymal stem cell osteoblast differentiation but inhibits adipocyte differentiation

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    Qiaoli Gu

    2012-01-01

    Full Text Available Background: Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric and has effects on bone health and fat formation. The bone marrow mesenchymal stem cells (MSCs are multipotent cells capable of differentiating into osteoblasts and adipocytes. Osteoblast differentiation of MSCs can be a result of upregulation of heme oxygenase (HO-1 expression. Curcumin can potently induce HO-1 expression. Objective: The present study describes the effects of curcumin on rat MSC (rMSCs differentiation into osteoblasts and adipocytes. Materials and Methods: Rat bone marrow MSCs were isolated and treated with or without curcumin. Osteoblast differentiation was confirmed and determined by alkaline phosphatase (ALP activity, mineralized nodule formation, the expression of Runx2 (runt-related transcription factor 2 and osteocalcin. Adipocyte differentiation was determined by Oil red O staining and the expression of peroxisome proliferator-activated receptor-γ 2 (PPARγ2 and CCAAT/enhancer-binding protein (C/EBP α. Results: Curcumin increased ALP activity and osteoblast-specific mRNA expression of Runx2 and osteocalcin when rMSCs were cultured in osteogenic medium. In contrast, curcumin decreased adipocyte differentiation and inhibited adipocyte-specific mRNA expression of PPARγ2 and C/EBPα when rMSCs were cultured in adipogenic medium. HO-1 expression was increased during osteogenic differentiation of rMSCs. Conclusions: These findings demonstrate that curcumin can promote osteogenic differentiation of rMSCs and inhibit adipocyte formation. The effect of curcumin on osteogenic differentiation of rMSCs is correlated with HO-1 expression.

  20. Boron Accelerates Cultured Osteoblastic Cell Activity through Calcium Flux.

    Science.gov (United States)

    Capati, Mark Luigi Fabian; Nakazono, Ayako; Igawa, Kazunari; Ookubo, Kensuke; Yamamoto, Yuya; Yanagiguchi, Kajirou; Kubo, Shisei; Yamada, Shizuka; Hayashi, Yoshihiko

    2016-12-01

    A low concentration of boron (B) accelerates the proliferation and differentiation of mammalian osteoblasts. The aim of this study was to investigate the effects of 0.1 mM of B on the membrane function of osteoblastic cells in vitro. Genes involved in cell activity were investigated using gene expression microarray analyses. The Ca(2+) influx and efflux were evaluated to demonstrate the activation of L-type Ca(2+) channel for the Ca(2+) influx, and that of Na(+)/K(+)-ATPase for the Ca(2+) efflux. A real-time PCR analysis revealed that the messenger RNA (mRNA) expression of four mineralization-related genes was clearly increased after 3 days of culture with a B-supplemented culture medium. Using microarray analyses, five genes involved in cell proliferation and differentiation were upregulated compared to the control group. Regarding the Ca(2+) influx, in the nifedipine-pretreated group, the relative fluorescence intensity for 1 min after adding B solution did not increase compared with that for 1 min before addition. In the control group, the relative fluorescence intensity was significantly increased compared with the experimental group (P < 0.05). Regarding the Ca(2+) efflux, in the experimental group cultured in 0.1 mM of B-supplemented medium, the relative fluorescence intensity for 10 min after ouabain treatment revealed a significantly lower slope value compared with the control group (P < 0.01). This is the first study to demonstrate the acceleration of Ca(2+) flux by B supplementation in osteoblastic cells. Cell membrane stability is related to the mechanism by which a very low concentration of B promotes the proliferation and differentiation of mammalian osteoblastic cells in vitro.

  1. Effect of fluoride on expression of insulinlike growth factor-1 and its receptor of rat osteoblasts

    Institute of Scientific and Technical Information of China (English)

    喻茂娟

    2013-01-01

    Objective To explore the influence of fluorine on mRNA and protein expression of the insulin-like growth factor-1 (IGF-1) and its receptor of rat osteoblasts.Methods Osteoblasts were isolated from rat bone by enzyme digestion.Different fluorine concentration[0 (con-

  2. The effect of enamel matrix derivative on spreading, proliferation, and differentiation of osteoblasts cultured on zirconia.

    Science.gov (United States)

    Wada, Yoshiyuki; Mizuno, Morimichi; Nodasaka, Yoshinobu; Tamura, Masato

    2012-01-01

    This study investigated the effect of enamel matrix derivative (EMD) on spreading, proliferation, and differentiation of osteoblasts cultured on zirconia disks with smooth and rough surfaces. EMD was added to the culture medium or coated on zirconia disks that had machined (smooth) or sandblasted (rough) surfaces. The effects of EMD on cell proliferation of MC3T3-E1 osteoblastic cells were examined using a hemocytometer. Osteoblastic differentiation was examined by histologic analysis of alkaline phosphatase (ALP) activity and the degree of mineralization. ALP activity was also measured quantitatively. Scanning electron microscopic analysis was performed to observe cell morphology. Enzyme-linked immunosorbent assay of osteocalcin and reverse-transcriptase polymerase chain reaction of osteocalcin, osteopontin, and type 1 collagen were performed to investigate the expression of osteoblast-related genes. The addition of EMD to the medium enhanced the spreading, proliferation, and differentiation of osteoblasts cultured on zirconia. However, when it was coated on zirconia, EMD reduced osteoblastic spreading and adhesion in the early stage of culture, although it enhanced proliferation and differentiation of osteoblasts in later stages. A promotive effect of EMD on osteocalcin mRNA expression, mineralization, and ALP activity of osteoblasts cultured on the rough surface was observed. EMD may contribute to treatment with zirconia implants via its promotion of osteoblastic proliferation and activity. However, the procedure for application of EMD may be a crucial factor for the outcome of implants.

  3. The effect of enamel matrix proteins on the spreading, proliferation and differentiation of osteoblasts cultured on titanium surfaces.

    Science.gov (United States)

    Miron, Richard J; Oates, Christine J; Molenberg, Aart; Dard, Michel; Hamilton, Douglas W

    2010-01-01

    Modifications of implant surface topography and chemistry have proven a means to enhance osseointegration, a process that ensures the stability of bone-contacting devices, including titanium dental implants. The commercial product Emdogain is an enamel matrix derivative (EMD) extracted from porcine teeth commonly used in periodontal surgery, where it has been shown to potentiate regeneration of bone. The aim of the present study was to evaluate the effect of EMD on the attachment, proliferation and differentiation of osteoblasts on titanium surfaces in vitro. Pickled (smooth) and SLA (roughened) titanium discs were coated with EMD or left uncoated. Primary rat calvarial osteoblasts were cultured on each surface from 1h to 4 weeks. EMD significantly increased cell spreading and proliferation at time points ranging from 3 to 7 days on both topographies. Alkaline phosphatase activity was significantly increased on EMD-coated titanium compared with titanium alone. Moreover, there was a 6 fold increase in levels of mRNA encoding bone sialoprotein and osteocalcin in osteoblasts cultured on EMD-coated titanium surfaces compared with uncoated surfaces. We conclude that coating of titanium with EMD enhances the proliferation and differentiation of osteoblasts irrespective of the titanium substratum topography.

  4. Spatial distribution of osteoblast activating peptide in the rat stomach.

    Science.gov (United States)

    Noreldin, Ahmed E; Sogabe, Maina; Yamano, Yoshiaki; Uehara, Masato; Mahdy, Mohamed A A; Elnasharty, Mohamed A; Sayed-Ahmed, Ahmed; Warita, Katsuhiko; Hosaka, Yoshinao Z

    2016-03-01

    Osteoblast activating peptide (OBAP) was previously reported to be expressed in the rat stomach and to have a vital role in osteogenesis, but its distribution in rat stomach has not been determined. Thus, the aim of the present study was to identify the cell types expressing OBAP in the rat stomach. The stomachs of twelve 10-to-11-week-old male Jc1:SD rats were used. Samples were collected for immunohistochemistry, immunoelectron microscopy and dot blot assay. Immunohistochemical investigation revealed that OBAP was distributed mainly in parietal cells without any expression in chief cells, X/A-like cells or enterochromaffin-like cells. Moreover, OBAP-immunopositive cells were observed mainly in the upper and lower parts of the gastric gland. Significantly high optical density of immunopositive cells was observed in the upper and lower gastric gland regions. The dot blot assay confirmed that OBAP is secreted by parietal cells and that it is present in the gastric gland lumen. Immunoelectron microscopy demonstrated that OBAP was confined to the mitochondrial inner membrane within parietal cells and that the number of mitochondria in the upper and lower parts of the gastric epithelium was significantly larger than the number in the middle part of the gastric epithelium. Based on the results, it was concluded that OBAP is mainly produced by mitochondria of parietal cells in the upper and lower parts of the gastric epithelium. Moreover, the presence of OBAP in the gastric gland lumen suggests an exocrine mechanism of release. Copyright © 2015 Elsevier GmbH. All rights reserved.

  5. Treatment of osteonecrosis of the femoral head using autologous cultured osteoblasts: a case report

    Directory of Open Access Journals (Sweden)

    Kim Seok-Jung

    2008-02-01

    Full Text Available Abstract Introduction Osteonecrosis of the femoral head is a progressive disease that leads to femoral head collapse and osteoarthritis. Our goal in treating osteonecrosis is to preserve, not to replace, the femoral head. Case presentation We present the case of a patient with bilateral osteonecrosis of the femoral head treated with autologous cultured osteoblast injection. Conclusion Although our experience is limited to one patient, autologous cultured osteoblast transplantation appears to be effective for treating the osteonecrosis of femoral head.

  6. Indoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells.

    Science.gov (United States)

    Nii-Kono, T; Iwasaki, Y; Uchida, M; Fujieda, A; Hosokawa, A; Motojima, M; Yamato, H; Kurokawa, K; Fukagawa, M

    2007-04-01

    Skeletal resistance to parathyroid hormone (PTH) is well known to the phenomenon in chronic renal failure patient, but the detailed mechanism has not been elucidated. In the process of analyzing an animal model of renal failure with low bone turnover, we demonstrated decreased expression of PTH receptor (PTHR) accompanying renal dysfunction in this model. In the present study, we focused on the accumulation of uremic toxins (UTx) in blood, and examined whether indoxyl sulfate (IS), a UTx, is associated with PTH resistance. We established primary osteoblast cultures from mouse calvariae and cultured the cells in the presence of IS. The intracellular cyclic adenosine 3',5' monophosphate (cAMP) production, PTHR expression, and free radical production in the primary osteoblast culture were studied. We found that the addition of IS suppressed PTH-stimulated intracellular cAMP production and decreased PTHR expression in this culture system. Free radical production in osteoblasts increased depending on the concentration of IS added. Furthermore, expression of organic anion transporter-3 (OAT-3) that is known to mediate cellular uptake of IS was identified in the primary osteoblast culture. These results suggest that IS taken up by osteoblasts via OAT-3 present in these cells augments oxidative stress to impair osteoblast function and downregulate PTHR expression. These finding strongly suggest that IS accumulated in blood due to renal dysfunction is at least one of the factors that induce skeletal resistance to PTH.

  7. The expression of insulin-like growth factor-Ⅰ mRNA and polypeptide in rat osteoblasts with exposure to parathyroid hormone

    Institute of Scientific and Technical Information of China (English)

    张克勤; 陈家伟; 王美莲; 汪承亚; 李光富; 郑肇熙; 赵人铮

    2003-01-01

    Objective To investigate the insulin-like growth factor-Ⅰ (IGF-Ⅰ) gene and polypeptide expression in cultured rat osteoblast (ROB) and the role of IGF-Ⅰ in mediating the cell-to-cell communication by mimicking the pharmacokinetics of parathyroid hormone (PTH).Methods The ROB was cultured with three kinds of treatment: (1) Control (Ctr), the cells were cultured without PTH during the first 6 hours and the subsequent 42 hours in a 48-hour cycle; (2) Intermittent exposure to PTH (Itm), the cells were cultured with PTH during the first 6 hours, but without PTH in the subsequent 42 hours; and (3) Continuous exposure to PTH (Ctu), the cells were cultured with PTH during the first 6 hours and the subsequent 42 hours.Results The bone-forming activities of ROB were increased in Itm and inhibited in Ctu. The IGF-Ⅰ mRNA content in Itm cells was elevated only during the first 6 hours and that in Ctu cells was elevated at any time during an incubation cycle. The free IGF-Ⅰ concentration in the medium of Itm cells was generally higher and that of the Ctu cells was generally lower compared with those of the Ctr cells. The IGF-Ⅰ antibody significantly reduced the alkaline phosphatase activity within the cells of Ctr and Itm.Conclusions PTH rapidly and constantly stimulates the IGF-Ⅰ gene transcription of osteoblast. There was an obvious discrepancy between the IGF-Ⅰ mRNA content within the osteoblast and the free IGF-Ⅰ level around the osteoblast in either mode of PTH action. The IGF-Ⅰ might be important for osteoblast-osteoblast communication and bone-forming activity, not only in intermittent PTH administration, but also in the physiological functioning of osteoblasts.

  8. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    Science.gov (United States)

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  9. The expression of TGF-βmRNA and its biological function in rat osteoblasts with exposure to raloxifene

    Institute of Scientific and Technical Information of China (English)

    Guo Ling-cen; Liu Jia-yin; Zhang Ke-qin; Zhang Hong-mei; Gong Jian

    2007-01-01

    Objective: To investigate the effects of raloxifene and estradiol on the proliferation, differentiation and the expression of transforming growth factor-β(TGF-β) of osteoblasts in vitro.Methods: Different doses of raloxifene and estradiol were added into the medium of the second generation osteoblasts obtained from the skull of newborn SD rats.The following parameters including cell proliferation,activity of alkaline phosphatase(ALP), the levels of type I collagen(Col-I) mRNA and TGF-β1 mRNA in different groups were measured and analyzed.Results: Raloxifene and 17-βestradiol(17-β E2 ) showed no significant effect on stimulating the proliferation of osteoblasts (P>0.05 vs the control).However, raloxifene could significantly improve ALP activity and Col-I mRNA expression in high consistency group (P<0.01) in dose-dependent manner.Raloxifene group in 10-8 mol/L increased the expression of TGF-β1 mRNA (vs the control, P < 0.05).Estradiol significantly increased ALPactivity, Col-I mRNA expression and TGF-β1 mRNA expression (P<0.05 or P<0.01 vs the control).Conclusions: Both of raloxifene and estradiol could stimulate differentiation of osteoblasts and expression of bone matrix, but showed no effect on proliferation of cultured osteoblasts.The expressions of TGF-β1 mRNA were different, which might imply their different mechanisms by means of estrogen receptor.

  10. Effects of Shenggu Injection (生骨注射液) on mRNA Expression of Vascular Endothelia Growth Factor in Rat Osteoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective: To study the effects of Shenggu injection (生骨注射液,SGI) on mRNA expression of vascular endothelia growth factor (VEGF) in rat osteoblasts in vitro and to explore its possible molecular mechanisms in promoting fracture healing. Methods: Rat osteoblasts cultured in vitro were stimulated with SGI according to the protocol. The expression levels of VEGF mRNA in the cells in every group were examined by reverse-transcriptase ploymerase chain reaction (RT-PCR). Results: When osteoblasts were stimulated with different concentrations of SGI for 5 days, the expression of VEGF mRNA peaked with 1 mg/ml SGI on the 5th day. When treated with 1 mg/ml SGI from the 1st to the 5th day, the expression of VEGF mRNA increased gradually with the increase of culturing time. Conclusion: SGI could promote significantly the expression of VEGF mRNA in rat osteoblasts in vitro. The levels of expression of VEGF mRNA changed along with different concentrations and stimulating time of SGI.

  11. Zinc enhances bone metabolism in ovariectomized rats and exerts anabolic osteoblastic/adipocytic marrow effects ex vivo.

    Science.gov (United States)

    Li, Binbin; Liu, Hao; Jia, Shengnan

    2015-02-01

    Investigations of bone mass and marrow adiposity are critical for defining the role of zinc (Zn) in bone metabolism. Rats used for study were grouped as follows: control (sham), ovariectomy (OVX), ovariectomy + estradiol (OVX-E), ovariectomy + Zn treatment (OVX-Zn). Bone mineral density (BMD) was quantified (microCT); serum osteocalcin, adiponectin, RANKL, and TRAP levels were assayed (ELISA); and biochemical determinations of serum alkaline phosphatase (ALP), calcium (Ca), and phosphorus (P) were done. Cells derived from bone mesenchymal stem cell (BMSC) isolates of respective test groups were compared, identifying primary osteoblasts by MTT assay and adipocytes by Oil Red O stain. Osteocalcin and adiponectin levels in culture supernatants were determined by ELISA. Zn supplementation resulted in a modest increase in BMD, but serum osteocalcin and ALP activity increased significantly (P < 0.01, both). Serum levels of RANKL and TRAP were lower in OVX-Zn (vs OVX) rats (P < 0.01), whereas serum concentrations of adiponectin, Ca, and P did not differ by group. Osteocalcin level was significantly upregulated ex vivo (P < 0.01) in the supernatant of cultured OVX-Zn (vs OVX) cells, accompanied by a slight upturn in osteoblastic differentiation. However, Oil Red O uptake and adiponectin level in supernatant were sharply diminished in cultured OVX-Zn (vs OVX) cells (P < 0.01). Overall, we concluded that Zn contributes to bone mass by marginally stimulating differentiation and proliferation of osteoblasts and by effectively inhibiting osteoclastic and adipocytic differentiation of BMSCs.

  12. Behaviour of moderately differentiated osteoblast-like cells cultured in contact with bioactive glasses

    Directory of Open Access Journals (Sweden)

    Hattar S.

    2002-12-01

    Full Text Available Bioactive glasses have been shown to stimulate osteogenesis both in vivo and in vitro. However, the molecular mechanisms underlying this process are still poorly understood. In this study, we have investigated the behaviour of osteoblast-like cells (MG63, cultured in the presence of bioglass particles. Three types of granules were used: 45S5registered bioactive glass, 45S5registered granules preincubated in tris buffer and 60S non-reactive glass, used as control. Phase contrast microscopy permitted step-by-step visualization of cell cultures in contact with the particles. Ultrastructural observations of undecalcified sections revealed direct contacts of the cells and an electron-dense layer located at the periphery of the material. Protein synthesis was evaluated biochemically and showed a gradual increase throughout the culture time in the three types of cultures. Alkaline phosphatase was detected in situ, in clusters of packed cells either in contact with the material or in the background cell layer. Semi-quantitative RT-PCR analysis of the main osteoblastic markers showed that gene expression was maintained in all three cultures. The fact that osteocalcin was not detected, supports the fact that the MG63 cell line is composed of less differentiated osteogenic cells rather than mature osteoblasts. We also demonstrated for the first time in this cell line, the expression of Msx-2, Dlx-3 and Dlx-7 homeogenes, known to regulate in vivo foetal skeletogenesis as well as adult skeletal regeneration. However, no significant differences could be recognised in the expression pattern of bone markers between the three types of cultures. Yet these preliminary results indicate that bioactive glasses provided a suitable environment for the growth and proliferation of osteoblasts in vitro, since no drastic changes in phenotype expression of pre-osteoblasts was noted.

  13. Identification of Rorβ targets in cultured osteoblasts and in human bone

    Energy Technology Data Exchange (ETDEWEB)

    Roforth, Matthew M., E-mail: roforth.matthew@mayo.edu; Khosla, Sundeep, E-mail: khosla.sundeep@mayo.edu; Monroe, David G., E-mail: monroe.david@mayo.edu

    2013-11-01

    Highlights: •We examine the gene expression patterns controlled by Rorβ in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Rorβ mRNA levels increase in aged, human bone biopsies. •Rorβ may affect osteoblast activity by modulation of these pathways. -- Abstract: Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

  14. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Dao-Cai [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 291st Hospital of P.L.A, Baotou (China); Li, De-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ji, Hui-Cang [Military Sanatorium of Retired Cadres, Baotou (China); Rao, Guo-Zhou [Center of Laboratory, School of Stomatology, Xi' an Jiaotong University, Xi' an (China); Liang, Li-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ma, Ai-Jie [Xi' an Technology University, Xi' an (China); Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China)

    2012-04-05

    In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

  15. Decreased osteoblasts and increased osteoclasts in rats after coal dust exposure

    Directory of Open Access Journals (Sweden)

    Izaak Zoelkarnain Akbar

    2016-02-01

    Full Text Available Bone remodeling is a physiological process of cortical and trabecular bone reconstruction, with initial bone resorption, by osteoclasts and concurrent bone formation by osteoblasts. Oxidative stress due to coal dust exposure is not only found in the lungs, but also in the circulation or systemically. The aim of this study was to determine the effect of oxidative stress from coal dust exposure on the number of osteoblasts and osteoclasts in rats. In this experimental study, four groups were evaluated: control; coal dust exposure at 6.25 mg/m3 for 28 days; coal dust exposure at 12.5 mg/m3 for 28 days; coal dust exposure at 25 mg/m3 for 28 days (all exposures were given daily for one hour. Circulatory oxidative stress was measured by malondialdehyde level. Osteoblast and osteoclast numbers were counted by light microscopic examination of distal femoral cross-sections stained with hematoxylin eosin. This study showed that malondialdehyde levels were significantly increased in coal dust exposure groups, in comparison with the control group (p0.05. Coal dust exposure increased malondialdehyde level and osteoclast numbers, and decreased osteoblast numbers, but no correlation was found between oxidative stress (caused by coal dust exposure and osteoblast and osteoclast numbers.

  16. Culturing rat hippocampal neurons.

    Science.gov (United States)

    Audesirk, G; Audesirk, T; Ferguson, C

    2001-01-01

    Cultured neurons are widely used to investigate the mechanisms of neurotoxicity. Embryonic rat hippocampal neurons may be grown as described under a wide variety of conditions to suit differing experimental procedures, including electrophysiology, morphological analysis of neurite development, and various biochemical and molecular analyses.

  17. Effects of Zhuangjinxugu decoction-containing serum on the proliferation of rat osteoblasts cultured in vitro%壮筋续骨汤含药血清体外培养大鼠成骨细胞的增殖

    Institute of Scientific and Technical Information of China (English)

    王力; 涂宏; 杨凤云; 孔令山; 鲍杰伟; 郑甦

    2011-01-01

    背景:壮筋续骨汤具有促进胫骨骨折愈合的作用.目的:观察壮筋续骨汤含药血清对体外培养大鼠成骨细胞增殖的影响.方法:大鼠灌胃给予壮筋续骨汤后制备含药血清.取第2代生长状况良好出生48 h内的SD大鼠成骨细胞,对照组和壮筋续骨汤组分别加入5%,10%,20%的正常SD大鼠血清和含药血清培养72 h.结果与结论:MTT法检测发现壮筋续骨汤含药血清对成骨细胞的增殖有明显的促进作用,以10%壮筋续骨汤组作用最明显(P < 0.05).流式细胞仪检测各组细胞DNA合成期(S期)的比例,发现壮筋续骨汤组处于S期的细胞比例明显大于对照组 (P < 0.05).说明壮筋续骨汤含药血清能促进成骨细胞DNA的合成,使更多的细胞进入S期,促进体外培养的成骨细胞增殖.%BACKGROUND: Zhuangjinxugu decoction has been shown to promote the healing of tibial fracture.OBJECTIVE: To investigate the effects of Zhuangjinxugu decoction-containing serum on proliferation of rat fibroblasts cultured invitro .METHODS: Rats were intragastrically administered with Zhuangjinxugu decoction to prepare drug-containing serum. Passage 2well growing SD rat fibroblasts were harvested within 48 hours after rat birth. Rats from the control and Zhuangjinxugu decoctiongroup were intragastrically administered with 5%, 10%, 20% normal SD rat serum and Zhuangjinxugu decoction-containingserum and then cultured for 72 hours.RESULTS AND CONCUSION: Cellular proliferation in each group was detected by MTT and results showed that Zhuangjinxugudecoction-containing serum greatly promote the proliferation of fibroblasts, in particular 10% Zhuangjinxugu decoction-containingserum (P < 0.05). The proportion of cells at DNA synthesis (S stage) was detected by flow cytometry, and the proportion of cellsat the S stage was greater in the Zhuangjinxugu decoction group than in the control group (P < 0.05). These findings suggest thatZhuangjinxugu deconction

  18. Apoptosis Induced by Zinc Deficiency in Rat Osteoblast: Possible Involvement of Protein Kinase C

    Institute of Scientific and Technical Information of China (English)

    CEN XIAO-BO; WANG RUI-SHU; AND WANG HANG

    1999-01-01

    Rat osteoblasts were isolated from the 21-day fetal rat calvarias. The cells were grown in DMEM plus 10% FBS, and were treated for 24 h. With 10 μmol/L TPEN or 10 μmol/L TPEN supplemented with 10 μmol/L Zn2+ . Apoptosis of osteoblasts were measured by flow cytometry, electron microscopy and DNA fragmentation analyzed by gel electrophoresis. In addition, IP3 production and PKC activity were measured in order to show whether they are involved in apoptosis in osteoblast induced by zinc deficiency. The results showed that 10 μmol/L TPEN could induce apoptosis in osteoblast in 24 h. But cells treated with 10 μmol/L TPEN supplemented with 10 μmol/L Zn2+showed no apoptotic changes in 24 h. TPEN significantly reduced the formation of IP3 and PKC activity after 24 h incubation. No differences were observed between the cells treated with TPEN supplemented with Zn2 + simultaneously and the untreated cells. It can be inferred that apoptosis induced by zinc deficiency may be due to the decreased activity of PKC which is impaired by reduced formation of IP3.

  19. Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts.

    Science.gov (United States)

    DeNichilo, Mark O; Shoubridge, Alexandra J; Panagopoulos, Vasilios; Liapis, Vasilios; Zysk, Aneta; Zinonos, Irene; Hay, Shelley; Atkins, Gerald J; Findlay, David M; Evdokiou, Andreas

    2016-03-01

    The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

  20. Enhanced and suppressed mineralization by acetoacetate and β-hydroxybutyrate in osteoblast cultures.

    Science.gov (United States)

    Saito, Akihiro; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Chikazu, Daichi; Yamamoto, Matsuo; Kamijo, Ryutaro

    2016-04-29

    It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

  1. Inositol hexakisphosphate inhibits mineralization of MC3T3-E1 osteoblast cultures.

    Science.gov (United States)

    Addison, William N; McKee, Marc D

    2010-04-01

    Inositol hexakisphosphate (IP6, phytic acid) is an endogenous compound present in mammalian cells and tissues. Differentially phosphorylated forms of inositol are well-documented to have important roles in signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit soft tissue calcification (specifically renal and vascular calcification) by binding extracellularly to calcium oxalate and calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent inhibition of mineralization without affecting cell viability, proliferation or collagen deposition. Osteoblast differentiation markers, including tissue-nonspecific alkaline phosphatase activity, bone sialoprotein and osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly increased protein and mRNA levels of osteopontin, a potent inhibitor of crystal growth and matrix mineralization. Inositol alone (without phosphate), as well as inositol hexakis-sulphate, a compound with a high negative charge similar to IP6, had no effect on mineralization or osteopontin induction. Binding of IP6 to mineral crystals from the osteoblast cultures, as well as to synthetic hydroxyapatite crystals, was confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by binding to growing crystals through negatively charged phosphate groups and by induction of inhibitory osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of osteopontin gene expression.

  2. Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells.

    Science.gov (United States)

    Nakamura, Toshiaki; Shinohara, Yukiya; Momozaki, Sawako; Yoshimoto, Takehiko; Noguchi, Kazuyuki

    2013-10-18

    Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2+FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9+FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.

  3. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

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    Dao-Cai Sun

    2012-06-01

    Full Text Available In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP concentration, and ELISA for the concentration of type I collagen (COL-I in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05. The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

  4. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds

    Science.gov (United States)

    Ishaug, S. L.; Crane, G. M.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

  5. Lecithin blended polyamide-6 high aspect ratio nanofiber scaffolds via electrospinning for human osteoblast cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Nirmala, R. [Bio-nano System Engineering, College of Engineering, Chonbuk National University, Jeonju, 561 756 (Korea, Republic of); Park, Hye-Min [Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); Navamathavan, R. [School of Advanced Materials Engineering, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); Kang, Hyung-Sub [Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); El-Newehy, Mohamed H. [Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Kim, Hak Yong, E-mail: khy@jbnu.ac.kr [Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Center for Healthcare Technology and Development, Chonbuk National University, Jeonju, 561 756 (Korea, Republic of)

    2011-03-12

    In this study, we focused on the preparation and characterization of lecithin blended polyamide-6 nanofibers via an electrospinning process for human osteoblastic (HOB) cell culture applications. The morphological, structural characterizations and thermal properties of polyamide-6/lecithin nanofibers were determined by using scanning electron microscopy (SEM), field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA). SEM images revealed that the nanofibers were well-oriented with good incorporation of lecithin. FT-IR results indicated the presence of amino groups of lecithin in the blended nanofibers. TGA analysis revealed that the onset degradation temperature decreased with increasing lecithin content in the blended nanofibers. The morphological features of cells attached on polyamide-6/lecithin nanofibers were confirmed by SEM. The adhesion, viability and proliferation properties of osteoblast cells on the polyamide-6/lecithin blended nanofibers were analyzed by in vitro cell compatibility test. This study demonstrated the non-cytotoxic behavior of electrospun polyamide-6/lecithin nanofibers for the osteoblast cell culture.

  6. SCANNING ELECTRON MICROSCOPIC STUDY OF FETAL CHICKEN CALVARIAL OSTEOBLAST-LIKE CELLS CULTURED IN VITRO

    Institute of Scientific and Technical Information of China (English)

    柴本甫; 汤雪明; 徐荣辉; 朱雅萍

    1993-01-01

    Three types of osteoblast-like cells with different cnfigurations could be ob-tained through culturing fetal chicken calvaria in vitro. They were spindle-shaped cells,globular cells, and polygonal or squamous cells. With passage of culture time, there werechanges in configuration so that the spindle-shaped cells and the globular cells turnedgradually into squamous cells, in quantity which increased greatly to produce confluenceand multi-layer formation of cells, and in function as evidenced by emergence ofintracytoplasmic granules, reflecting collagen synthesis.

  7. Calcification in human osteoblasts cultured in medium conditioned by the prostatic cancer cell line PC-3 and prostatic acid phosphatase.

    Science.gov (United States)

    Kimura, G; Sugisaki, Y; Masugi, Y; Nakazawa, N

    1992-01-01

    A medium that had been conditioned by PC-3 cells stimulated the calcification of a human osteoblastic cell line, Tak-10, in a nonmitogenic culture. The calcification of the osteoblasts was stimulated maximally at a 25% concentration of the conditioned medium. Calcification activity was markedly enhanced by the addition of both prostatic acid phosphatase (PAP) and its substrate, alpha-glycerophosphate, to the medium; however, PAP added alone did not enhance this activity. These results suggest that human prostatic carcinoma cells produce a factor that stimulates the calcification of the human osteoblasts. Results have also suggested that PAP is a requisite for osteogenesis provided that its substrates are abundant in the medium.

  8. Feeding blueberry diets in early life prevent senescence of osteoblasts and bone loss in ovariectomized adult female rats.

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    Jian Zhang

    Full Text Available BACKGROUND: Appropriate nutrition during early development is essential for maximal bone mass accretion; however, linkage between early nutrition, childhood bone mass, peak bone mass in adulthood, and prevention of bone loss later in life has not been studied. METHODOLOGY AND PRINCIPAL FINDINGS: In this report, we show that feeding a high quality diet supplemented with blueberries (BB to pre-pubertal rats throughout development or only between postnatal day 20 (PND20 and PND34 prevented ovariectomy (OVX-induced bone loss in adult life. This protective effect of BB is due to suppression of osteoblastic cell senescence associated with acute loss of myosin expression after OVX. Early exposure of pre-osteoblasts to serum from BB-fed rats was found to consistently increase myosin expression. This led to maintenance osteoblastic cell development and differentiation and delay of cellular entrance into senescence through regulation of the Runx2 gene. High bone turnover after OVX results in insufficient collagenous matrix support for new osteoblasts and their precursors to express myosin and other cytoskeletal elements required for osteoblast activity and differentiation. CONCLUSIONS/SIGNIFICANCE: These results indicate: 1 a significant prevention of OVX-induced bone loss from adult rats can occur with only 14 days consumption of a BB-containing diet immediately prior to puberty; and 2 the molecular mechanisms underlying these effects involves increased myosin production which stimulates osteoblast differentiation and reduces mesenchymal stromal cell senescence.

  9. The effect of caffeine on osteoblast proliferation after tooth extraction in Wistar rats

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    Budi Yuwono

    2006-03-01

    Full Text Available Caffeine is the most well-known substance which consumed by most people daily. Behind its popularity as favorable drinks and food, this substance also known can inhibit the post extraction wound healing by decreasing the proliferation of osteoblast cells through the increase of intracellular cyclic Adenosine Mono Phosphate (cAMP. The objective of this study was done to observe the effect of caffeine intake toward the number of osteoblast cells during the wound healing of post dental extraction in Wistar’s rats. This study was an experimental laboratory research and the post test-only control group design was used for the statistical evaluation. The samples used were 24 healthy 3 months old male Wistar’s rats, with approximately 200 grams of body weight and devided into 4 groups. Three groups were taken and represented as a treated group (P and the rest of one group was used as a control group (KO. Caffeine diet with a dosage of 3.78 mg/100 ml grams of body weight/cc was given for 7 days in group P1, P2 for 14 days, and 21 days in group P3 and the diet was given orally using an oral sonde. Teeth extractions of the right first molar in the lower jaw were done in all groups according to the interval time had been scheduled. Seven days of post-extraction time was waiting in all groups before the sample being decapitated for further histological examination in the post extracted sites. A Hematoxillin and Eosin staining was used and the number of osteoblast cells were counted under light microscopy with 400 times magnification. One-way ANOVA and Least Significant Difference (LSD test were used for the statistical evaluation. The result of the study shown a significant decrease of the number of osteoblast cells in caffeine consumed group of 7, 14, and 21 days observed (p < 0.05. This study conclude that the duration time of caffeine consumed had been interfered significantly with the osteoblast cell proliferation during the wound healing after

  10. Hydroxyapatite combined with hyaluronic acid metronidazole gel increased the quantity of osteoblasts in the alveolar bone wistar rat

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    Ernie Maduratna Setiawatie

    2015-12-01

    Full Text Available Background: Bone graft material have been used extensively in bone healing and periodontal treatment. Alloplast such as hydroxyapatite are frequently used to repair and reconstruct bone defects. By merely applying hydroxyapatite for the treatment of bone is not fully effective yet to produce new bone regeneration. Locally applied high molecular hyaluronic acid (HA has been shown to stimulate differentiation and migration of mesenchymal cells. Recent studies on regenerative surgical procedures indicate that reduction of bacterial burden at the wound site may improve the clinical outcome of regenerative therapy. Metronidazole has the greatest bacteriostatic effect. A clinical application of HA metronidazole gels during the surgical therapy may reduce the bacterial contamination of surgical wound site. Purpose: The purpose of this study was to examine the effect of combination hydroxyapatite and HA metronidazole gel 1% on osteoblast cell number after wound healing process in the wistar rats incisor tooth extraction socket. Method: Twenty seven wistar rats were divided randomly into 3 groups. The first group consisted of wistar rats given hydroxyapatite were subjected to the mandibular incisor extraction socket. The second group were given hydroxyapatite combined with HA metronidazole gel 1%. The control group were filled with blood. Wistar rats were euthanized on day 14 and then preparation for histological examination was stained using hematoxylin-eosin and then the numbers of the osteoblasts were calculated. Result: The differences in each group were tested by one way Anova test (α=0.05. The numbers of osteoblasts in each group had a significant difference (p<0.05: the highest numbers of osteoblasts were found in the group that was given hydroxyapatite combined with HA metronidazole, followed by the lower numbers of osteoblasts in the hydroxyapa ite group and the lowest numbers of osteoblasts were found in the control group. Conclusion

  11. Intermittent versus continuous stretching effects on osteoblast-like cells in vitro.

    NARCIS (Netherlands)

    Winter, L.C.; Walboomers, X.F.; Bumgardner, J.D.; Jansen, J.A.

    2003-01-01

    The objective of this study was to quantify and compare stretch-mediated responses of primary rat osteoblast-like cells to uniform cyclic strain applied intermittently or continuously. Primary rat osteoblast-like cells were seeded and cultured in silicone rubber dishes for 2 days. They were then sub

  12. A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro.

    Science.gov (United States)

    He, Ling; Lin, Ye; Hu, Xiulian; Zhang, Yu; Wu, Hui

    2009-11-01

    The purpose of this study was to evaluate the effect of biologic characteristics of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) on proliferation and differentiation of rat osteoblasts. Blood samples were collected from 14 healthy volunteers (7 male) with a mean age of 23.2 +/- 2.24 years. PRP and PRF were prepared with standard protocols. The exudates of PRP and PRF were collected at the time points of 1, 7, 14, 21, and 28 days. The levels of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor beta1 (TGF-beta1) were quantified in PRP and PRF. Then the exudates of PRP and PRF were used to culture rat calvaria osteoblasts. The biologic characteristics of osteoblasts were analyzed in vitro for 14 days. PRP released the highest amounts of TGF-beta1 and PDGF-AB at the first day, followed by significantly decreased release at later time points. PRF released the highest amount of TGF-beta1 at day 14 and the highest amount of PDGF-AB at day 7. Exudates of PRP collected at day 1 and exudates of PRF collected at day 14 expressed maximum alkaline phosphatase (ALP) activity, though no significance was shown. Cells treated with exudates of PRF collected at day 14 reached peak mineralization significantly more than both negative control and positive control groups. PRF is superior to PRP, from the aspects of expression of ALP and induction of mineralization. PRF released autologous growth factors gradually and expressed stronger and more durable effect on proliferation and differentiation of rat osteoblasts than PRP in vitro.

  13. 血清素对体外培养大鼠成骨细胞增殖分化和矿化功能的影响%Effects of serotonin on proliferation, differentiation and mineralization of cultured rat osteoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    代守前; 施秀; 韦永中; 殷国勇; 余利鹏

    2013-01-01

    目的 探讨不同浓度血清素对体外培养大鼠成骨细胞(OBs)增殖、分化和矿化功能的影响. 方法 在新生SD大鼠颅骨OBs培养液中分别加入不同浓度血清素:0 mol/L组(空白对照组)、10-9 mol/L组、10-8 mol/L组、10-7 mol/L组、10-6 mol/L组、10-5 mol/L组,观察不同浓度血清素对OBs的增殖能力(CCK-8法)、分化能力(Western法检测碱性磷酸酶蛋白水平、pNPP法检测碱性磷酸酶活性和ELISA法检测骨钙素蛋白表达水平)和矿化能力(茜素红染色)的影响.并用荧光定量聚合酶链反应方法检测OBs分化早、晚期血清素受体亚型的mRNA表达水平. 结果 不同浓度血清素均可抑制OBs的增殖、分化和矿化,这种抑制作用在低浓度时具有时间依赖性,而在高浓度时作用减弱,呈现“V”形趋势.5-HT1A、5-HT1B、5-HT1D、5-HT2A、5-HT2B、5-HT2C等受体在OBs上均有表达,其中5-HT2A和5-HT1B受体是表达最多的两种受体亚型. 结论 体外OBs表达血清素受体,血清素可抑制OBs的增殖、分化和矿化,提示血清素能够调节骨代谢.%Objective To investigate the effects of serotonin at different concentrations on proliferation,differentiation and mineralization of cultured primary rat osteoblasts (OBs) in vitro.Methods The culture medium with different serotonin concentrations (from 109 to 105 mol/L) and the osteoblasts from the skull of newborn SD rats were mixed to observe respectively the proliferation (using CCK-8 method),the differentiation (using detections of alkaline phosphatase osteocalcin protein levels and alkaline phosphatase activity),and the mineralization (using ARS staining and OD562) of the OBs.The experiments were performed in 6 groups:blank control,10-9 mol/L,10-8 mol/L,10-7 mol/L,10-6 mol/L and 10-5 mol/L groups.In addition,real-time RT-PCR was used to detect the mRNA expression levels of serotonin receptor subtypes both in early and late differentiation stages of OBs.Results Different

  14. Effects of bone morphogenetic protein-7 stimulation on osteoblasts cultured on different biomaterials.

    Science.gov (United States)

    Açil, Yahya; Springer, Ingo N G; Broek, Vanessa; Terheyden, Hendrik; Jepsen, Søren

    2002-01-01

    The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 microl osteoblasts (at a density of 3.3 x 10(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.

  15. Effect of Beta-Blockers on Number of Osteoblasts and Osteoclasts in Alveolar Socket Following Tooth Extraction in Wistar Rats

    Science.gov (United States)

    Sadr, Katayoun; Aghbali, Amirala; Sadr, Makan; Abachizadeh, Hamed; Azizi, Maryam; Mesgari Abbasi, Mehran

    2017-01-01

    Statement of the Problem: Various researchers have suggested the use of β2-adrenergic receptor antagonists in prevention or treatment of bone resorption. Purpose: This study aimed to evaluate the effect of β2-adrenergic receptor antagonists on number of osteoclasts and osteoblasts involved in the healing of extraction socket of maxillary first molar in rats. Materials and Method: Maxillary first molars of 40 rats were extracted and divided into two groups. The test group received 0.1 mg/kg propranolol intraperitoneally daily. The controls received normal saline. At days 7, 14, 21 and 28 post-extraction, 5 rats were euthanized from each group. Maxillary bone was resected and the mean number of osteoblasts and osteoclasts in tooth socket was measured. Results: After 1 week, the number of osteoclasts in the controls was significantly higher than the test group. A significant increase in the number of osteoclasts in both groups at week 1 was observed compared to the following weeks (p< 0.05). The number of osteoblasts in the controls at second week reached its maximum rate but stayed constant in the 3rd and 4th weeks. Osteoblasts in the test group increased similar to the controls but reached its maximum at 3rd week and showed a significant increase compared to the controls (p< 0.05). Conclusion: β2 adrenergic receptor antagonists decrease the number of osteoclasts and increase the number of osteoblasts during extraction socket healing. PMID:28280758

  16. Effect of Beta-Blockers on Number of Osteoblasts and Osteoclasts in Alveolar Socket Following Tooth Extraction in Wistar Rats

    Directory of Open Access Journals (Sweden)

    Katayoun Sadr

    2017-03-01

    Full Text Available Statement of the Problem: Various researchers have suggested the use of β2-adrenergic receptor antagonists in prevention or treatment of bone resorption. Purpose: This study aimed to evaluate the effect of β2-adrenergic receptor antagonists on number of osteoclasts and osteoblasts involved in the healing of extraction socket of maxillary first molar in rats. Materials and Method: Maxillary first molars of 40 rats were extracted and divided into two groups. The test group received 0.1 mg/kg propranolol intraperitoneally daily. The controls received normal saline. At days 7, 14, 21 and 28 post-extraction, 5 rats were euthanized from each group. Maxillary bone was resected and the mean number of osteoblasts and osteoclasts in tooth socket was measured. Results: After 1 week, the number of osteoclasts in the controls was significantly higher than the test group. A significant increase in the number of osteoclasts in both groups at week 1 was observed compared to the following weeks (p< 0.05. The number of osteoblasts in the controls at second week reached its maximum rate but stayed constant in the 3rd and 4th weeks. Osteoblasts in the test group increased similar to the controls but reached its maximum at 3rd week and showed a significant increase compared to the controls (p< 0.05. Conclusion: β2 adrenergic receptor antagonists decrease the number of osteoclasts and increase the number of osteoblasts during extraction socket healing.

  17. Role of p38MAPK in signal transduction of low density ESW and Iow dose intermittent rhPTH1 -34's action on bone formation of cultured rat osteoblasts%p38MAPK在低能ESW和间歇rhPTH1-34促进ROB成骨的信号转导中的作用

    Institute of Scientific and Technical Information of China (English)

    王李; 刘长剑; 罗宗键

    2011-01-01

    [ Objective] To investigate the role of p38MAPK in the signal path way of low density ESW and low dose intermittent rhPTH1 -34 stimulation's action on rat osteoblasts. [ Methods] After stimulations of 0. 18 rmJ/mm2 ESW and 10-11 mol/L intermittent rhPTHI - 34,the specific inhibitor of p38MAPK was added for finding the role of p38MAPK in the intracellular signal path way. The rat osteoblasts were collected, and cultured was detected, proliferation of these cells was observed by MTI and bone formation by measuring ALP activity. The expression of p - p38MAPK was detected by Western blot. [ Results] Enhaning effect on bone formation by ESW can be significantly inhibited by SB203580, a inhibitor of p38MAPK( P <0.05 ). But the enhancement of ROBs' bone formation by intermittent rhPTHI -34 can not be inhibited by SB203580. Suitable ESW stimulation can improve the expression of p38MAPK, but intermittent rhPTH1 - 34 stimulation can not. [ Conclusions] Suitable ESW stimulation can enhance bone formation of ROB by activation of p38MAPK. The enhancement of ROBs' bone formation by intermittent rhlPFH1 -34 stimulation dosen' t depend on activation of p38MAPK.%[目的]探讨低能体外冲击波(ESW)和间歇人重组甲状旁腺素(rhPTH1-34)刺激引起的体外培养大鼠成骨细胞(ROB)成骨的细胞内信号转导中p38MAPK的作用.[方法]分别用有效的低能ESW刺激和间歇rhPTH1-34作用于ROB,并设立加入p38MAPK抑制剂SB203580组,来观察ROB增殖、成骨指标及Western Blot检测的p38MAPK磷酸化激活变化.[结果]p38MAPK抑制剂SB203580能明显抑制120次0.18 mJ/mm2ESW刺激引起的ROB细胞增殖和成骨作用(P0.05).ESW应力刺激可促进P-p38MAPK表达,但间歇rhPTH1-34不能促进P-p38MAPK表达.[结论]适当的ESW应力刺激可通过激活p38MAPK促进体外培养ROB增殖和成骨;但p38MAPK并不参与10-11 mol/L间歇rhPTH1-34刺激引起的ROB细胞增殖和成骨的细胞信号转导.

  18. Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures.

    Science.gov (United States)

    Hoac, Betty; Kiffer-Moreira, Tina; Millán, José Luis; McKee, Marc D

    2013-04-01

    Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and β-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10μM. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as

  19. Daily low-intensity pulsed ultrasound-mediated osteogenic differentiation in rat osteoblasts

    Institute of Scientific and Technical Information of China (English)

    Akito Suzuki; Tadahiro Takayama; Naoto Suzuki; Michitomo Sato; Takeshi Fukuda; Koichi Ito

    2009-01-01

    There were few studies investigating the effects of the mechanical stimulation provided by daily low-intensity pulsed ultrasound (LIPUS) treatment. LIPUS is known to accelerate bone mineralization and regeneration; however, the precise cellular mechanism is unclear. Our purpose was to determine how daily LIPUS treat-ment affected cell viability, alkaline phosphatase activity, osteogenesis-related gene expression, and mineralized nodule formation in osteoblasts. The typical osteoblastic cell line ROS 17/2.8 cells were cul-tured in the absence or presence of LIPUS stimulation. Daily LIPUS treatments (1.5 MHz; 20 min) were admi-nistered at an intensity of 30 mW/cm2 for 14 days. Expression of osteogenesis-related genes was examined at mRNA levels using real-time polymerase chain reac-tion and at protein levels using western blotting analy-sis. LIPUS stimulation did not affect the rate of cell viability. Alkaline phosphatase activity was increased after 10 days of culture with daily LIPUS stimulation. LIPUS significantly increased the expression of mRNAs encoding Runx2, Msx2, DIx5, osterix, bone sialoprotein,and bone morphogenetic protein-2, whereas it signifi-cantly reduced the expression of mRNA encoding the transcription factor AJ18. Mineralized nodule for-mation was markedly increased on Day 14 of LIPUS stimulation. LIPUS stimulation directly affected osteo-genic cells, leading to mineralized nodule formation. LIPUS is likely to have a fundamental influence on key functional activities of osteoblasts in alveolar bone.

  20. Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

    Science.gov (United States)

    Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

  1. Behavior of MC3T3-E1 Osteoblast Cultured on Chitosan Modified with Polyvinylpyrrolidone

    Institute of Scientific and Technical Information of China (English)

    XI Jing; GAO Yuan; KONG Lijun; GONG Yandao; ZHAO Nanming; ZHANG Xiufang

    2005-01-01

    The physical and chemical properties of four kinds of modified chitosan materials made by blending chitosan with polyvinylpyrrolidone (PVP) were investigated. All four of these modified chitosan materials were hydrophilic with water contact angles ranging from 59° to 69°. Fourier transform-infrared spectra of the modified materials showed a new band at 1288 cm-1, implying formation of a surface physical interpenetrating network structure. Enzyme linked immunosorbent assay results indicated that much less fibronectin was adsorbed on the modified materials than on only chitosan. The viability of MC3T3-E1 osteoblasts cultured on the materials was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide assay. The results show that adding PVP10000 into the chitosan promotes adhesion of MC3T3-E1 osteoblasts on the modified materials, but has no effect on cell growth and proliferation; while adding PVP40000 reduces cell adhesion, growth, and proliferation. The results suggest that the increased hydrophilicity of the material surface does not always improve its biocompatibility, which will influence the selection and design of biomaterials.

  2. Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts.

    Science.gov (United States)

    Kirschner, Gyöngyi; Balla, Bernadett; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Kósa, János Pál; Lakatos, Péter

    2016-09-01

    Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

  3. Biocompatibility of three bioabsorbable membranes assessed in FGH fibroblasts and human osteoblast like cells culture.

    Science.gov (United States)

    Soares, Michelle Pereira Costa Mundim; Soares, Paulo Vinícius; Pereira, Analice Giovani; Moura, Camilla Christian Gomes; Soares, Priscila Barbosa Ferreira; Naves, Lucas Zago; de Magalhães, Denildo

    2014-08-06

    Specific physical and chemical features of the membranes may influence the healing of periodontal tissues after guided tissue regeneration (GTR). The aim of the present investigation was to analyze the biological effects of three bioabsorbable membranes. The hypothesis is that all tested membranes present similar biological effects. Human osteoblast like-cells (SaOs-2) and gingival fibroblasts FGH (BCRJ -RJ) were cultured in DMEM medium. The viability of the cells cultured on the membranes was assesses using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Quantitative determination of activated human Transforming Growth Factor beta 1 (TGF-β1) on the supernatants of the cell culture was observed. Samples were examined using scanning electron microscope (SEM). SaOs2, in 24 hours, PLA group showed higher values when compared to other groups (P statistical significance values when compared two times. In 4 h and 24 h, for the fibroblasts group, significantly difference was found to PLA membrane, when compared with the other groups (p statistically significant difference (p analysis of culture supernatants of fibroblasts, in 24 hours, only PLA group presented significant difference (p = 0,008). The biomaterials analyzed did not show cytotoxicity, since no membrane presented lower results than the control group. PLA membrane presented the best performance due to its higher cell viability and absorbance levels of proliferation. Both collagen membranes showed similar results either when compared to each other or to the control group.

  4. Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

    Science.gov (United States)

    Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

    1994-01-01

    Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

  5. Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

    Science.gov (United States)

    Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

    1994-01-01

    Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

  6. In vitro effects of Choukroun's PRF (platelet-rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in primary cultures.

    Science.gov (United States)

    Dohan Ehrenfest, David M; Diss, Antoine; Odin, Guillaume; Doglioli, Pierre; Hippolyte, Marie-Pascale; Charrier, Jean-Baptiste

    2009-09-01

    The objective of this study was to analyze the effects of Choukroun's PRF (platelet-rich fibrin), a leucocyte and platelet concentrate clinically usable as fibrin membrane or clot, on human primary cultures of gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts. For the proliferation study, these cells were cultivated with or without a PRF membrane originating from the same donor as for the cells. For osteoblasts and fibroblasts, dose-dependent effect was assessed (using 2 membranes). Cell counts and cytotoxicity tests were performed at 3, 7, 14, and 21 days, and even 28 days for osteoblasts. More osteoblast cultures were prepared in differentiation conditions according to 3 modalities (without PRF, with PRF, with PRF the first day and differentiation medium applied only after the first week of culture). Osteoblast differentiation was analyzed using Von Kossa staining and alkaline phosphatase, DNA and total cell proteins dosage. PRF induced a significant and continuous stimulation of proliferation in all cell types. It was dose dependent during all the experiment with osteoblasts, but only on day 14 with fibroblasts. Moreover, PRF induced a strong differentiation in the osteoblasts, whatever the culture conditions. The analysis of osteoblast cultures in differentiation conditions with PRF, using light and scanning electron microscopy, revealed a starting mineralization process in the PRF membrane itself after 14 days. Moreover, PRF leucocytes seemed to proliferate and interact with osteoblasts. Cultures with PRF are always cocultures with leucocytes. These "chaperone leucocytes" could be the source of differential geographic regulation within the culture and explain the double contradictory effect proliferation/differentiation observed on osteoblasts.

  7. Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol

    DEFF Research Database (Denmark)

    Kassem, M; Kveiborg, Marie; Eriksen, E F

    2000-01-01

    Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-...

  8. Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol

    DEFF Research Database (Denmark)

    Kassem, M; Kveiborg, Marie; Eriksen, E F

    2000-01-01

    Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-...

  9. Evaluation of bioactivity of octacalcium phosphate using osteoblastic cell aggregates on a spheroid culture device

    Directory of Open Access Journals (Sweden)

    Takahisa Anada

    2016-03-01

    Full Text Available Much attention has been paid to three-dimensional cell culture systems in the field of regenerative medicine, since three-dimensional cellular aggregates, or spheroids, are thought to better mimic the in vivo microenvironments compared to conventional monolayer cultured cells. Synthetic calcium phosphate (CaP materials are widely used as bone substitute materials in orthopedic and dental surgeries. Here we have developed a technique for constructing a hybrid spheroid consisting of mesenchymal stem cells (MSCs and synthetic CaP materials using a spheroid culture device. We found that the device is able to generate uniform-sized CaP/cell hybrid spheroids rapidly and easily. The results showed that the extent of osteoblastic differentiation from MSCs was different when cells were grown on octacalcium phosphate (OCP, hydroxyapatite (HA, or β-tricalcium phosphate (β-TCP. OCP showed the greatest ability to increase the alkaline phosphatase activity of the spheroid cells. The results suggest that the spheroids with incorporated OCP may be an effective implantable hybrid consisting of scaffold material and cells for bone regeneration. It is also possible that this CaP–cell spheroid system may be used as an in vitro method for assessing the osteogenic induction ability of CaP materials.

  10. Icariin enhances differentiation and maturation of rat calvarial osteoblasts in collagen hydrogel three-dimensional culture%淫羊藿苷促进胶原水凝胶三维立体培养成骨细胞的成熟分化

    Institute of Scientific and Technical Information of China (English)

    谢艳芳; 王鸣刚; 陈克明; 石文贵; 周建; 高玉海

    2015-01-01

    Objective:To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts ( ROB ) in collagen hydrogel three-dimensional culture.Methods: ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture.The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy .ROB were treated with icariin at the concentration of 1 ×10 -4 , 1 ×10 -5 , 1 ×10 -6 and 1 ×10 -7 mol/L respectively .The activity of alkaline phosphatase (ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24 , 36 , 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2 ( BMP-2 ) , Runt-related transcription factor 2 ( RUNX-2 ) and Osterix were detected by real time RT-PCR.The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting .Results: ROB were cultured in collagen hydrogel successfully .FDA/PI staining , HE staining , and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously .Icariin at final concentration of 1 ×10 -5 , 1 ×10 -6 and 1 ×10 -7 mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB , and 1 ×10 -6 mol/L was the optimal concentration.Besides,icariin(1 ×10 -6 mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group .Conclusion:Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly .%目的:研究淫羊藿苷对胶原水凝胶三维立体培养的新生大鼠颅骨成骨细胞( ROB)分化和成熟的影响。方法:取新生SD 大鼠颅骨,酶消化法培养ROB,将传代1次的ROB包埋于2 mg/mL的Ⅰ型鼠尾胶原

  11. In vitro culture and identification of osteoblasts%成骨细胞的体外培养与鉴定

    Institute of Scientific and Technical Information of China (English)

    王勇平; 廖燚; 蒋垚

    2011-01-01

    BACKGROUND: The methods of osteoblast culture in vitro have certain limitations.OBJECTIVE: To summarize osteoblast source, in vitro culture method, culture condition, and identification signs.METHODS: A computer-based retrieval was performed in PubMed and Wanfang databases to search manuscripts publishedbetween 2000 and 2010 with the key words” osteoblast, cell culture, identification” in English and Chinese languages. The papersdescribing in vitro culture and identification of osteoblasts were included. Repetitive research and Meta analysis were excluded.RESULTS AND CONCLUSION: With the development of cell culture techniques in vitro , osteoblasts from animal bone,periosteum, bone marrow and non-bone tissue have been successfully cultured. Studies have shown that these osteoblasts havetypical characteristics and good biological properties. Currently, the cultured osteoblasts are common experimental model in vitroto study physiology, pathology, and repair and have become the basis for studying osteoblast growth and metabolism and bonetissue engineering.%背景:成骨细胞的体外培养方法均存在一定的局限性.目的:对体外培养成骨细胞的来源,成骨细胞的培养方法及培养条件,鉴定成骨细胞的标志进行总结.方法:以"Osteoblast,cell culture,identification"为检索词,检索PubMed 数据库(2000/2010),以"成骨细胞、细胞培养、鉴定"为检索词,检索万方数据库(2000/2010),文献检索语种限制为英文和中文.纳入与成骨细胞的体外培养及鉴定密切相关的研究,排除重复性研究和Meta 分析后对文献进行综述.结果与结论:随着体外细胞培养技术的发展,人们已经从许多动物的骨、骨膜、骨髓及骨外组织中成功培养了成骨细胞,经鉴定具有典型成骨细胞的特征及良好的生物学特性,目前体外培养成骨细胞为常用的体外实验模型,成为研究骨生理、病理及修复的重要手段,也成为研究成骨细胞生长代谢及骨组织工程的基础.

  12. In vitro osteoblastic differentiation and identification of rat bone marrow mesenchymal stem cells by whole bone marrow adherent culture%全骨髓贴壁培养大鼠骨髓间充质干细胞体外成骨的定向诱导及鉴定

    Institute of Scientific and Technical Information of China (English)

    刘斌钰; 李宁毅; 樊功为; 袁荣涛; 金晓明; 陈立强

    2007-01-01

    107 L-1 in 6-well culture plate. Then, the cells were divided into experimental group and control group. Inducing culture medium was added to experimental group, and the same amount of basic culture medium was added to control group. ① Cell differentiation and calcium tuberculation were observed under the inverted microscope. ② Biological characteristics of induced cells were detected by calcium tubercle Von Kossa and alizarin Bordeaux. ③ALP activity was detected by diazo salt staining. ④Human core binding factor alpha subunit-1 (Cbf α-1), osteocalcin (OCN) and osteoblast-specific Osterix (OSX) mRNA expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: ① Induction and differentiation results of cells. ② Biological characteristics of cells induced by rat BMSCs. ③ ALP activity. ④ Cbf α-1, OCN and OSX expressions.RESULTS: ①Inducing culture medium was added in the serial subcultivation. About 9 days later, cell clones were connected to each other. On about 21 to 28 days, some pykno-round mineralized tubercles appeared. Meanwhile,control cells were connected to each other, but they did not form the tubercle. ② In the experimental group, when MSCs were induced for 21 to 28 days, obvious round or oval calcified tubercles were seen by naked eyes. The results of Von Kossa staining exhibited black sediments, and those of alizarin Bordeaux staining exhibited salmon tubercles. Calcium tubercles were not found in the control group. ③The ALP activity after 2 weeks of induction was obviously increased in the experimental group, but was relatively weak in the control group. ④In the experimental group,Cbf α-1, OCN and OSX expressions were significantly increased after induction.CONCLUSION: After being in vitro induced and differentiated by whole bone marrow adherent culture, rat BMSCs exhibited morphological and biological characteristics similar to typical osteoblasts.

  13. β-Caryophyllene promotes osteoblastic mineralization, and suppresses osteoclastogenesis and adipogenesis in mouse bone marrow cultures in vitro

    Science.gov (United States)

    Yamaguchi, Masayoshi; Levy, Robert M.

    2016-01-01

    Osteoporosis is induced by the reduction in bone mass through decreased osteoblastic osteogenesis and increased osteoclastic bone resorption, and it is associated with obesity and diabetes. Osteoblasts and adipocytes are derived from bone marrow mesenchymal stem cells. The prevention of osteoporosis is an important public health concern in aging populations. β-caryophyllene, a component of various essential oils, is a selective agonist of the cannabinoid receptor type 2 and exerts cannabimimetic anti-inflammatory effects in animals. The present study aimed to identify the effect of β-caryophyllene on adipogenesis, osteoblastic mineralization and osteoclastogenesis in mouse bone marrow cell cultures in vitro. Bone marrow cells obtained from mouse femoral tissues were cultured in the presence of β-caryophyllene (0.1–100 µM) in vitro. The results revealed that β-caryophyllene stimulated osteoblastic mineralization, and suppressed adipogenesis and osteoclastogenesis. Thus, β-caryophyllene may be used as a therapeutic agent for the prevention and treatment of osteoporosis. PMID:28105093

  14. Surface modification of parylene-N films for the culture of osteoblast-like cells (MG-63)

    Science.gov (United States)

    Liaqat, Usman; Ko, Hyuk; Suh, Hwal; Lee, Misu; Pyun, Jae-Chul

    2016-08-01

    The influence of microenvironments on the culture of osteoblast-like cells (MG-63) has been investigated using parylene films with different surfaces, such as parylene-N film, UV-modified parylene-N film, functional parylene film with amine groups (parylene-A), and UV-modified parylene-A film. In this work, parylene-N film was found to induce dramatic changes in cell adhesion and cell viability before and after UV-treatment with respect to the culture of osteoblast-like cells (MG-63). The influences of such a chemical environment on cell culture were investigated in relation to the cell proliferation (viability and proliferation rate) and the cell physiology (cell cycle, protein synthesis, and differentiation) of cells grown on parylene-N film, UV-modified parylene-N film, parylene-A film, and UV-modified parylene-A film in comparison with cells grown on a polystyrene surface.

  15. Surface modification of parylene-N films for the culture of osteoblast-like cells (MG-63)

    Energy Technology Data Exchange (ETDEWEB)

    Liaqat, Usman [Graduate Program of Nano Science and Technology, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Ko, Hyuk [Department of Materials Science and Engineering, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Suh, Hwal [Graduate Program of Nano Science and Technology, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Department of Medical Engineering, College of Medicine, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul, 120-749 (Korea, Republic of); Lee, Misu [Division of Life Sciences, College of Life Science and Bioengineering, Incheon National University, Incheon 406-772 (Korea, Republic of); Pyun, Jae-Chul, E-mail: jcpyun@yonsei.ac.kr [Department of Materials Science and Engineering, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of)

    2016-08-15

    Highlights: • Osteoblast-like cells (MG-63) was cultured on differently modified surfaces of parylene films. • Proliferation of MG-63 was observed to be far increased on UV-treated parylene-N film. • The influences of UV-treatment were found out on cell viability, proliferation rate and cell cycle. • The influence was estimated to be negligible on the protein synthesis, cell differentiation. • The UV-treated parylene-N was demonstrated to be effectively used for the culture of MG-63. - Abstract: The influence of microenvironments on the culture of osteoblast-like cells (MG-63) has been investigated using parylene films with different surfaces, such as parylene-N film, UV-modified parylene-N film, functional parylene film with amine groups (parylene-A), and UV-modified parylene-A film. In this work, parylene-N film was found to induce dramatic changes in cell adhesion and cell viability before and after UV-treatment with respect to the culture of osteoblast-like cells (MG-63). The influences of such a chemical environment on cell culture were investigated in relation to the cell proliferation (viability and proliferation rate) and the cell physiology (cell cycle, protein synthesis, and differentiation) of cells grown on parylene-N film, UV-modified parylene-N film, parylene-A film, and UV-modified parylene-A film in comparison with cells grown on a polystyrene surface.

  16. Substance P Promotes the Proliferation, but Inhibits Differentiation and Mineralization of Osteoblasts from Rats with Spinal Cord Injury via RANKL/OPG System

    Science.gov (United States)

    Li, Hao; Chen, Liang; Han, Li-Ren; Yang, Xiao-Fei

    2016-01-01

    Spinal cord injury (SCI) causes a significant amount of bone loss, which results in osteoporosis (OP). The neuropeptide substance P (SP) and SP receptors may play important roles in the pathogenesis of OP after SCI. To identify the roles of SP in the bone marrow mesenchymal stem cell derived osteoblasts (BMSC-OB) in SCI rats, we investigated the expression of neurokinin-1 receptors (NK1R) in BMSC-OB and the effects of SP on bone formation by development of BMSC-OB cultures. Sixty young male Sprague-Dawley rats were randomized into two groups: SHAM and SCI. The expression of NK1R protein in BMSC-OB was observed using immunohistochemistry and Western blot analysis. The dose- and time-dependent effects of SP on the proliferation, differentiation and mineralization of BMSC-OB and the expression of osteoblastic markers by in vitro experiments. The expression of NK1R in BMSC-OB was observed on plasma membranes and in cytoplasm. One week after osteogenic differentiation, the expression of NK1R was significantly increased after SCI at mRNA and protein levels. However, this difference was gradually attenuated at 2 or 3 weeks later. SP have the function to enhance cell proliferation, inhibite cell differentiation and mineralization at a proper concentration and incubation time, and this effect would be inhibited by adding SP or NK1R antagonist. The expression of RANKL/OPG was significantly increased in tibiae after SCI. Similarly, the RANKL/OPG expression in SCI rats was significantly increased when treating with 10−8 M SP. SP plays a very important role in the pathogenesis of OP after SCI. The direct effect of SP may lead to increased bone resorption through the RANKL/OPG axis after SCI. In addition, high expression of SP also results in the suppression of osteogenesis in SCI rats. Then, the balance between bone resorption and bone formation was broken and finally osteoporosis occurred. PMID:27764190

  17. Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics

    Directory of Open Access Journals (Sweden)

    Mansur Halai

    2014-09-01

    Full Text Available In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow–derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow–derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

  18. Cadmium induces apoptosis in primary rat osteoblasts through caspase and mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Zhao, Hongyan; Liu, Wei; Wang, Yi; Dai, Nannan; Gu, Jianhong; Yuan, Yan; Liu, Xuezhong; Bian, Jianchun; Liu, Zong-Ping

    2015-01-01

    Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs.

  19. Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs

    Energy Technology Data Exchange (ETDEWEB)

    Feyen, J.H.; van der Wilt, G.; Moonen, P.; Di Bon, A.; Nijweide, P.J.

    1984-12-01

    The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)2 D3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with (/sup 14/C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of (/sup 3/H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF1 alpha, TXB2, PGF2 alpha, PGE2, PGD2 and PGB2, the latter being the most abundant one. Indomethacin (10(-5) M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH)2D3 was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B2 and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH)2D3.

  20. Effect of osteoblastic culture conditions on the structure of poly(DL-lactic-co-glycolic acid) foam scaffolds

    Science.gov (United States)

    Goldstein, A. S.; Zhu, G.; Morris, G. E.; Meszlenyi, R. K.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1999-01-01

    Poly(DL-lactic-co-glycolic acid) (PLGA) foams are an osteoconductive support that holds promise for the development of bone tissue in vitro and implantation into orthopedic defects. Because it is desirable that foams maintain their shape and size, we examined a variety of foams cultured in vitro with osteoblastic cells. Foams were prepared with different porosities and pore sizes by the method of solvent casting/porogen leaching using 80, 85, and 90 wt% NaCl sieved with particle sizes of 150-300 and 300-500 microm and characterized by mercury intrusion porosimetry. Foams seeded with cells were found to have volumes after 7 days in static culture that decreased with increasing porosity: the least porous exhibited no change in volume while the most porous foams decreased by 39 +/- 10%. In addition, a correlation was observed between decreasing foam volume after 7 days in culture and decreasing internal surface area of the foams prior to seeding. Furthermore, foams prepared with the 300-500 microm porogen had lower porosities, greater mean wall thicknesses between adjacent pores, and larger volumes after 7 days in culture than those prepared with the smaller porogen. Two culture conditions for maintaining cells, static and agitated (in a rotary vessel), were found to have similar influences on foam size, cell density, and osteoblastic function for 7 and 14 days in culture. Finally, we examined unseeded foams in aqueous solutions of pH 3.0, 5.0, and 7.4 and found no significant decrease in foam size with degradation. This study demonstrates that adherent osteoblastic cells may collapse very porous PLGA foams prepared by solvent casting/particulate leaching: a potentially undesirable property for repair of orthopedic defects.

  1. Effect of osteoblastic culture conditions on the structure of poly(DL-lactic-co-glycolic acid) foam scaffolds

    Science.gov (United States)

    Goldstein, A. S.; Zhu, G.; Morris, G. E.; Meszlenyi, R. K.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1999-01-01

    Poly(DL-lactic-co-glycolic acid) (PLGA) foams are an osteoconductive support that holds promise for the development of bone tissue in vitro and implantation into orthopedic defects. Because it is desirable that foams maintain their shape and size, we examined a variety of foams cultured in vitro with osteoblastic cells. Foams were prepared with different porosities and pore sizes by the method of solvent casting/porogen leaching using 80, 85, and 90 wt% NaCl sieved with particle sizes of 150-300 and 300-500 microm and characterized by mercury intrusion porosimetry. Foams seeded with cells were found to have volumes after 7 days in static culture that decreased with increasing porosity: the least porous exhibited no change in volume while the most porous foams decreased by 39 +/- 10%. In addition, a correlation was observed between decreasing foam volume after 7 days in culture and decreasing internal surface area of the foams prior to seeding. Furthermore, foams prepared with the 300-500 microm porogen had lower porosities, greater mean wall thicknesses between adjacent pores, and larger volumes after 7 days in culture than those prepared with the smaller porogen. Two culture conditions for maintaining cells, static and agitated (in a rotary vessel), were found to have similar influences on foam size, cell density, and osteoblastic function for 7 and 14 days in culture. Finally, we examined unseeded foams in aqueous solutions of pH 3.0, 5.0, and 7.4 and found no significant decrease in foam size with degradation. This study demonstrates that adherent osteoblastic cells may collapse very porous PLGA foams prepared by solvent casting/particulate leaching: a potentially undesirable property for repair of orthopedic defects.

  2. [Establishment of osteoblast primary cilia model removed by chloral hyrate].

    Science.gov (United States)

    Ma, Xiao-ni; Shi, Wen-gui; Xie, Yan-fang; Ma, Hui-ping; Ge, Bao-feng; Zhen, Ping; Chen, Ke-ming

    2015-06-01

    To establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field. Three 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining. Compared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01). 4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.

  3. Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

    Science.gov (United States)

    Rath, Subha N; Strobel, Leonie A; Arkudas, Andreas; Beier, Justus P; Maier, Anne-Kathrin; Greil, Peter; Horch, Raymund E; Kneser, Ulrich

    2012-10-01

    In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine.

  4. Culture of cryopreserved rat hepatocyte

    Institute of Scientific and Technical Information of China (English)

    Haitao Yin; Gaojun Teng; Lifeng Wang; Baorui Liu; Xiaoping Qian

    2006-01-01

    Objective: To study the method of cryopreserving rat hepatocytes and double collagen gel culture measurement after its cryopreservation. Methods: Rat hepatocytes, isolated by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved in double collagen gel with culture medium added by epidermal growth factor(EGF).The expression of cell function and cellular morphology were examined during culture. Results: The hepatocytes cryopreserved in double collagen gel concluding EGF showed good morphology and biological characteristics. After thawing, the MTT metabolism and protein synthesis of hepatocytes in sandwich ± EGF groups were better than those in control group. And the morphology and function of hepatocytes in sandwich group was better than that in EGF group(P < 0.05). Conclusion: Double collagen gel culture can keep hepatocyte's activities. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.

  5. Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

    Science.gov (United States)

    Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

    1999-01-01

    Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

  6. Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

    Science.gov (United States)

    McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

    1996-01-01

    Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

  7. Study on biocompatibility of PDLLA/HA/DBM with co-cultured human osteoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    郭乔楠; 赵建华; 卢佳友

    2003-01-01

    Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDLLA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen Ⅰ expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of biomaterial and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.

  8. The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells

    DEFF Research Database (Denmark)

    Henriksen, Zanne; Hiken, Jeffrey F; Steinberg, Thomas H;

    2006-01-01

    Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine...... assessed by video imaging of Fura-2 loaded cells after 1, 2 and 4 months culture. The P2Y2 receptor and the gap junction protein Cx43 were assessed by Western blot and real-time PCR. In resting conditions, P2Y mediated ICW prevailed and spread rapidly to about 13 cells. P2Y receptor desensitization by ATP......, but as cells differentiate in culture, gap-junction-mediated ICW become more prominent. These results suggest that P2Y receptor-mediated and gap junction-mediated mechanisms of intercellular calcium signaling may play different roles during differentiation of bone-forming cells....

  9. Possible recruitment of osteoblastic precursor cells from hypertrophic chondrocytes during initial osteogenesis in cartilaginous limbs of young rats.

    Science.gov (United States)

    Franzen, A; Oldberg, A; Solursh, M

    1989-08-01

    The appearance of the bone phenotype during rat embryogenesis was studied by in situ hybridization using a cDNA clone to osteopontin. Radiolabeled sense and antisense RNA probes were prepared from the osteopontin cDNA by in vitro transcription. The probes were used to hybridize paraffin sections of the cartilaginous diaphysis from embryonic rats at day 17 of gestation. The hybridization pattern was analyzed by autoradiography. Hybridization with the antisense probe gave patterns of silver grain labeling, indicating the presence of osteopontin mRNA among the hypertrophic chondrocytes. No silver grains could be detected in the corresponding region following hybridization of consecutive sections with the sense probe, showing the specificity of the technique being used. Whether these results indicate that the osteopontin gene is transiently expressed by hypertrophic chondrocytes or that osteopontin is an early marker for osteoblastic precursor cells will have to be explored further.

  10. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells.

    Science.gov (United States)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen; Stark, G Björn

    2010-01-01

    Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR-alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells but not in immortalized osteoblastic cell lines. Functional inhibition of gap junctional communication between HUVECs and hOBs by 18alpha-glycyrrhetinic acid had no effect on HUVEC-mediated PDGFR-alpha downregulation, whereas inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the HUVEC-mediated reduction in osteoblastic PDGFR-alpha expression. To delineate the molecular mechanism underlying the PDGFR-alpha downregulation, we examined the effect of HUVEC co-cultivation on osteoblastic PDGFR-alpha promoter activity as well as mRNA stability. Co-cultivation of HUVECs with hOBs significantly shortened the half-life of osteoblastic PDGFR-alpha mRNA, but did not decrease its promoter activity. In summary, our data show that PDGFR-alpha is downregulated in hOBs by co-cultivation with human primary endothelial cells through a p38 MAPK-dependent post-transcriptional mechanism.

  11. Biocompatibility evaluation in vitro. Part I: Morphology expression and proliferation of human and rat osteoblasts on the biomaterials

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The biocompatibility evaluation of calcium phosphate based biomaterials is performed by tissue culture in vitro model. Three kinds of bioceramic materials which are potential to deal with bone trauma and/or conduct tissue growth are recommodated. The biological research results show that human and animal osteoblast cells anchor the materials surface in two hours in culture. Confocal laser scanning microscopy (CLSM) demonstrated the normal cell distribution and proliferation on both of dense and porous biomaterials. Hydroxyapatite and tricalcium phosphate stimulate cell proliferation. However, DNA and protein synthesis were considerably limited and the apoptosis phenomenon would be present on the hydroxyapatite (HA) materials by adding Al, Mg elements. Several important methods of biocompatibility evaluation of implant materials are described and the related biological molecular techniques such as tissue culture, cell transfection, cellular DNA stain, and Lowry assay are involved in the present research.

  12. Biocompatibility and osteoconductivity of injectable bone xenograft, hydroxyapatite and hydroxyapatite-chitosan on osteoblast culture

    Directory of Open Access Journals (Sweden)

    Bachtiar EW

    2010-12-01

    Full Text Available Background: Bone graft in the form of injectable paste gives several advantages over the powder form as it could be placed in the defect area that has limited accessibility. Purpose: The purpose of this study was to assess biocompatibility and osteoconductivity of an injectable bone xenograft (IBX, injectable hydroxyapatite (IHA and injectable hydroxyapatite-chitosan (IHA-C on osteoblastic cell line (MG-63. Methods: Three concentrations (0.25%, 0.5% and 1.0% of IBX, IHA and IHA-C were supplemented with DMEM culture medium. The viability cells were measured by MTT assay 4 hour after incubation. ALP activity was measured at day 1, 3, 5 and 7. Calcium deposition was tested at day 3 and day 7 by means of Von Kossa staining. Results: MTT assay showed that the viability cells of all the test groups were above 100% compared to the control group. The cell viability of the 0.25% IHA paste was significantly higher (115.02% ± 4.37%, p < 0.05 compared with IBX paste and IHA-C in all concentrations tested. The highest level of ALP secretion of all test groups was found on the fifth day of exposure. The highest level of ALP in the IBX paste group was 0.25% concentration while the highest level of ALP in the IHA-C and IHA paste group was 1% and 0.25%, respectively. In addition, the highest calcium deposition was shown on IHA 1% at day 7 (p > 0.05. Conclusion: It was suggested that adequate biocompatibility and osteoconductivity was evident for all injectable pastes tested.Latar belakang: Bahan tandur tulang dalam bentuk pasta injeksi memiliki kelebihan dibandingkan bila bahan tersebut berupa bubuk, karena lebih mudah diaplikasikan pada daerah yang sulit dijangkau. Tujuan: Penelitian ini bertujuan untuk mengamati sifat biokompatibilitas dan osteokonduktifitas biomaterial tandur tulang dalam bentuk injectable bone xenograft (IBX, injectable hydroxyapatite (IHA dan injectable hydroxyapatite-chitosan (IHA-C pada galur sel osteoblas (MG-63. Metode: Bahan tandur

  13. Cultured Human Periosteum-Derived Cells Can Differentiate into Osteoblasts in a Perioxisome Proliferator-Activated Receptor Gamma-Mediated Fashion via Bone Morphogenetic Protein signaling.

    Science.gov (United States)

    Chung, Jin-Eun; Park, Jin-Ho; Yun, Jeong-Won; Kang, Young-Hoon; Park, Bong-Wook; Hwang, Sun-Chul; Cho, Yeong-Cheol; Sung, Iel-Yong; Woo, Dong Kyun; Byun, June-Ho

    2016-01-01

    The differentiation of mesenchymal stem cells towards an osteoblastic fate depends on numerous signaling pathways, including activation of bone morphogenetic protein (BMP) signaling components. Commitment to osteogenesis is associated with activation of osteoblast-related signal transduction, whereas inactivation of this signal transduction favors adipogenesis. BMP signaling also has a critical role in the processes by which mesenchymal stem cells undergo commitment to the adipocyte lineage. In our previous study, we demonstrated that an agonist of the perioxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipocyte differentiation, stimulates osteoblastic differentiation of cultured human periosteum-derived cells. In this study, we used dorsomorphin, a selective small molecule inhibitor of BMP signaling, to investigate whether BMP signaling is involved in the positive effects of PPARγ agonists on osteogenic phenotypes of cultured human periosteum-derived cells. Both histochemical detection and bioactivity of ALP were clearly increased in the periosteum-derived cells treated with the PPARγ agonist at day 10 of culture. Treatment with the PPARγ agonist also caused an increase in alizarin red S staining and calcium content in the periosteum-derived osteoblasts at 2 and 3 weeks of culture. In contrast, dorsomorphin markedly decreased ALP activity, alizarin red S staining and calcium content in both the cells treated with PPARγ agonist and the cells cultured in osteogenic induction media without PPARγ agonist during the culture period. In addition, the PPARγ agonist clearly increased osteogenic differentiation medium-induced BMP-2 upregulation in the periosteum-derived osteoblastic cells at 2 weeks of culture as determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunocytochemical analyses. Although further study will be needed to clarify the mechanisms of PPARγ-regulated osteogenesis

  14. Thermo-responsive poly(N-isopropylacrylamide)-grafted hollow fiber membranes for osteoblasts culture and non-invasive harvest.

    Science.gov (United States)

    Zhuang, Meiling; Liu, Tianqing; Song, Kedong; Ge, Dan; Li, Xiangqin

    2015-10-01

    Hollow fiber membrane (HFM) culture system is one of the most important bioreactors for the large-scale culture and expansion of therapeutic cells. However, enzymatic and mechanical treatments are traditionally applied to harvest the expanded cells from HFMs, which inevitably causes harm to the cells. In this study, thermo-responsive cellulose acetate HFMs for cell culture and non-invasive harvest were prepared for the first time via free radical polymerization in the presence of cerium (IV). ATR-FTIR and elemental analysis results indicated that the poly(N-isopropylacrylamide) (PNIPAAm) was covalently grafted on HFMs successfully. Dynamic contact angle measurements at different temperatures revealed that the magnitude of volume phase transition was decreased with increasing grafted amount of PNIPAAm. And the amount of serum protein adsorbed on HFMs surface also displayed the same pattern. Meanwhile osteoblasts adhered and spread well on the surface of PNIPAAm-grafted HFMs at 37 °C. And Calcein-AM/PI staining, AB assay, ALP activity and OCN protein expression level all showed that PNIPAAm-grafted HFMs had good cell compatibility. After incubation at 20 °C for 120 min, the adhering cells on PNIPAAm-grafted HFMs turned to be round and detached after being gently pipetted. These results suggest that thermo-responsive HFMs are attractive cell culture substrates which enable cell culture, expansion and the recovery without proteolytic enzyme treatment for the application in tissue engineering and regenerative medicine.

  15. Fabrication and characterization of PDLLA/pyrite composite bone scaffold for osteoblast culture

    Indian Academy of Sciences (India)

    Lifang Zhang; Yanyan Zheng; Chengdong Xiong

    2015-06-01

    A series of highly interconnected porous poly(D,L-lactide acid) (PDLLA)/pyrite (Zi-Ran-Tong, FeS2) scaffold containing 5–20% of pyrite was fabricated by particle leaching combined with the thermal-induced phase separation method. Pyrite (FeS2, named as Zi-Ran-Tong in Chinese medicine), as a traditional Chinesemedicine, has been used in the Chinese population to treat bone diseases and to promote bone healing. The mechanical properties of the PDLLA scaffold were significantly enhanced after the addition of pyrite. The osteoblastic ROS17/2.8 cell line was used and seeded on the PDLLA/pyrite scaffold to study its potential to support the growth of osteoblastic cells and to estimate the optimal dose of pyrite for bone tissue engineering. The effects of pyrite on cell proliferation and differentiation were evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide and alkaline phosphatase activity assay. The cells on the porous composite scaffold formed a continuous layer on the outer and inner surface observed by scanning electron microscopy and fluorescence microscope. The results strongly suggested that the PDLLA/pyrite composite scaffold could stimulate the growth of ROS17/2.8 cells in vitro and it could be potentially used as a scaffold for bone tissue engineering.

  16. Effects of Chitin Whiskers on Physical Properties and Osteoblast Culture of Alginate Based Nanocomposite Hydrogels.

    Science.gov (United States)

    Huang, Yao; Yao, Mengyu; Zheng, Xing; Liang, Xichao; Su, Xiaojuan; Zhang, Yu; Lu, Ang; Zhang, Lina

    2015-11-09

    Novel nanocomposite hydrogels composed of polyelectrolytes alginate and chitin whiskers with biocompatibility were successfully fabricated based on the pH-induced charge shifting behavior of chitin whiskers. The chitin whiskers with mean length and width of 300 and 20 nm were uniformly dispersed in negatively charged sodium alginate aqueous solution, leading to the formation of the homogeneous nanocomposite hydrogels. The experimental results indicated that their mechanical properties were significantly improved compared to alginate hydrogel and the swelling trends were inhibited as a result of the strong electrostatic interactions between the chitin whiskers and alginate. The nanocomposite hydrogels exhibited certain crystallinity and hierarchical structure with nanoscale chitin whiskers, similar to the structure of the native extracellular matrix. Moreover, the nanocomposite hydrogels were successfully applied as bone scaffolds for MC3T3-E1 osteoblast cells, showing their excellent biocompatibility and low cytotoxicity. The results of fluorescent micrographs and scanning electronic microscope (SEM) images revealed that the addition of chitin whiskers into the nanocomposite hydrogels markedly promoted the cell adhesion and proliferation of the osteoblast cells. The biocompatible nanocomposite hydrogels have potential application in bone tissue engineering.

  17. Effects of masticatory movement on cranial bone mass and micromorphology of osteocytes and osteoblasts in developing rats.

    Science.gov (United States)

    Kawakami, Toshikazu; Takise, Sadafumi; Fuchimoto, Takafumi; Kawata, Hiroshi

    2009-01-01

    In order to evaluate the influence of masticatory movement on cranial bone mineral density (BMD) and osteocyte and osteoblast micromorphology, we conducted a study in rats fed with solid feed (n=10) and powdered feed (n=10). Cranial BMD was measured by dual X-ray absorptiometry (DXA). Osteocyte morphology was evaluated by light microscopy. In addition, some of the tissue was treated with EDTA-KOH to digest the bone matrix and prepare osteocyte samples. Micromorphology of the osteocytes was examined by scanning electron microscopy (SEM). Bone mineral content (BMC) was significantly higher in the solid feed group (1.86 +/- 0.11 g) than in the powdered feed group (1.63 +/- 0.09 g) (p micromorphology.

  18. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G. [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada); Werstuck, Geoff H., E-mail: Geoff.Werstuck@taari.ca [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

  19. Mineral trioxide aggregate-based endodontic sealer stimulates hydroxyapatite nucleation in human osteoblast-like cell culture.

    Science.gov (United States)

    Salles, Loise Pedrosa; Gomes-Cornélio, Ana Lívia; Guimarães, Felipe Coutinho; Herrera, Bruno Schneider; Bao, Sonia Nair; Rossa-Junior, Carlos; Guerreiro-Tanomaru, Juliane Maria; Tanomaru-Filho, Mario

    2012-07-01

    The main purpose of this study was to evaluate the biocompatibility and bioactivity of a new mineral trioxide aggregate (MTA)-based endodontic sealer, MTA Fillapex (MTA-F; Angelus, Londrina, Brazil), in human cell culture. Human osteoblast-like cells (Saos-2) were exposed for 1, 2, 3, and 7 days to MTA-F, Epiphany SE (EP-SE; SybronEndo, Orange, CA), and zinc oxide-eugenol sealer (ZOE). Unexposed cultures were the control group (CT). The viability of the cells was assessed by MTT assay and the morphology by scanning electron microscopy (SEM). The bioactivity of MTA-F was evaluated by alkaline phosphatase activity (ALP) and the detection of calcium deposits in the culture with alizarin red stain (ARS). Energy-dispersive X-ray spectroscopy (EDS) was used to chemically characterize the hydroxyapatite crystallites (HAP). Saos-2 cells were cultured for 21 days for ARS and SEM/EDS. ARS results were expressed as the number of stained nodules per area. Statistical analysis was performed with analysis of variance and Bonferroni tests (P CT > EP-SE > ZOE). SEM/EDS analysis showed hydroxyapatite crystals only in the MTA-F and CT groups. In the MTA-F group, crystallite morphology and chemical composition were different from CT. After setting, the cytotoxicity of MTA-F decreases and the sealer presents suitable bioactivity to stimulate HAP crystal nucleation. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  20. Communication between osteoblasts stimulated by electromagnetic fields

    Institute of Scientific and Technical Information of China (English)

    ZHANG JianBao; ZHANG XiaoJun

    2007-01-01

    Pulsed electromagnetic field can affect the proliferation of osteoblasts, but the mechanism is obscure yet. The communication between osteoblasts, isolated from calvaria bone of newborn SD rats and stimulated with the rectangular electromagnetic field of 15 Hz and 4 mT, was studied. Our results showed that the osteoblasts radiated a kind of light after they were stimulated with the electromagnetic field and it is the light that promotes the proliferation of un-stimulated osteoblasts.

  1. Osteoblast integration of dental implant materials after challenge by sub-gingival pathogens:a co-culture study in vitro

    Institute of Scientific and Technical Information of China (English)

    Bingran Zhao; Henny C van der Mei; Minie Rustema-Abbing; Henk J Busscher; Yijin Ren

    2015-01-01

    Sub-gingival anaerobic pathogens can colonize an implant surface to compromise osseointegration of dental implants once the soft tissue seal around the neck of an implant is broken. In vitro evaluations of implant materials are usually done in monoculture studies involving either tissue integration or bacterial colonization. Co-culture models, in which tissue cells and bacteria battle simultaneously for estate on an implant surface, have been demonstrated to provide a better in vitro mimic of the clinical situation. Here we aim to compare the surface coverage by U2OS osteoblasts cells prior to and after challenge by two anaerobic sub-gingival pathogens in a co-culture model on differently modified titanium (Ti), titanium-zirconium (TiZr) alloys and zirconia surfaces. Monoculture studies with either U2OS osteoblasts or bacteria were also carried out and indicated significant differences in biofilm formation between the implant materials, but interactions with U2OS osteoblasts were favourable on all materials. Adhering U2OS osteoblasts cells, however, were significantly more displaced from differently modified Ti surfaces by challenging sub-gingival pathogens than from TiZr alloys and zirconia variants. Combined with previous work employing a co-culture model consisting of human gingival fibroblasts and supra-gingival oral bacteria, results point to a different material selection to stimulate the formation of a soft tissue seal as compared to preservation of osseointegration under the unsterile conditions of the oral cavity.

  2. Response of Cytoskeleton of Murine Osteoblast Cultures to Two-step Freezing

    Institute of Scientific and Technical Information of China (English)

    Bao-Lin LIU; John McGRATH

    2005-01-01

    Understanding the ultrastructural response of cells to the freezing process is important for designing cryopreservation strategies for cells and tissues. The cellular structures of attached cells are targets of cryopreservation-induced damage. Specific fluorescence staining was used to assess the status of the actin filaments (F-actin) of murine osteoblasts attached to hydroxyapatite discs and plastic coverslips for a two-step freezing process. The F-actin of dead cells was depolymerized and distorted in the freezing process,whereas that of live cells had little change. The results suggest that the cytoskeleton may support the robustness of cells during cryopreservation. The present study helps to investigate the damage mechanism of attached cells during the freezing process.

  3. An advanced tri-culture model to evaluate the dynamic interplay among osteoblasts, osteoclasts, and endothelial cells.

    Science.gov (United States)

    Pagani, Stefania; Torricelli, Paola; Veronesi, Francesca; Salamanna, Francesca; Cepollaro, Simona; Fini, Milena

    2018-01-01

    The dynamic metabolism and the numerous roles of bone tissue necessitate a suitable in vitro model to represent them. In order to investigate the interaction among the several cell types composing bone microenvironment, we studied a tri-culture model including human osteoblasts (OBs), osteoclasts (OCs), and endothelial cells (HUVEC). While OBs are essential for bone deposition and OCs for bone resorption, the vasculature is necessary to provide growth factors, nutrients, and oxygen in the mature tissue. The results of this study showed a strong mutual influence between OBs, OCs, and HUVEC in term of proliferation, viability, and activity (release of ALP, Coll I, OPG, RANKL, VEGF, CTSK, TGFβ, and IL-6). The behavior of the single cultures demonstrated to be different compared to the bi- or tri-cultures and depending on the cell types involved: the coexistence of OBs and OCs stimulated the synthetic activity of both cell types, while the presence of HUVEC induced a stimulating role for OBs but mainly an inhibitory effect for OC. In addition, evidence of the effects of OBs and OCs on HUVEC is highlighted by their morphology: regular and able to "sketch" little vessels in presence of OBs, more disorganized and heterogeneous in presence of OCs. Taken together, these observations well characterize an advanced cellular model to be used as starting point for mimicking bone microenvironment in vivo, thus reducing the use of animals in the preclinical phase and offering a more reliable tool to test new and innovative biomaterials. © 2017 Wiley Periodicals, Inc.

  4. Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Rattan, Suresh; Clark, Brian F.C.

    2001-01-01

    of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging......Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long......-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage ( 90% lifespan completed). We found a significant reduction in mRNA levels...

  5. Thermo-responsive poly(N-isopropylacrylamide)-grafted hollow fiber membranes for osteoblasts culture and non-invasive harvest

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, Meiling, E-mail: zhuangmeiling2006@126.com; Liu, Tianqing, E-mail: liutq@dlut.edu.cn; Song, Kedong, E-mail: kedongsong@dlut.edu.cn; Ge, Dan, E-mail: gedan@dlut.edu.cn; Li, Xiangqin, E-mail: xiangqinli@163.com

    2015-10-01

    Hollow fiber membrane (HFM) culture system is one of the most important bioreactors for the large-scale culture and expansion of therapeutic cells. However, enzymatic and mechanical treatments are traditionally applied to harvest the expanded cells from HFMs, which inevitably causes harm to the cells. In this study, thermo-responsive cellulose acetate HFMs for cell culture and non-invasive harvest were prepared for the first time via free radical polymerization in the presence of cerium (IV). ATR-FTIR and elemental analysis results indicated that the poly(N-isopropylacrylamide) (PNIPAAm) was covalently grafted on HFMs successfully. Dynamic contact angle measurements at different temperatures revealed that the magnitude of volume phase transition was decreased with increasing grafted amount of PNIPAAm. And the amount of serum protein adsorbed on HFMs surface also displayed the same pattern. Meanwhile osteoblasts adhered and spread well on the surface of PNIPAAm-grafted HFMs at 37 °C. And Calcein-AM/PI staining, AB assay, ALP activity and OCN protein expression level all showed that PNIPAAm-grafted HFMs had good cell compatibility. After incubation at 20 °C for 120 min, the adhering cells on PNIPAAm-grafted HFMs turned to be round and detached after being gently pipetted. These results suggest that thermo-responsive HFMs are attractive cell culture substrates which enable cell culture, expansion and the recovery without proteolytic enzyme treatment for the application in tissue engineering and regenerative medicine. - Highlights: • PNIPAAm-grafted HFMs exhibited thermoresponsive characteristic. • The OB cells could adhere and spread well on the surface of PNIPAAm-grafted HFMs. • PNIPAAm-grafted HFMs do not significantly impact ALP activity and OCN protein expression level of OB cells. • Cell could be detached from PNIPAAm-grafted HFMs when temperature decreased from 37 °C to 20 °C.

  6. Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

    Science.gov (United States)

    Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

    2000-01-01

    Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

  7. Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors.

    Science.gov (United States)

    Chen, Jiaxuan; Dosier, Christopher R; Park, Jung Hwa; De, Subhendu; Guldberg, Robert E; Boyan, Barbara D; Schwartz, Zvi

    2016-01-01

    1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein-2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three-dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild-type (WT), Pdia3-silenced (Sh-Pdia3) and VDR-silenced (Sh-VDR) pre-osteoblastic MC3T3-E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε-caprolactone)-collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro-computed tomography (μCT) showed that the mineralized volume of untreated Sh-Pdia3 and Sh-VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh-VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3.

  8. Effect of contact with titanium alloys on the proliferation of mouse osteoblastic cells in culture.

    Science.gov (United States)

    Onuki, Hiroyuki; Sakagami, Hiroshi; Kobayashi, Masahiko; Hibino, Yasushi; Yokote, Yoshiko; Nakajima, Hiroshi; Shimada, Jun

    2010-01-01

    This study was aimed at studying the effect of contact with titanium alloy plates of different surface textures on the proliferative capability of mouse osteoblastic MC3T3-E1 cells. First, the proliferation characteristics of MC3T3-E1 cells were investigated. MC3T3-E1 cells showed a high capacity for proliferation and survived for a long period even under nutritionally starved conditions. During logarithmic cell growth, the consumption of Ser, Gln, Val, Ile and Leu increased time-dependently. Contact with an hydoxyapatite (HA)-coated titanium alloy plate resulted in the increase in the recovery of cells from the plate by trypsin, and an increase in the consumption of these amino acids, suggesting enhanced cell proliferation. On the contrary, contact with the sandblasted and anodized titanium alloy plates resulted in the reduction of the recovery of the cells from the plate, but a slight increase in the amino acid consumption, suggesting the tight adhesion of the cells to the plates. This study demonstrates that the present method, based on the amino acid consumption of the cells, is useful for monitoring the cell proliferative capability, without detachment of the cells from the plate. This method may be applicable to the study of the interaction between cells and metal plates.

  9. Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis

    Science.gov (United States)

    ABUNA, Rodrigo Paolo Flores; STRINGHETTA-GARCIA, Camila Tami; FIORI, Leonardo Pimentel; DORNELLES, Rita Cassia Menegati; ROSA, Adalberto Luiz; BELOTI, Marcio Mateus

    2016-01-01

    ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population. PMID:27556209

  10. Ectopic bone formation by marrow stromal osteoblast transplantation using poly(DL-lactic-co-glycolic acid) foams implanted into the rat mesentery

    Science.gov (United States)

    Ishaug-Riley, S. L.; Crane, G. M.; Gurlek, A.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat marrow stromal cells and implanted into the rat mesentery to investigate in vivo bone formation at an ectopic site. Cells were seeded at a density of 6.83 x 10(5) cells/cm2 onto polymer foams having pore sizes ranging from either 150 to 300 to 710 microns and cultured for 7 days in vitro prior to implantation. The polymer/cell constructs were harvested after 1, 7, 28, or 49 days in vivo and processed for histology and gel permeation chromatography. Visual observation of hematoxylin and eosin-stained sections and von Kossa-stained sections revealed the formation of mineralized bonelike tissue in the constructs within 7 days postimplantation. Ingrowth of vascular tissue was also found adjacent to the islands of bone, supplying the necessary metabolic requirements to the newly formed tissue. Mineralization and bone tissue formation were investigated by histomorphometry. The average penetration depth of mineralized tissue in the construct ranged from 190 +/- 50 microns for foams with 500-710-microns pores to 370 +/- 160 microns for foams with 150-300-microns pores after 49 days in vivo. The mineralized bone volume per surface area and total bone volume per surface area had maximal values of 0.28 +/- 0.21 mm (500-710-microns pore size, day 28) and 0.038 +/- 0.024 mm (150-300-microns, day 28), respectively. As much as 11% of the foam volume penetrated by bone tissue was filled with mineralized tissue. No significant trends over time were observed for any of the measured values (penetration depth, bone volume/surface area, or percent mineralized bone volume). These results suggest the feasibility of bone formation by osteoblast transplantation in an orthotopic site where not only bone formation from transplanted cells but also ingrowth from adjacent bone may occur.

  11. Ectopic bone formation by marrow stromal osteoblast transplantation using poly(DL-lactic-co-glycolic acid) foams implanted into the rat mesentery

    Science.gov (United States)

    Ishaug-Riley, S. L.; Crane, G. M.; Gurlek, A.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat marrow stromal cells and implanted into the rat mesentery to investigate in vivo bone formation at an ectopic site. Cells were seeded at a density of 6.83 x 10(5) cells/cm2 onto polymer foams having pore sizes ranging from either 150 to 300 to 710 microns and cultured for 7 days in vitro prior to implantation. The polymer/cell constructs were harvested after 1, 7, 28, or 49 days in vivo and processed for histology and gel permeation chromatography. Visual observation of hematoxylin and eosin-stained sections and von Kossa-stained sections revealed the formation of mineralized bonelike tissue in the constructs within 7 days postimplantation. Ingrowth of vascular tissue was also found adjacent to the islands of bone, supplying the necessary metabolic requirements to the newly formed tissue. Mineralization and bone tissue formation were investigated by histomorphometry. The average penetration depth of mineralized tissue in the construct ranged from 190 +/- 50 microns for foams with 500-710-microns pores to 370 +/- 160 microns for foams with 150-300-microns pores after 49 days in vivo. The mineralized bone volume per surface area and total bone volume per surface area had maximal values of 0.28 +/- 0.21 mm (500-710-microns pore size, day 28) and 0.038 +/- 0.024 mm (150-300-microns, day 28), respectively. As much as 11% of the foam volume penetrated by bone tissue was filled with mineralized tissue. No significant trends over time were observed for any of the measured values (penetration depth, bone volume/surface area, or percent mineralized bone volume). These results suggest the feasibility of bone formation by osteoblast transplantation in an orthotopic site where not only bone formation from transplanted cells but also ingrowth from adjacent bone may occur.

  12. Reconstruction of rat calvarial defects with human mesenchymal stem cells and osteoblast-like cells in poly-lactic-co-glycolic acid scaffolds

    Directory of Open Access Journals (Sweden)

    C Zong

    2010-09-01

    Full Text Available Human mesenchymal stem cells (hMSCs can be used for xenogenic transplantation due to their low immunogenicity, high proliferation rate, and multi-differentiation potentials. Therefore, hMSCs are an ideal seeding source for tissue engineering. The present study evaluates the reconstruction effects of hMSCs and osteoblast-like cells differentiated from hMSCs in poly-lactic-co-glycolic acid (PLGA scaffolds on the calvarial defect of rats. Two bilateral full-thickness defects (5mm in diameter were created in the calvarium of nonimmunosuppressed Sprague-Dawley rats. The defects were filled by PLGA scaffolds with hMSCs (hMSC Construct or with osteoblast-like cells differentiated from hMSCs (Osteoblast Construct. The defects without any graft (Blank Defect or filled with PLGA scaffold without any cells (Blank Scaffold were used as controls. Evaluation was performed using macroscopic view, histology and immunohistochemical analysis respectively at 10 and 20 weeks after transplantation. In addition, fluorescent carbocyanine CM-Dil was used to track the implanted cells in vivo during transplantation. The results showed that while both hMSC Construct and Osteoblast Construct led to an effective reconstruction of critical-size calvarial defects, the bone reconstruction potential of hMSC Construct was superior to that of Osteoblast Construct in non-autogenous applications. Our findings verify the feasibility of the use of xenogenic MSCs for tissue engineering and demonstrate that undifferentiated hMSCs are more suitable for bone reconstruction in xenotransplantation models.

  13. Coculture of osteoblasts and endothelial cells: optimization of culture medium and cell ratio

    NARCIS (Netherlands)

    Ma, J.; Beucken, J.J. van den; Yang, F.; Both, S.K.; Cui, F.Z.; Pan, J.; Jansen, J.A.

    2011-01-01

    Vascularization strategies in cell-based bone tissue engineering depend on optimal culture conditions. The present study aimed to determine optimal cell culture medium and cell ratio for cocultures of human marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) in view of

  14. Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

    Science.gov (United States)

    Rinker, Torri E; Hammoudi, Taymour M; Kemp, Melissa L; Lu, Hang; Temenoff, Johnna S

    2014-03-01

    Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on the neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts at high glucose levels. In contrast, MSCs showed no reduction of viability or clonogenicity when cultured with adipocytes under high glucose conditions, and the adipogenic gene expression indicates that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided a novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis.

  15. [Effects of serum of Bushen Huoxue prescription (Chinese characters) on classic Wnt/β-catenin signaling pathways of osteoblasts].

    Science.gov (United States)

    Xu, Bing; Jin, Hong-ting; Wang, Xiao-feng; Xia, Lu-wei; Tong, Pei-jian

    2015-06-01

    To observe effects of serum Bushen Huoxue prescription(Chinese characters) on classic Wnt/β-catenin signaling pathways of osteoblasts, and explore mechanism of Bushen Huoxue prescription (Chinese characters) for preventing osteoporosis. Twenty health female rats were randomly divided into two groups, including Bushen Huoxue (Chinese characters) group and saline group,10 in each group. Bushen Huoxue (Chinese characters) group and Saline group were gavaged Bushen Huoxue and saline every day for 1 week. Bushenhuoxue containing serum and saline containing serum were got according to methods of serum preparation of drug-containing. The osteoblasts was cultured with neonatal rat skull according to Enzyme Consumer Law, and was identified by Wright-Giemsa staining (R-J) and alkaline phosphatase staining (ALP). The third generation of osteoblasts was divided into three groups, including saline group, normal group,Bushen Huoxue (Chinese characters) group. Each group were added to 15% appropriate medium. ALP activity of osteoblasts and osteoblasts proliferation rate were tested, mineralized nodules were observed, the expression of β-catenin, Runx2, Osx mRNA of osteoblasts were tested by RT-PCR. There was blue granules in cytoplasm, cell nucleus was flint with 1 to 3 nucleoli showed by R-J staining, morphology of osteoblasts were cultured. ALP staining showed cytoplasm with purple granules, the results showed that the cultured cell was osteoblasts. The content of ALP in Bushen Huoxue (Chinese characters)group was (6.272±0.131) ,appreciation rate was (0.81? 0.172), and could significantly improve differentiation and proliferation activity of osteoblasts compared with Saline group (P0.05). Bushen Huoxue (Chinese characters)group could obviously promote differentiation, proliferation and mineralization of osteoblasts through activation of Wnt, β-catenin signaling pathway. It suggested that the mechanism of action of Bushen Huoxue (O'f f Il.t)particle clould prevent

  16. Osteogenic effects of D(+)β-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) on osteoblasts and bone marrow stromal cells of intact and prednisone-treated rats

    Institute of Scientific and Technical Information of China (English)

    Liao CUI; Yu-yu LIU; Tie WU; Chun-mei AI; Huai-qing CHEN

    2009-01-01

    Aim: Previous studies have shown that D(+)β-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) has anabolic effects on prednisone (GC)-induced osteoporosis in rats. The current study aims to investigate the molecular mechanism of SAA's impact on osteogenesis and adipogenesis in bone marrow stromal cells in intact and GC-treated rats. Methods: For in vitro study, newborn rat calvaria osteoblasts (rOBs) and rat bone marrow stromal cells (rMSCs) were isolated, identified and cultured with SAA at different concentrations to evaluate SAA's influence on osteogenesis and adipogenesis. In addition, 3-month-old Sprague-Dawley (SD) male rats were treated with distilled water, prednisone alone (3.0mg·kg-1·d-1)or prednisone (3.0mg·kg-1·d-1)and SAA (25mg·kg-1·d-1)for 45d.At the end point,the different groups of rMSCs were isolated by density-gradient centrifugation and cultured. Results: (1) At 0.1-10.0 mg/L, SAA increased ALP activity, type I collagen (Coil-I) mRNA and OPG mRNA expression and stimulated nodule mineralization of rObs. SAA (0.5 mg/L) also significantly increased the ALP activity of rMSCs without a need for osteogenesis-inducing medium. At 5.0 mg/L, SAA decreased the number of adipocytes with less lipid droplet formation from the rMSCs, which typically undergo adipocyte induction. (2) Coll-I expression was markedly decreased, whereas lipoprotein lipase (LPL) mRNA expression increased by 98% when compared with the first generation of rMSCs in GC-treated rats. The SAA-treated rats demonstrated an over 2-fold increase in CoU-I expression when compared with intact rats and further showed a significant decrease in LPL expression when compared with GC-treated rats. When rMSCs were co-cultured with SAA (0.5 mg/L) in vitro, SAA did not affect Coll-I and LPL gene expression in intact rats but significantly increased Coll-I and decreased LPL gene expression in GC-treated rats.Conclusion: SAA protected bone from GC-induced bone marrow impairment by stimulating

  17. Minyak ikan Lemuru (Sardinella longicep menurunkan apoptosis osteoblas pada tulang alveolaris tikus wistar (Fish oil of Lemuru (Sardinella longicep reduced the osteoblast apoptosis in wistar rat alveolar bone

    Directory of Open Access Journals (Sweden)

    Didin Erma Indahyani

    2013-12-01

    Full Text Available Background: Periodontal disease is caused by periodontopatogen bacteria resulting the alveolar bone damage. The decrease of osteoblasts and the increased of osteoclasts can cause bone destruction. The decrease of osteoblasts, due to a disturbance of differentiation, proliferation and apoptosis. Inflammatory mediators are prostaglandin E2 (PGE2, interleukin-1 (IL-1, IL-6 also tumor necrosis alpha (TNF-α stimulates osteoblast apoptosis through gene expression, signaling molecules and receptor-forming osteoblasts. Fish oil of Lemuru, which is widely encountered in Indonesian coast, containing n-3 poly unsaturated fatty acids (n-3 PUFAs are quite high. Consumption of fish oil shown to reduce the expression of PGE2, IL-1, IL-6 and TNF-α. Purpose: The purpose of this study was to examine the effect of Lemuru (Sardinella longicep fish oil on osteoblast apoptosis of rat alveolar bone induced periodontal infection. Methods: Thirty Wistar rats, male, age 5 days, divided into 3 groups: group I rats induced with normal saline, group II rats induced by LPS, and group III rats induced with lemuru fish oil and LPS. Each group was divided into 2 sub-groups that would be sacrified at 13 days and 21 days of age. Fish oil was given at a dose 1ml/300-350 grams. Lipopolysaccharide (LPS induced with the purpose to cause periodontal infection in the maxillary buccal fold molar region with dose 5μl LPS/PBS 0.03 ml. After decapitation and decalcification, the maxilla was cut in 5μm thickness. Apoptosis was analyzed on DNA and detected by TUNEL reaction (transferase-mediated digoxigenin-deoxy-UTP nick end labeling. Results: The results showed that apoptosis of osteoblast cells was significantly smaller in rats induced by Lemuru fish oil. Conclusion: The study showed that Lemuru fish oil reduced the osteoblast apoptosis of rats alveolar bone induced periodontal infection by LPS.Latar belakang: Penyakit periodontal akibat bakteri peridontopatogen, menyebabkan

  18. Effect of BMP-2 and BMP-7 homodimers and a mixture of BMP-2/BMP-7 homodimers on osteoblast adhesion and growth following culture on a collagen scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Laflamme, Claude; Rouabhia, Mahmoud [Groupe de recherche en ecologie buccale, Faculte de medecine dentaire, Universite Laval, Quebec City, Quebec G1K 7P4 (Canada)], E-mail: Mahmoud.rouabhia@fmd.ulaval.ca

    2008-03-01

    In the present study, we studied the involvement of BMP-2 and BMP-7 as homodimers and as a mixture of homodimers in bone regeneration using an engineered bone model. The engineered bone model consisted of a collagen scaffold populated with osteoblasts that acted as a carrier for the BMPs. BMP-2, BMP-7 and a mixture of BMP-2/BMP-7 were used at final concentrations of 10 and 100 ng ml{sup -1}. Osteoblasts seeded onto a collagen scaffold were cultured for 24 h before being stimulated with the BMPs. Four days later, osteoblast adhesion to and growth on the scaffold were assessed. Osteocalcin, IL-6, metalloproteinase (MMP-2 and MMP-9) and protease inhibitor (TIMP-1 and TIMP-2) mRNA and protein levels were measured. Our results showed that the BMP-2, BMP-7 and a mixture of BMP-2/BMP-7 all promoted osteoblast growth on the collagen scaffold, with the mixture of BMP-2/BMP-7 enhancing the most growth. BMP-2 and the mixture of BMP-2/BMP-7 enhanced osteocalcin (an osteoblast differentiation marker) mRNA expression and protein secretion, likely via the IL-6 pathway given that IL-6 secretion was upregulated by BMP-7 and a mixture of BMP-2/BMP-7. BMPs promote extracellular matrix production by inhibiting MMP-2 mRNA and increasing TIMP-1 and TIMP-2 mRNA expressions and protein secretions. BMP-2, BMP-7 and the mixture of BMP-2/BMP-7 could promote bone regeneration via different mechanisms involving IL-6 and MMP inhibitors.

  19. The effect of Ginkgo biloba extract treatment in the Bcl-2 expression by osteoblasts in the femoral trabecular bone of Wistar rats with glucocorticoid-induced osteoporosis

    Directory of Open Access Journals (Sweden)

    Leda M.F. Lucinda

    2014-06-01

    Full Text Available Evaluate the effect of the extract of Ginkgo biloba L., Ginkgoaceae (EGb in the Bcl-2 expression by osteoblasts in the femoral trabecular bone of Wistar rats with glucocorticoid-induced osteoporosis. Rats were divided into five groups: osteoporosis; EGb1 (28 mg/kg; EGb2 (56 mg/kg; alendronate (0.2 mg/animal and control. The treatments were conducted for 20 or 30 days. The Bcl-2 expression by osteoblasts cells was evaluated in the femoral trabecular bone. The control group was compared with the osteoporosis-induced group (Student's t-test. The other groups were analyzed by ANOVA test followed by Tukey's test (p < 0.05. The percentage of Bcl-2 expression was reduced, when the control group (17.95 ± 3.45 20 days; 21.11 ± 3.43 30 days was compared with the osteoporosis group (10.64 ± 3.30 20 days; 9.72 ± 2.84 30 days. Nevertheless, this percentage increased in the EGb2 group (18.58 ± 3.41 20 days; 16.51 ± 1.80 30 days when compared to the osteoporosis group. The EGb increased the expression of the anti-apoptotic protein, suggesting a decrease in osteoblast apoptosis.

  20. The antiarrhythmic peptide analog rotigaptide (ZP123) stimulates gap junction intercellular communication in human osteoblasts and prevents decrease in femoral trabecular bone strength in ovariectomized rats

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

    2005-01-01

    and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined 4 wk after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body......Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in noncardiac tissue are unknown. The purpose...... of this study was to examine whether antiarrhythmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass...

  1. Mesothelial cell differentiation into osteoblast- and adipocyte-like cells.

    Science.gov (United States)

    Lansley, Sally M; Searles, Richelle G; Hoi, Aina; Thomas, Carla; Moneta, Helena; Herrick, Sarah E; Thompson, Philip J; Newman, Mark; Sterrett, Gregory F; Prêle, Cecilia M; Mutsaers, Steven E

    2011-10-01

    Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0-26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies.

  2. 体外培养成骨细胞的影响因素%Factors influencing the in vitro culture of osteoblasts

    Institute of Scientific and Technical Information of China (English)

    金黎明; 刘万顺; 韩宝芹; 杨艳; 田文杰; 范圣第

    2006-01-01

    OBJECTIVE: Osteoblasts are essential for osteogenesis and bone metabolism, the in vitro culture of osteoblasts is the foundation for studies on bone metabolism and osteogenetic mechanism. Therefore, it is of great significance to study the related factors affecting it.DATA SOURCES: Related literature about the influencing factors of in vitro culture of osteoblasts were searched for in Medline from January 1980 to December 2004 with retrieval words of "osteoblasts, culture in vitro, in fluencing factors", with the language limited to English. Meanwhile, it was also searched in the CBM between January 1995 and December 2004 with the retrieval words of "osteoblasts, in vitro culture, influencing factors",with the language of the articles limited to Chinese.STUDY SELECTION: After preliminary examination, literature that met the need of this study was searched for the full text. Inclusion criteria: Factors influencing the in vitro culture of osteoblasts included ① physical factors; ② microelements; ③ growth factors; and ④ hormones.Reviews were removed from this study because of summary or repetitive research.DATA EXTRACTION: A total of 105 articles related to the influencing factors of the in vitrocul ture of osteoblasts were obtained, articles of repetitive and similar researchwere removed; thereby 17 articles were included in the study.DATA SYNTHESIS: ① Physical factors: Ionizing radiation, microgravity,external force, and oxygen pressure. ② Microelements: microelement deficiency would hinder the skeletal growth, or even lead to malformation. Osteoblastic proliferation is closely related to some microelements, mainly including zinc, aluminum, fluorine, copper, manganese, calcium, and magnesium. ③ Growth factors closely related to osteogenesis mainly consist of bone morphogenetic protein, platelet-derived growth factor, fibroblast growth factor, transforming growth factor-beta, insulin like growth factor,and osteogenic growth peptide (OGP). ④ Hormones

  3. Co-Culture of S. epidermidis and Human Osteoblasts on Implant Surfaces: An Advanced In Vitro Model for Implant-Associated Infections.

    Directory of Open Access Journals (Sweden)

    Sarah Zaatreh

    Full Text Available Total joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings.For initial surface adhesion, human primary osteoblasts (hOB were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R®, and PALACOS R® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04, and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU. The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces.After 2 days, we observed approximately 104 CFU/ml biofilm-bound S. epidermidis (103 CFU/ml initial population on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 105 CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10

  4. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures

    DEFF Research Database (Denmark)

    Justesen, J; Dokkedahl, Karin Stenderup; Eriksen, E F

    2002-01-01

    Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the ......Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP...

  5. Influence of titanium ion on mineral formation and properties of osteoid nodules in rat calvaria cultures.

    Science.gov (United States)

    Liao, H; Wurtz, T; Li, J

    1999-11-01

    The effect of the addition of titanium ion (Ti) on osteoblast function and the mineralization of osteoid nodules in rat calvaria cultures was characterized. Concentrations of 10 ppm of Ti or more inhibited cell proliferation; 5 ppm or less either had no effect or stimulated proliferation. The number of nodules formed was not influenced by 5 ppm of Ti, but mineral deposition in nodules was suppressed, as revealed by von Kossa staining. Likewise, 5 ppm of Ti inhibited the incorporation of [(45)Ca] in cultures during nodule formation even if the Ti was withdrawn from the medium when mineralization was initiated. In order to test whether the synthesis of osteoid components was affected, the expression of osteonectin (OSN), osteopontin (OPN), osteocalcin (OSC), and alkaline phosphatase (ALP) mRNAs as well as ALP enzyme activity was analyzed. The expression of OSN and OPN mRNAs was reduced dramatically, but OSC mRNA was little affected by 5 ppm of Ti. Ti delayed the development of ALP mRNA expression and enzyme activity relative to the controls. Thus Ti treatment changed the proportional composition of cellular mRNA contributing the osteoblast phenotype.

  6. Stretch-mediated responses of osteoblast-like cells cultured on titanium-coated substrates in vitro

    NARCIS (Netherlands)

    Walboomers, X.F.; Habraken, W.J.E.M.; Feddes, B.; Winter, L.C.; Bumgardner, J.D.; Jansen, J.A.

    2004-01-01

    Cyclic stretching experiments on osteoblast-like cells have proven to be a useful tool in understanding the underlying mechanisms of load transduction at the bone-implant surface. However, most experimental setups use silicone rubber substrates, which are atypical for orthopedic and dental implant m

  7. The effect of light-emitting diode irradiation at different wavelengths on calcification of osteoblast-like cells in 3D culture.

    Science.gov (United States)

    Chintavalakorn, Rochaya; Tanglitanont, Tatsanee; Khantachawana, Anak; Viravaidya-Pasuwat, Kwanchanok; Santiwong, Peerapong

    2015-08-01

    This study aimed to investigate the effect of four different light-emitting diode (LED) wavelengths on calcification and proliferation of osteoblast-like cells in vitro. MC3T3-E1 cells were seeded within three-dimensional collagen scaffolds and irradiated daily by LED light with peak emission wavelengths of 630-, 680-, 760- and 830-nm at constant fluency of 3.1 J/cm(2) (irradiance intensity 2 mW/cm(2)). Cultures were measured for calcium content at day 0, 7, 14, 21, 28, 35 and 42. The significant enhancement in calcium content was observed at the early stage of culture (days 7 and 14) (plight irradiation on osteoblastic cell calcification. Only 680-nm irradiated samples revealed a significant enhancement of calcium content until the late stages of culture (from days 21 to 42) (p<;0.001). The cyclin D mRNA expression that was investigated 3 hours after stimulation at day3 also show that the 680-nm LED irradiation can enhance cyclin D expression more than others. For enhancing bone mineralization, LED irradiation at the 680-nm is more effective than those at 630-, 760- and 830-nm. Further studies should be investigated in order to obtain the most effective parameters of LLLI on bone regeneration in clinical setting.

  8. Increased osteoblast adhesion on nanophase Ti6Al4V

    Institute of Scientific and Technical Information of China (English)

    JI WeiPing; HAN Pei; ZHAO OhangLi; JIANG Yao; ZHANG XiaoNong

    2008-01-01

    The objective of the present study was to prepare a novel nanostructured surface of Ti6Al4V alloy by the severe plastic deformation (SPD) and the chemical treatment process and to evaluate the adhesion of osteoblast on the nanophase titanium alloy. In the in vitro study, the primary cultured osteoblasts of neonatal rat calvaria were cultured on the nanophase and the as-received smooth Ti6Al4V substrates. Then osteoblasts adhesion behaviors on different substrates were observed by the fluorescence microscopy, scanning electron microscopy (SEM) and RT-PCR analysis. The results of our research showed increased osteoblast adhesion on the nanophase titanium alloy compared with the as-received case. On the nanophase substrate, the presence of extensive filopodia, strong cellular adhesion and early cellular confluency could be observed. In addition, the expression of the adhesion-related integrin β1 mRNA was also higher on the nanophase substrate. It suggested that the nano technology could be further considered for orthopedic implant applications.

  9. Kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling

    Directory of Open Access Journals (Sweden)

    Guo Ava

    2012-04-01

    Full Text Available Abstract Background Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, chemically resemble estrogen and some have been used as estrogen substitutes. Kaempferol, a flavonol derived from the rhizome of Kaempferia galanga L., is a well-known phytoestrogen possessing osteogenic effects that is also found in a large number of plant foods. The herb K. galanga is a popular traditional aromatic medicinal plant that is widely used as food spice and in medicinal industries. In the present study, both the estrogenic and osteogenic properties of kaempferol are evaluated. Methods Kaempferol was first evaluated for its estrogenic properties, including its effects on estrogen receptors. The osteogenic properties of kaempferol were further determined its induction effects on specific osteogenic enzymes and genes as well as the mineralization process in cultured rat osteoblasts. Results Kaempferol activated the transcriptional activity of pERE-Luc (3.98 ± 0.31 folds at 50 μM and induced estrogen receptor α (ERα phosphorylation in cultured rat osteoblasts, and this ER activation was correlated with induction and associated with osteoblast differentiation biomarkers, including alkaline phosphatase activity and transcription of osteoblastic genes, e.g., type I collagen, osteonectin, osteocalcin, Runx2 and osterix. Kaempferol also promoted the mineralization process of osteoblasts (4.02 ± 0.41 folds at 50 μM. ER mediation of the kaempferol-induced effects was confirmed by pretreatment of the osteoblasts with an ER antagonist, ICI 182,780, which fully blocked the induction effect. Conclusion Our results showed that kaempferol stimulates osteogenic differentiation of cultured osteoblasts by acting through the estrogen receptor signaling.

  10. Osteoblastic and osteoclastic differentiation of human mesenchymal stem cells and monocytes in a miniaturized three-dimensional culture with mineral granules.

    Science.gov (United States)

    Gamblin, Anne Laure; Renaud, Audrey; Charrier, Céline; Hulin, Philippe; Louarn, Guy; Heymann, Dominique; Trichet, Valérie; Layrolle, Pierre

    2014-12-01

    The pathologies of the skeleton have a significant socioeconomic impact on our population. Although therapies have improved the treatment of osteosarcoma and osteoporosis, their efficacy still remains limited. In this context, we developed a miniaturized 3-D culture model of bone cells on calcium phosphate ceramics. Human bone marrow mesenchymal stem cells (MSCs) were three-dimensionally cultured on particles of biphasic calcium phosphate (BCP, 125-200μm) in osteogenic media. The MSCs seeded on the BCP particles adhered and proliferated, producing abundant collagenous extracellular matrix (ECM). Light and confocal laser scanning microscopy showed that the MSCs created bridges between the BCP particles and formed a 3-D structure. Energy dispersive X-ray analysis in a scanning electron microscope confirmed the mineralization of the collagen matrix. The 96-well sized bone constructs were tested by immunohistology and transcription analysis, proving cell differentiation. Both techniques corroborated the osteoblastic differentiation with high production of bone sialoprotein and osteocalcin. Peripheral blood CD14-positive monocytes (MOs) were pre-differentiated into osteoclasts prior to seeding on the 3-D constructs. Multinucleated and tartrate-resistant acid phosphatase-positive cells were also identified at the surface of the 3-D constructs after 90days of culture. In addition, cell viability within these constructs was measured by flow cytometry. In summary, we have developed a miniaturized 3-D culture of bone cell precursors with osteoblasts and osteoclasts. This 3-D culture may make it possible to test the effects of new drugs for bone healing, osteoporosis and osteosarcomas, in more appropriate cell-cell and cell-matrix interactions than conventional 2-D cultures.

  11. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xinhua [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Wang, Xiaoyuan [Department of Nephrology, Xi An Honghui Hospital, Xi an (China); Hu, Xiongke; Chen, Yong; Zeng, Kefeng [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Zhang, Hongqi, E-mail: zhq9699@126.com [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China)

    2015-07-01

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression.

  12. Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

    Science.gov (United States)

    Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

    2016-02-17

    Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. The angiogenic behaviors of human umbilical vein endothelial cells (HUVEC) in co-culture with osteoblast-like cells (MG-63) on different titanium surfaces.

    Science.gov (United States)

    Shi, Bin; Andrukhov, Oleh; Berner, Simon; Schedle, Andreas; Rausch-Fan, Xiaohui

    2014-08-01

    Interaction between osteogenesis and angiogenesis plays an important role in implant osseointegration. In the present study we investigated the influence of titanium surface properties on the angiogenic behaviors of endothelial cells grown in direct contact co-culture with osteoblasts. Human umbilical vein endothelial cells (HUVECs) and osteoblast-like cells (MG-63 cells) were grown in direct co-culture on the following titanium surfaces: acid-etched (A), hydrophilic A (modA), coarse-gritblasted and acid-etched (SLA) and hydrophilic SLA (SLActive). Cell proliferation was evaluated by cell counting combined with flow cytometry. The expression of von Willebrand Factor (vWF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), E-Selectin, as well as vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR in HUVECs and VEGF in MG-63 were measured by qPCR. The dynamic behavior of endothelial cells was recorded by time-lapse microscopy. Proliferation of HUVECs was highest on A, followed by SLA, modA and SLActive surfaces. The expression of vWF, TM, EPCR, E-Selectin and Flt-1 in HUVECs was significantly higher on A than on all other surfaces. The expression of KDR in HUVECs grown on A surface was below detection limit. VEGF expression in MG-63 cells was significantly higher on SLActive vs SLA and modA vs A surfaces. Time-lapse microscopy revealed that HUVECs moved quickest and formed cell clusters earlier on A surface, followed by SLA, modA and SLActive surface. In co-culture conditions, proliferation and expression of angiogenesis associated genes in HUVECs are promoted by smooth hydrophobic Ti surface, which is in contrast to previous mono-culture studies. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  14. Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis

    Science.gov (United States)

    Scott, D. K.; Brakenhoff, K. D.; Clohisy, J. C.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS).

  15. Rat fetal ventral mesencephalon grown as solid tissue cultures

    DEFF Research Database (Denmark)

    Höglinger, G U; Sautter, J; Meyer, Morten;

    1998-01-01

    Free-floating roller tube (FFRT) cultures of fetal rat and human nigral tissue are a means for tissue storage prior to grafting in experimental Parkinson's disease. In the present study, FFRT cultures prepared from embryonic-day-14 rat ventral mesencephalon were maintained for 4, 8, 12, or 16 days...

  16. Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.

    Science.gov (United States)

    Simão, Ana Maria S; Beloti, Márcio M; Cezarino, Rodrigo M; Rosa, Adalberto Luiz; Pizauro, João M; Ciancaglini, Pietro

    2007-04-01

    Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.

  17. Co-culture of adipose-derived stem cells and osteoblasts under different conditions%不同培养条件下脂肪干细胞与成骨细胞的共培养

    Institute of Scientific and Technical Information of China (English)

    张扬; 刘大诚; 杨效宁

    2014-01-01

    BACKGROUND:After co-culture with osteoblasts, bone marrow stem cells can be induced to differentiate into osteoblasts. Whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts or not? OBJECTIVE:To observe whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts. METHODS:Adipose-derived stem cells and osteoblasts were isolated from New Zealand white rabbits. Then, passage 3 adipose-derived stem cells were co-cultured with passage 2 osteoblasts in 10%or 5%fetal bovine serum for 14 days. RESULTS AND CONCLUSION:After 7 days of co-culture, some adipose-derived stem cells became round in the two groups. After 14 days of co-culture, adipose-derived stem cells highly differentiated and differentiated cells were similar to mature osteoblasts that were positive for alkaline phosphatase staining and alizarin red staining. The mRNA expression of type I col agen and osteocalcin increased in both two group, especial y in the 10%fetal bovine serum group. These findings indicate that adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts induced by high-concentration serum culture.%背景:成骨细胞与骨髓干细胞共培养后可以诱导骨髓干细胞向成骨细胞分化,成骨细胞与脂肪干细胞共培养是否也能诱导向成骨细胞分化呢?目的:观察脂肪干细胞与成骨细胞共培养后能否向成骨细胞分化。方法:分离新西兰大白兔脂肪干细胞和成骨细胞,待脂肪干细胞生长至3代,成骨细胞生长至2代时,进行共培养。根据培养时血清浓度不同分为10%胎牛血清共培养组和5%胎牛血清共培养组,共培养14 d。结果与结论:共培养7 d后,2组脂肪干细胞均出现部分变圆。14 d后,脂肪干细胞高度分化与成熟成骨细胞相似,碱性磷酸酶染色阳性、茜素红染色阳性,其Ⅰ型胶原和骨钙素mRNA表达均增高,以10%胎

  18. Three-dimensional dynamic culture of pre-osteoblasts seeded in HA-CS/Col/nHAP composite scaffolds and treated with α-ZAL

    Institute of Scientific and Technical Information of China (English)

    Lu Liu; Hao Li; Xizheng Zhang; Yong Guo; Xuezhong Chen; Ruixin Li; Zhihong Li; Liang Wang; Zongming Wan; Jianyu Li; Qingxin Hao

    2012-01-01

    Pre-osteoblast MC3T3-E1 cells were cultured in hyaluronie acid-modified chitosan/collagen/nano-hydroxyapatite (HA-CS/Col/nHAP) composite scaffolds and treated with phytoestrogen α-zearalanol (α-ZAL) to improve bone tissue formation for bone tissue engineering.Perfusion and dynamic strain were applied to three-dimensional (3D) cultured cells,which simulates mechanical microenvironment in bone tissue and solves mass transfer issues.The morphology of cell-scaffold constructs in vitro was then examined and markers of osteogenesis were assessed by immunohistochemistry staining and western blotting.The results showed that cells expanded their pseudopodia in an irregular manner and dispersed along the walls in 3D-dynamic culture.Osteogenic phenotype was increased or maintained by enhanced collagen I (COLI) levels,decreased osteopontin expression and having little effect on osteocalcin expression during the 12 days of in vitro culture.In response to o-ZAL,the cell-scaffold constructs showed inhibited cellular proliferation,enhanced the alkaline phosphatase (ALP) activity and increased ratio of osteoprotegerin to receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL).Application of perfusion and dynamic strain to cells-scaffold constructs treated with α-ZAL represents a promising approach in the studies of osteogenesis stimulation of bone tissue engineering.

  19. Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Brot, C

    2000-01-01

    Effective bone remodeling requires the coordination of bone matrix deposition by osteoblastic cells, which may occur via soluble mediators or via direct intercellular communication. We have previously identified two mechanisms by which rat osteoblastic cell lines coordinate calcium signaling among...

  20. The observation of hypoxia inducible factor-1α in osteoblasts cultured under intermittent hypoxia%间断性低氧环境下成骨细胞低氧诱导因子-1α表达变化的观察

    Institute of Scientific and Technical Information of China (English)

    弓煦; 高雪梅; 李成杰

    2012-01-01

    Objective To investigate the expression of hypoxia inducible factor-1α(HIF-1α) in osteoblasts cultured under intermittenthypoxia.Methods The osteoblasts of the Sprague-Dawley rat were divided into three groups:① The control group were cultured under 21%O2 and 5%CO2 for 25 h; ② The continuous hypoxia group were cultured under 2%O2 and 5%CO2 for 3 h and 25 h;③ The intermittent hypoxia group:the osteoblasts cultured undernormoxic conditions (2%O2 and 5%CO2) and hypoxia conditions (21%O2 and 5%CO2 ),alternating every hour.The first hour was in hypoxia condition.The osteoblasts cultured for 1 h,3 h,5 h,9 h,13 h,25 h.The expression of HIF-1α in three groups was investigated by reverse transcription polymerase chain reaction,RT-PCR.Results There was more expression of HIF-1α in the 25 h than the 3 h in the continuous hypoxia group.In the intermittent hypoxia group,there was expression of HIF-1α in all groups.The expression of HIF-1α in 1 h and 5 h groups was less than 3 h,9 h,13 h,25 h groups.Conclusions Intermittent hypoxia may lead to an increase on the expression of HIF-1α in the osteoblasts. It may affect the bone metabolism of the patients with obstructive sleep apnea hypopnea syndrome.%目的 观察间断性低氧环境下成骨细胞低氧诱导因子-1α(hypoxia inducible factor,HIF-1α)表达变化,试探讨睡眠呼吸暂停低通气综合征患者骨代谢特点.方法 将SD大鼠(Sprague-Dawley rat,SD rat)成骨细胞分为3组进行培养:①常氧组:常氧条件(21%O2,5%CO2)培养25h提取总RNA;②持续性低氧组:低氧条件(2%O2,5%CO2)下培养,分别在第3h、25 h提取总RNA;③间断性低氧组:交替在低氧条件(2%O2,5% CO2)和常氧条件(21%O2,5%CO2)培养,每隔1h交替,第1h为低氧培养,分别在第1h、3h、5h、9h、13h、25 h提取总RNA.反转录聚合酶链反应(RT-PCR)检测以上样本HIF-1α的表达.结果 在持续性低氧条件下,H1F-1α表达随时间增加而增加.间断性

  1. Acidosis is a key regulator of osteoblast ecto-nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity.

    Science.gov (United States)

    Orriss, Isabel R; Key, Michelle L; Hajjawi, Mark O R; Millán, José L; Arnett, Timothy R

    2015-12-01

    Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi ) to pyrophosphate (PPi ) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi , a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto-nucleotidases. This study investigated the expression and activity of ecto-nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto-nucleotidases including NTPdase 1-6 (ecto-nucleoside triphosphate diphosphohydrolase) and NPP1-3 (ecto-nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 >  alkaline phosphatase > ecto-5-nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8-fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto-nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5-fold, P acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  2. Tissue engineering of composite grafts: Cocultivation of human oral keratinocytes and human osteoblast-like cells on laminin-coated polycarbonate membranes and equine collagen membranes under different culture conditions.

    Science.gov (United States)

    Glaum, R; Wiedmann-Al-Ahmad, M; Huebner, U; Schmelzeisen, R

    2010-05-01

    In complex craniomaxillofacial defects, the simultaneous reconstruction of hard and soft tissue is often necessary. Until now, oral keratinocytes and osteoblast-like cells have not been cocultivated on the same carrier. For the first time, the cocultivation of human oral keratinocytes and human osteoblast-like cells has been investigated in this study. Different carriers (laminin-coated polycarbonate and equine collagen membranes) and various culture conditions were examined. Human oral keratinocytes and human osteoblast-like cells from five patients were isolated from tissue samples, seeded on the opposite sides of the carriers and cultivated for 1 and 2 weeks under static conditions in an incubator and in a perfusion chamber. Proliferation and morphology of the cells were analyzed by EZ4U-tests, light microscopy, and scanning electron microscopy. Cocultivation of both cell-types seeded on one carrier was possible. Quantitative and qualitative growth was significantly better on collagen membranes when compared with laminin-coated polycarbonate membranes independent of the culture conditions. Using perfusion culture in comparison to static culture, the increase of cell proliferation after 2 weeks of cultivation when compared with the proliferation after 1 week was significantly lower, independent of the carriers used. In conclusion, the contemporaneous cultivation of human oral keratinocytes and human osteoblast-like cells on the same carrier is possible, a prerequisite for planned in vivo studies. As carrier collagen is superior to laminin-coated polycarbonate membranes. Regarding the development over time, the increase of proliferation rate is lower in perfusion culture. Examinations of cellular differentiation over time under various culture conditions will be subject of further investigations.

  3. 芦丁含药血清对体外培养大鼠颅骨成骨细胞增殖与分化成熟的影响%Effects of the serum of rats administered rutin on the proliferation, differentiation and maturation of rat calvarial osteoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    张福琴

    2011-01-01

    serums were supplemented into the culture media of rat calvarial osteoblasts by 2.5%, 5% and 10% respectively. The proliferation of ROB was analyzed by MTT reduction assay. The osteogenic differentiation and maturation were assayed by measuring alkaline phosphatase activity(ALP)and numbering the mineralized bone nodules. Results The value of A570 in 2.5% and 5% SR group were Significantly [(0.678±0.242)%、(0.796±0.155)%]higher than those of the control. 10% SR was toxic to ROB. 5% SR was stronger (63.35±5.20)μg/μl than 2.5% SR in increasing ALP activity and the number of mineralized bone nodules which were significantly higher in SR group than in the control. Conclusion The metabolites of rutin after oral administration stimulate the proliferation and osteogenic differentiation of osteoblasts. Because so far, the vast majority of the Chinese herbal medicine containing Rutin are oral. The metabolites of rutin after oral administration are likely to be the effective in anti-osteoporosis. However, the role of specific forms and modalities have yet to be studied in depth.

  4. Serotonin regulates osteoblast proliferation and function in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Dai, S.Q.; Yu, L.P. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Shi, X. [Department of Obstetrics and Gynecology, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Wu, H. [Emergency Department, The First Affiliated Hospital, Soochow University, Suzhou (China); Shao, P.; Yin, G.Y.; Wei, Y.Z. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China)

    2014-08-01

    The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT{sub 1A}, 5-HT{sub 1B}, 5-HT{sub 1D}, 5-HT{sub 2A}, 5-HT{sub 2B}, and 5-HT{sub 2C}) were found to exist in rat osteoblasts. Of these, 5-HT{sub 2A} and 5-HT{sub 1B} receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

  5. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation of h......-life of osteoblastic PDGFR-alpha mRNA, but did not decrease its promoter activity. In summary, our data show that PDGFR-alpha is downregulated in hOBs by co-cultivation with human primary endothelial cells through a p38 MAPK-dependent post-transcriptional mechanism.......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation...... of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

  6. Preliminary results of implantation in animal model and osteoblast culture evaluation of prototypes of biomimetic multispiked connecting scaffold for noncemented stemless resurfacing hip arthroplasty endoprostheses.

    Science.gov (United States)

    Uklejewski, Ryszard; Rogala, Piotr; Winiecki, Mariusz; Kędzia, Andrzej; Ruszkowski, Piotr

    2013-01-01

    We present the new fixation method for RHA (resurfacing hip arthroplasty) endoprostheses by means of the biomimetic multispiked connecting scaffold (MSC-Scaffold). Such connecting scaffold can generate new type of RHA endoprostheses, that is stemless and fixed entirely without cement. The preprototypes of this MSC-Scaffold were manufactured with modern additive laser additive technology (SLM). The pilot surgical implantations in animal model (two laboratory swine) of MSC-Scaffold preprototypes have showed after two months neither implant loosening, migration, and nor other early complications. From the results of performed histopathological evaluation of the periscaffold spikes bone tissue and 10-day culture of human osteoblasts (NHOst) we can conclude that (1) the scaffolding effect was obtained and (2) to improve the osseointegration of the scaffold spikes, their material surface should be physicochemically modified (e.g., with hydroxyapatite). Some histopathological findings in the periscaffold domain near the MSC-Scaffold spikes bases (fibrous connective tissue and metallic particles near the MSC-Scaffold spikes bases edges) prompt considering the necessity to optimize the design of the MSC-Scaffold in the regions of its interspike space near the spikes bases edges, to provide more room for new bone formation in this region and for indispensable post-processing (glass pearl blasting) after the SLM manufacturing.

  7. Selective Estrogen Receptor Modulator (SERM)-like Activities of Diarylheptanoid, a Phytoestrogen from Curcuma comosa, in Breast Cancer Cells, Pre-osteoblast Cells, and Rat Uterine Tissues.

    Science.gov (United States)

    Thongon, Natthakan; Boonmuen, Nittaya; Suksen, Kanoknetr; Wichit, Patsorn; Chairoungdua, Arthit; Tuchinda, Patoomratana; Suksamrarn, Apichart; Winuthayanon, Wipawee; Piyachaturawat, Pawinee

    2017-05-03

    Diarylheptanoids from Curcuma comosa, of the Zingiberaceae family, exhibit diverse estrogenic activities. In this study we investigated the estrogenic activity of a major hydroxyl diarylheptanoid, 7-(3,4 -dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound 092) isolated from C. comosa. The compound elicited different transcriptional activities of estrogen agonist at low concentrations (0.1-1 μM) and antagonist at high concentrations (10-50 μM) using luciferase reporter gene assay in HEK-293T cells. In human breast cancer (MCF-7) cells, compound 092 showed an anti-estrogenic activity by down-regulating ERα-signaling and suppressing estrogen-responsive genes, whereas it attenuated the uterotrophic effect of estrogen in immature ovariectomized rats. Of note, compound 092 promoted mouse pre-osteoblastic (MC3T3-E1) cell differentiation and the related bone markers, indicating its positive osteogenic effect. Our findings highlight a new, nonsteroidal, estrogen agonist/antagonist of catechol diarylheptanoid from C. comosa, which is scientific evidence supporting its potential as a dietary supplement to prevent bone loss with low risk of breast and uterine cancers in postmenopausal women.

  8. Induction of cAMP-dependent protein kinase A activity in human skin fibroblasts and rat osteoblasts by extremely low-frequency electromagnetic fields

    Energy Technology Data Exchange (ETDEWEB)

    Thumm, S.; Glock, S.; Haemmerle, H. [Natural and Medical Sciences Institute Reutlingen, University of Tuebingen (NMI), Markwiesenstrasse 55, D-72770 Reutlingen (Germany); Loeschinger, M.; Rodemann, H.P. [Section of Radiobiology and Molecular Environmental Research, University of Tuebingen, Roentgenweg 11, D-72076 Tuebingen (Germany)

    1999-09-01

    Sinusoidal extremely low-frequency electromagnetic fields (ELF-EMF; 7-8 mT, 20 Hz) have already been shown to inhibit proliferation and to accelerate terminal differentiation of human skin fibroblasts in vitro. In order to elucidate the underlying processes of signal transduction, we analysed the activity of cAMP-dependent protein kinase (PKA). EMF exposure for 60 min resulted in an increased PKA activity in human skin fibroblasts (2-fold) and rat embryonic osteoblasts (1.7-fold). Long-term exposure for up to 7 days with a constant 1 h-on/1 h-off EMF exposure rhythm indicated a transient stimulation of PKA activity during the first two exposure rhythms followed by a decrease to the baseline levels of sham-exposed controls. Based on these results, we postulate that a modulation of proliferation and differentiation processes in cells of mesenchymal origin is triggered by an immediate and transient EMF-induced increase in PKA activity. (orig.)

  9. Production of compartmented cultures of rat sympathetic neurons.

    Science.gov (United States)

    Campenot, Robert B; Lund, Karen; Mok, Sue-Ann

    2009-01-01

    The compartmented culture, in which primary neurons plated in a proximal compartment send their axons under silicone grease barriers and into left and right distal compartments, has enhanced the experimental capabilities of neuronal cultures. Treatments can be applied separately to cell bodies/proximal axons or distal axons, and cell bodies/proximal axons and distal axons can be separately harvested and analyzed. Distal axons can be axotomized, and the neurons can be studied while their axons regenerate. Construction of the culture dishes requires 3 h for 48 cultures, and preparing the neurons also requires 3 h. Compartmented cultures provide enough cellular material for biochemical analyses such as immunoblotting. The uses of compartmented cultures have included studies of neurotrophic factor retrograde signaling, axonal transport, and axonal protein and lipid biosynthesis. Here we focus on sympathetic neurons cultured from neonatal rats and provide protocols for the production and some of the uses of compartmented cultures.

  10. 25-Hydroxy- and 1α,25-Dihydroxycholecalciferol Have Greater Potencies than 25-Hydroxy- and 1α,25-Dihydroxyergocalciferol in Modulating Cultured Human and Mouse Osteoblast Activities

    Science.gov (United States)

    Hulley, Philippa A.; Sabokbar, Afsie; Javaid, M. Kassim; Morovat, Alireza

    2016-01-01

    Despite differences in the phamacokinetics of 25-hydroxycholecalciferol (25(OH)D3) and 25-hydroxyergocalciferol (25(OH)D2) in man, the effects of these and their 1α-hydroxylated forms (1,25(OH)2D3 and 1,25(OH)2D2) on cellular activity of vitamin D-responsive cells have hardly been compared. We studied differences in the effects of these metabolites on cell number, gene transcription, protein expression and mineralisation of cultured human bone marrow-derived stromal cells (hBMSC) and rapidly mineralising mouse 2T3 osteoblasts. 50–1000 nM 25(OH) and 0.05–10 nM 1,25(OH)2 metabolites were used. At high concentrations, 25(OH)D2/D3 and 1,25(OH)2D2/D3 suppressed cell number in both human and mouse cells. The suppression was greater with cholecalciferol (D3) metabolites than with those of ergocalciferol (D2). In both cell types, 25(OH)D2 and 25(OH)D3 increased the expression of osteopontin, osteocalcin, collagen-1, receptor activator of nuclear factor kappa-B ligand, vitamin D receptor, CYP24A1 and CYP27B1 genes. Whereas there was little or no difference between the effects of 25(OH)D2 and 25(OH)D3 in hBMSCs, differences were observed in the magnitude of the effects of these metabolites on the expression of most studied genes in 2T3 cells. Alkaline phosphatase (ALP) activity was increased by 25(OH)D2/D3 and 1,25(OH)2D2/D3 in hBMSC and 2T3 cells, and the increase was greater with the D3 metabolites at high concentrations. In hBMSCs, mineralisation was also increased by 25(OH)D2/D3 and 1,25(OH)2D2/D3 at high concentrations, with D3 metabolites exerting a greater influence. In 2T3 cells, the effects of these compounds on mineralisation were stimulatory at low concentrations and inhibitory when high concentrations were used. The suppression at high concentrations was greater with the D3 metabolites. These findings suggest that there are differences in the effects of 25-hydroxy and 1α,25(OH)2 metabolites of D3 and D2 on human preosteoblasts and mouse osteoblasts, with

  11. Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Zunich Samantha M

    2012-07-01

    Full Text Available Abstract Background Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. Results In the presence of exogenous ascorbic acid (AA, a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis

  12. The effect of microgravity on 1,25-dihydroxyvitamin d3 signalling in osteoblasts

    Science.gov (United States)

    Coenegrachts, Lieve; Stockmans, Ingrid; Segers, Ilse; Bouillon, Roger; Carmeliet, Geert

    2007-09-01

    Microgravity encountered during space flight induces bone loss, as seen in both humans and rats. This type of bone loss is mainly caused by decreased bone formation due to reduced osteoblast proliferation and differentiation. Yet, the molecular alterations induced by microgravity during osteoblast differentiation are still enigmatic. Therefore, the effect of microgravity on the intracellular signalling pathway of 1,25-dihydroxyvita-min D3 was investigated during the Odissea Mission. The ligand 1,25-dihydroxyvitamin D3 interacts with the vitamin D receptor (VDR) and this complex binds to vitamin D response elements (VDRE) in the promoter region of target genes to stimulate or suppress gene transcription. To investigate the interaction of liganded VDR with VDRE, the mouse osteoblastic cell line, MC3T3, was stable transfected with a construct containing multiple VDREs of the rat osteocalcin promoter fused to growth hormone as reporter gene. Treatment of these transfectants with 1,25-dihydroxyvitamin D3 resulted in a time- and dose-dependent release of growth hormone in the culture medium. Space flight cultures responded to 1,25-dihydroxyvitamin D3 treatment with increased growth hormone production that was comparable with the induction observed in ground cultures. No 1g centrifuge was available during the space flight. These data indicate that microgravity for 5 days did not alter the interaction of VDR with the osteocalcin VDRE or the subsequent gene transcription.

  13. Biocompatibility evaluation in vitro. Part Ⅲ: Cytotoxicity expression of human and animal osteoblasts on the biomaterials

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Exclusion and imflammation in the clinic are observed for various reasons including material chemical composition, physical properties as well as macro- and micro-structure of the implants, surface condition of the implants, and also patient dependent factors. Cytotoxicity expression of cells is a central issue in current biocompatibility to screen the potential implant materials and drugs. This study was aimed at investigation reaction between the potential implant materials and surround tissue. Cytotoxicity of human and mt osteoblast in the material extracts was determinated by testing standards such as GHS assay, MTI assay, alkaline phosphatase activity assay, LDH assay, and Lowry assay. Research results demonstrated that compared with the control condition polystyrene culture plate both human and rat osteoblast cells have normal phenotypie expression in hydroxyapatite extract, and this expression was statistically restricted in hydroxyapatite-spinel extract. However, this restrict, e.g. cytotoxicity could be partially eliminated by immersion treatment of the materials in culture medium.

  14. The cytotoxicity of lead and uranium on rat osteoblastic cells is highly dependent on chemical speciation and cellular accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Milgram, S.; Carriere, M.; Thiebault, C.; Gouget, B. [CEA Saclay, CNRS - UMR9956, Lab Pierre Sue, F-91198 Gif Sur Yvette, (France); Malval, L. [INSERM, E366, Lab Biol Tissue Osseux, St Etienne, (France)

    2007-07-01

    Complete text of publication follows: Uranium (U) and lead (Pb), as other heavy metals, present a strong chemical toxicity. After blood contamination, U and Pb, complexed with proteins or inorganic molecules are conveyed to target organs, the skeleton being the major long-term storage site. Once in bones, both metals are incorporated in the hydroxyapatite matrix by substitution with calcium. They can thus be released during re-modelling, which explains in part their toxicity. Although the clinical effects of these metals are well known, the cellular mechanisms of their action are not well understood. To investigate the biological effects of U and Pb acute exposure on osteoblasts, ROS17/2.8 cells were exposed to Pb or U [0-1 mM] for 24 h. The most relevant chemical and physical states, namely the most likely forms (species) of the toxics in contact with cells after blood contamination were selected for cell exposure. For each metal species, Pb and U toxicity were assessed through cell viability assay. The results show that whatever the speciation, U chemical toxicity to bone cells is far lower than Pb toxicity. Pb appears to be cytotoxic when left free in the exposure medium or when it is complexed with bicarbonate, cysteine or citrate, but not with albumin or phosphate (an insoluble form of Pb). In order to explain these differences in sensitivity between different metals and metal chemical species, time-course and dose-response curves of cellular accumulation at lethal or sub-lethal doses were drawn by direct elemental analysis of metal concentrations in digested cell pellets, using Inductive Coupling Plasma Mass Spectroscopy. These showed a clear correlation between toxicity and cellular accumulation. Also, Pb induces an inhibition of ALP activity after 24 h exposure to sub-lethal doses, which is speciation-dependent and again correlates with cellular accumulation. Phenotypic effects of U are under investigation. In addition, electron-microscopic observation of

  15. Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

    Science.gov (United States)

    Zhang, Lingli; Hanagata, Nobutaka; Maeda, Megumi; Minowa, Takashi; Ikoma, Toshiyuki; Fan, Hongsong; Zhang, Xingdong

    2009-04-01

    Because calcium phosphate (Ca-P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca-P ceramics with different Ca-P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca-P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca-P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca-P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca-P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.

  16. Interactions among osteoblastic cells, Staphylococcus aureus and chitosan-immobilized titanium implants in a post-operative co-culture system: An in vitro study.

    Science.gov (United States)

    Ghimire, Niranjan; Foss, Berit L; Sun, Yuyu; Deng, Ying

    2015-10-21

    Biomaterial-related infections (BRIs) have become a major challenge in the field of orthopedic implants. In this study, we delved into the problem of BRI and attempted to reduce the possibility of BRI incidence via surface modification of titanium (Ti) with chitosan (SA-CS-Ti). To comprehensively evaluate the anti-infection potential of SA-CS-Ti, we first constructed a post-operative infection (POI) model with varying concentrations of bacteria (10(2) CFU/sample and 10(4) CFU/sample) and a constant number of SaOS-2 cells (10(5) /sample). Then, we biologically characterized the interactions between the SaOS-2 cells, bacteria, and different Ti implants using the POI model. The results from the osteoblastic cell and bacterial attachment tests demonstrated that the SA-CS-Ti surfaces exhibit superior osteogenic behavior relative to other Ti surfaces studied while showing significant anti-infective activities in the POI model with a low infection ratio (bacteria: cell ratio of 0.001:1) 30 minutes after infection. Additionally, the SA-CS-Ti surfaces showed significantly reduced (panti-fouling property. The significantly increased (p<0.05) sensitivity of S. aureus adhered to the SA-CS-Ti surfaces against cefazolin (1 mg/L treatment) and gentamicin (10 mg/L and 100 mg/L treatment) in the co-culture system augmented potential of SA-CS-Ti to be used as orthopedic implants. This article is protected by copyright. All rights reserved.

  17. Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes

    Science.gov (United States)

    Shalhoub, V.; Conlon, D.; Tassinari, M.; Quinn, C.; Partridge, N.; Stein, G. S.; Lian, J. B.

    1992-01-01

    To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the

  18. Kinetics of lactate and pyruvate transport in cultured rat myotubes

    DEFF Research Database (Denmark)

    von Grumbckow, Lena; Elsner, Peter; Hellsten, Ylva;

    1999-01-01

    Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The initial rate of decrease in intracellular pH (pHi) upon lactate or pyruvate incubation was used...

  19. 锌对铅染毒新生大鼠成骨细胞增殖分化的影响%Combined Effects of Lead and Zinc on Proliferation and Differentiation of osteoblasts in Newborn Rats

    Institute of Scientific and Technical Information of China (English)

    刘国军; 阎春生; 姚海艳; 他维玮; 魏万倩

    2012-01-01

    目的:探讨铅锌联合染毒对乳鼠颅骨成骨细胞增殖分化的影响.方法:分离并培养原代成骨细胞,加入不同浓度铅、锌培养48h,检测其对成骨细胞增殖的作用;用碱性磷酸酶试剂盒检测ALP活力.结果:在染铅48h后,当铅浓度≥10pμmol/L时,细胞增殖功能下降(P<0.05);加锌干预48h后,铅+锌组细胞增殖功能均高于各自单独染铅组,其中铅(1μmol/L、10 μmol/L)+锌(50μmol/L)组、铅(10)+锌(100)组与对照组间的差异具有统计学意义(P<0.05).铅干预48h后,100μ mol/L铅组的ALP活力显著下(P<0.05),给予锌干预的铅锌联合染毒组,各组ALP活力均有增加,其中铅(1μmol/L、10μmol/L)+锌(50μ mol/L)组ALP活力均高于对照组,而铅(100μmol/L)+锌(50μmol/L)组ALP活力低于对照组,差异均有统计学意义(P<0.05).结论:铅对成骨细胞有毒性作用,影响其增殖和分化功能;50μmol/L锌在一定程度上可以拮抗铅对成骨细胞增殖和分化功能的损伤,且对ALP活力的作用更显著,为铅中毒骨病的防治提供一定的科学依据.%To investigate the effects of lead and zinc on proliferation and differentiation of osteoblasts in newborn rats. Methods: OsteobLasts were isolated from the calvaria of newborn rats and cultured in α-MEM medium. Lead and Zinc acetate with different concentration was administrated and cultivated for 48h.The proliferation response to lead and zinc was determined by MTT methods. The activity of alkaline phosphatase (ALP) was measured by alkaline phosphatase kit. Results: Treated with different levels of lead (10 μmol/L and 100 μmol/L) for 48h, the cell proliferation decreased(P<0.05); Treated with zinc and lead for 48h, cell proliferation was higher than their single lead-treated group; differences were of statistical significance (P<0.05) between the lead (1 μmol/L) plus zinc (50μmol/L), lead(10μmol/L) plus zinc (50μmol/L), lead(10μmol/L) plus zinc (100μmol/L) and control

  20. 2-Dimensional MEMS dielectrophoresis device for osteoblast cell stimulation.

    Science.gov (United States)

    Zou, H; Mellon, S; Syms, R R A; Tanner, K E

    2006-12-01

    A fixed microelectrode device for cell stimulation has been designed and fabricated using micro-electro-mechanical systems (MEMS) technology. Dielectrophoretic forces obtained from non-uniform electric fields were used for manipulating and positioning osteoblasts. The experiments show that the osteoblasts experience positive dielectrophoresis (p-DEP) when suspended in iso-osmotic culture medium and exposed to AC fields at 5 MHz frequency. Negative dielectrophoresis (n-DEP) is obtained at 0.1 MHz. The viability of osteoblasts under dielectrophoresis has been investigated. The viability values for cells exposed to DEP are nearly three times higher than the control values, indicating that dielectrophoresis may have an anabolic effect on osteoblasts.

  1. Rat embryonic palatal shelves respond to TCDD in organ culture

    Energy Technology Data Exchange (ETDEWEB)

    Abbott, B.D.; Birnbaum, L.S. (National Institute of Environmental Health Sciences, Research Triangle Park, NC (USA))

    1990-05-01

    TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental contaminant, is teratogenic in mice, inducing cleft palate (CP) and hydronephrosis at doses which are not overtly maternally or embryo toxic. Palatal shelves of embryonic mice respond to TCDD, both in vivo and in organ culture, with altered differentiation of medial epithelial cells. By contrast, in the rat TCDD produces substantial maternal, embryonic, and fetal toxicity, including fetal lethality, with few malformations. In this study the possible effects of maternal toxicity on induction of cleft palate were eliminated by exposure of embryonic rat palatal shelves in organ culture. The shelves were examined for specific TCDD-induced alterations in differentiation of the medial cells. On Gestation Day (GD) 14 or 15 palatal shelves from embryonic F344 rats were placed in organ culture for 2 to 3 days (IMEM:F12 medium, 5% FBS, 0.1% DMSO) containing 0, 1 x 10(-8), 1 x 10(-9), 1 x 10(-10), or 5 x 10(-11) M TCDD. The medial epithelial peridermal cells degenerated on shelves exposed to control media or 5 x 10(-11) M TCDD. Exposure to 10(-10), 10(-9), and 10(-8) M TCDD inhibited this degeneration in 20, 36, and 60% of the shelves, respectively, and was statistically significant at the two highest doses. A normally occurring decrease in (3H)TdR incorporation was inhibited in some GD 15 shelves cultured with 10(-10) and 10(-9) M TCDD. The medial cells of TCDD-exposed shelves continued to express high levels of immunohistochemically detected EGF receptors. The altered differentiation of rat medial epithelium is similar to that reported for TCDD-exposed mouse medial cells in vivo and in vitro. However, in order to obtain these responses, the cultured rat shelves require much higher concentrations of TCDD than the mouse shelves.

  2. Primary cortical brain cells influence osteoblast activity.

    Science.gov (United States)

    Anissian, Lucas; Kirby, Michael; Stark, André

    2009-12-18

    The presence of neuropeptides and neuroreceptors in the bone have been reported in several studies. Bone turn-over seems to be controlled by the nervous system. The actual pathway or the control mechanism is still under investigation. In this study we investigate the changes in osteoblast cells if they are in co-culture with primary cortical brain cells. After seven days in co-culture with the primary fetal brain cells the osteoblast cells exhibited hypertrophic morphological changes and showed stronger ALP activity.

  3. Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro

    Institute of Scientific and Technical Information of China (English)

    Jie-li LI; Lin ZHAO; Bin CUI; Lian-fu DENG; Guang NING; Jian-min LIU

    2011-01-01

    Aim: Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro.Methods: Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression.The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alkaloid phosphotase (ALP) was assessed using a commercial ALP staining kit.Results: Microarray analyses revealed that 10 genes were up-regulated by NMDA (0.5 mmol/L) and down-regulated by MK801 (100μmol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 μmol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 μmol/L), the PKA inhibitor H-89 (20 nmol/L), or the PI3K inhibitor wortmannin (100 nmol/L) blocked the ALP activity increase caused by NMDA (0.5 mmol/L). Furthermore, NMDA (0.5 mmol/L) rapidly increased PI3K phosphorylation, which could be blocked by pretreatment of wortmannin (100 nmol/L).Conclusion: The results suggest that activation of NMDA receptors stimulates osteoblasts differentiation through PKA, PKC, and PI3K signaling pathways, which is a new role for glutamate in regulating bone remodeling.

  4. Magnitude-dependent response of osteoblasts regulated by compressive stress

    Science.gov (United States)

    Shen, Xiao-qing; Geng, Yuan-ming; Liu, Ping; Huang, Xiang-yu; Li, Shu-yi; Liu, Chun-dong; Zhou, Zheng; Xu, Ping-ping

    2017-01-01

    The present study aimed to investigate the role of magnitude in adaptive response of osteoblasts exposed to compressive stress. Murine primary osteoblasts and MC3T3-E1 cells were exposed to compressive stress (0, 1, 2, 3, 4, and 5 g/cm2) in 3D culture. Cell viability was evaluated, and expression levels of Runx2, Alp, Ocn, Rankl, and Opg were examined. ALP activity in osteoblasts and TRAP activity in RAW264.7 cells co-cultured with MC3T3-E1 cells were assayed. Results showed that compressive stress within 5.0 g/cm2 did not influence cell viability. Both osteoblastic and osteoblast-regulated osteoclastic differentiation were enhanced at 2 g/cm2. An increase in stress above 2 g/cm2 did not enhance osteoblastic differentiation further but significantly inhibited osteoblast-regualted osteoclastic differentiation. This study suggested that compressive stress regulates osteoblastic and osteoclastic differentiation through osteoblasts in a magnitude-dependent manner. PMID:28317941

  5. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Milam, K.M.; Byard, J.L.

    1985-06-30

    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  6. Long-term organ culture of adult rat colon

    DEFF Research Database (Denmark)

    1978-01-01

    . The effect of in vivo carcinogen pretreatment was also studied. The explant culture from control untreated animals showed good epithelial differentiation with crypts until 6 weeks. In contrast, the explants from animals pretreated with 4 weekly doses of azoxymethane consistently showed epithelial......Colon explants from adult rats were maintained in culture for over 3 months in our laboratories with good epithelial preservation and cellular differentiation. The light and transmission electron microscopic features of rat colon mucosa during the culture period are described. In all the explants...... that remained viable, there was an initial phase of degeneration of the surface and crypt cells, later these areas were repopulated in one week, showing well-formed crypts, goblet cells, and ultrastructural features such as extensive lateral interdigitations, microvilli and glycocalyx--typical of colon...

  7. Acidosis inhibits mineralization in human osteoblasts.

    Science.gov (United States)

    Takeuchi, Shoko; Hirukawa, Koji; Togari, Akifumi

    2013-09-01

    Osteoblasts and osteoclasts maintain bone volume. Acidosis affects the function of these cells including mineral metabolism. We examined the effect of acidosis on the expression of transcription factors and mineralization in human osteoblasts in vitro. Human osteoblasts (SaM-1 cells) derived from the ulnar periosteum were cultured with α-MEM containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate (calcifying medium). Acidosis was induced by incubating the SaM-1 cells in 10 % CO₂ (pH approximately 7.0). Mineralization, which was augmented by the calcifying medium, was completely inhibited by acidosis. Acidosis depressed c-Jun mRNA and increased osteoprotegerin (OPG) production in a time-dependent manner. Depressing c-Jun mRNA expression using siRNA increased OPG production and inhibited mineralization. In addition, depressing OPG mRNA expression with siRNA enhanced mineralization in a dose-dependent manner. Acidosis or the OPG protein strongly inhibited mineralization in osteoblasts from neonatal mice. The present study was the first to demonstrate that acidosis inhibited mineralization, depressed c-Jun mRNA expression, and induced OPG production in human osteoblasts. These results suggest that OPG is involved in mineralization via c-Jun in human osteoblasts.

  8. Isolation and Primary Culture of Rat Hepatocytes Using Kiwifruit Actinidin

    Directory of Open Access Journals (Sweden)

    Z. Shirvani Farsani

    2007-07-01

    Full Text Available Introduction & Objective: Isolation of cells from different tissues rely on proteolytic enzymes mainly collagenases that selectively digest collagen fibers of extra-cellular matrix. It is important to find new and proper collagenases from plant sources. In the present research actinidin, a cysteine protease abundant in Kiwifruit, was used to isolate and culture of rat hepatocytes. Materials & Methods: Different concentrations of actinidin was used to isolate rat hepatocytes by one or two-step perfusion method. The viability of the separated cells was examined by the trypan blue test. The isolated rat hepatocytes were cultured on collagen coated plates in William´s E medium. The morphology of hepatocytes was examined microscopically after staining with the Papanicolaou method.Results: Actinidin in the concentration of 0.4 mg/ml in two-step perfusion method properly isolated hepatocytes from rat liver. The viability of isolated hepatocytes that successfully cultured in collagen coated plates was 90-95 percent.Conclusion: These results showed that actinidin is a proper protease for isolation of hepatocytes. In addition, purification of this enzyme is simpler than the collagenases.

  9. Type II Na+-Pi cotransporters in osteoblast mineral formation: regulation by inorganic phosphate.

    Science.gov (United States)

    Lundquist, Patrik; Murer, Heini; Biber, Jürg

    2007-01-01

    During calcification of bone, large amounts of phosphate (P(i)) must be transported from the circulation to the osteoid. Likely candidates for osteoblast P(i) transport are the type II sodium-phosphate cotransporters NaPi-IIa and NaPi-IIb that facilitate transcellular P(i) flux in kidney and intestine, respectively. We have therefore determined the 'cotransporters' expression in osteoblast-like cells. We have also studied the 'cotransporters' regulation by P(i) and during mineralization in vitro. Phosphate uptake and cotransporter protein expression was investigated at early, late and mineralizing culture stages of mouse (MC3T3-E1) and rat (UMR-106) osteoblast-like cells. Both NaPi-IIa and NaPi-IIb were expressed by both osteoblast-like cell lines. NaPi-IIa was upregulated in both cell lines one week after confluency. After 7 days in 3mM P(i) NaPi-IIa was strongly upregulated in both cell lines. NaPi-IIb expression was unaffected by both culture stage and P(i) supplementation. The expression of both cotransporters was unaffected by P(i) deprivation. In vitro mineralization at 1.5mM P(i) was preceded by a three-fold increase in osteoblast sodium-dependent P(i) uptake and a corresponding upregulation of both NaPi-IIa and NaPi-IIb. Their expression thus seem regulated by phosphate in a manner consistent with their playing a role in transcellular P(i) flux during mineralization.

  10. Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

    Directory of Open Access Journals (Sweden)

    Barrio Daniel A

    2001-08-01

    Full Text Available Abstract Background The tissue accumulation of protein-bound advanced glycation endproducts (AGE may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS and nitric oxide synthase (NOS expression on these AGE-collagen mediated effects. Results AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP activity. In preosteoblastic MC3T3E1 cells (24-hour culture, proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. Conclusions These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

  11. Periosteum Metabolism and Nerve Fiber Positioning Depend on Interactions between Osteoblasts and Peripheral Innervation in Rat Mandible.

    Directory of Open Access Journals (Sweden)

    Cédric Mauprivez

    Full Text Available The sympathetic nervous system controls bone remodeling by regulating bone formation and resorption. How nerves and bone cells influence each other remains elusive. Here we modulated the content or activity of the neuropeptide Vasoactive Intestinal Peptide to investigate nerve-bone cell interplays in the mandible periosteum by assessing factors involved in nerve and bone behaviors. Young adult rats were chemically sympathectomized or treated with Vasoactive Intestinal Peptide or Vasoactive Intestinal Peptide10-28, a receptor antagonist. Sympathectomy depleted the osteogenic layer of the periosteum in neurotrophic proNerve Growth Factor and neurorepulsive semaphorin3a; sensory Calcitonin-Gene Related Peptide-positive fibers invaded this layer physiologically devoid of sensory fibers. In the periosteum non-osteogenic layer, sympathectomy activated mast cells to release mature Nerve Growth Factor while Calcitonin-Gene Related Peptide-positive fibers increased. Vasoactive Intestinal Peptide treatment reversed sympathectomy effects. Treating intact animals with Vasoactive Intestinal Peptide increased proNerve Growth Factor expression and stabilized mast cells. Vasoactive Intestinal Peptide10-28 treatment mimicked sympathectomy effects. Our data suggest that sympathetic Vasoactive Intestinal Peptide modulate the interactions between nervous fibers and bone cells by tuning expressions by osteogenic cells of factors responsible for mandible periosteum maintenance while osteogenic cells keep nervous fibers at a distance from the bone surface.

  12. Periosteum Metabolism and Nerve Fiber Positioning Depend on Interactions between Osteoblasts and Peripheral Innervation in Rat Mandible.

    Science.gov (United States)

    Mauprivez, Cédric; Bataille, Caroline; Baroukh, Brigitte; Llorens, Annie; Lesieur, Julie; Marie, Pierre J; Saffar, Jean-Louis; Biosse Duplan, Martin; Cherruau, Marc

    2015-01-01

    The sympathetic nervous system controls bone remodeling by regulating bone formation and resorption. How nerves and bone cells influence each other remains elusive. Here we modulated the content or activity of the neuropeptide Vasoactive Intestinal Peptide to investigate nerve-bone cell interplays in the mandible periosteum by assessing factors involved in nerve and bone behaviors. Young adult rats were chemically sympathectomized or treated with Vasoactive Intestinal Peptide or Vasoactive Intestinal Peptide10-28, a receptor antagonist. Sympathectomy depleted the osteogenic layer of the periosteum in neurotrophic proNerve Growth Factor and neurorepulsive semaphorin3a; sensory Calcitonin-Gene Related Peptide-positive fibers invaded this layer physiologically devoid of sensory fibers. In the periosteum non-osteogenic layer, sympathectomy activated mast cells to release mature Nerve Growth Factor while Calcitonin-Gene Related Peptide-positive fibers increased. Vasoactive Intestinal Peptide treatment reversed sympathectomy effects. Treating intact animals with Vasoactive Intestinal Peptide increased proNerve Growth Factor expression and stabilized mast cells. Vasoactive Intestinal Peptide10-28 treatment mimicked sympathectomy effects. Our data suggest that sympathetic Vasoactive Intestinal Peptide modulate the interactions between nervous fibers and bone cells by tuning expressions by osteogenic cells of factors responsible for mandible periosteum maintenance while osteogenic cells keep nervous fibers at a distance from the bone surface.

  13. Toxicity of iron oxide nanoparticles against osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Shi Sifeng [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China); Jia Jingfu [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Guo Xiaokui [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Zhao Yaping [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Liu Boyu [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Chen Desheng; Guo Yongyuan; Zhang Xianlong, E-mail: zhangxianlong20101@163.com [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China)

    2012-09-15

    Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. Here, we conducted an in vitro study to examine the interaction of magnetic iron oxide nanoparticles with human osteoblasts to evaluate the dose-related toxicity of the nanoparticles on osteoblasts. A transmission electron microscope was used to visualise the internalised magnetic nanoparticles in osteoblasts. The CCK-8 results revealed increased cell viability (107.5 % vitality compared with the control group) when co-cultured at a low concentration (20 {mu}g/mL) and decreased cell viability (59.5 % vitality in a concentration of 300 {mu}g/mL and 25.9 % in 500 {mu}g/mL) when co-cultured in high concentrations. The flow cytometric detection revealed similar results with 5.48 % of apoptosis in a concentration of 20 {mu}g/mL, 23.40 % of apoptosis in a concentration of 300 {mu}g/mL and 28.49 % in a concentration of 500 {mu}g/mL. The disrupted cytoskeleton of osteoblasts was also revealed using a laser scanning confocal microscope. We concluded that use of a low concentration of magnetic iron oxide nanoparticles is important to avoid damage to osteoblasts.

  14. Triptolide upregulates NGF synthesis in rat astrocyte cultures.

    Science.gov (United States)

    Xue, Bing; Jiao, Jian; Zhang, Lei; Li, Kai-Rong; Gong, Yun-Tao; Xie, Jun-Xia; Wang, Xiao-Min

    2007-07-01

    Triptolide (T10), an extract from the traditional Chinese herb, Tripterygium wilfordii Hook F (TWHF), has been shown to attenuate the rotational behavior induced by D: -amphetamine and prevent the loss of dopaminergic neurons in the substantia nigra in rat models of Parkinson's disease. To examine if the neuroprotective effect is mediated by its stimulation of production of neurotrophic factors from astrocytes, we investigated the effect of T10 on synthesis and release of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in rat astrocyte cultures. T10 did not affect the synthesis and release of either BDNF or GDNF. However, it significantly increased NGF mRNA expression. It also increased both intracellular NGF and NGF level in culture medium. These results indicate that the neuroprotective effect of T10 might be mediated, at least in part, via a stimulation of the production and release of NGF in astrocytes.

  15. Proteomic analysis of indium embryotoxicity in cultured postimplantation rat embryos.

    Science.gov (United States)

    Usami, Makoto; Nakajima, Mikio; Mitsunaga, Katsuyoshi; Miyajima, Atsuko; Sunouchi, Momoko; Doi, Osamu

    2009-12-01

    Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24h in the presence of 50 microM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

  16. Elevation of extracellular Ca2+ induces store-operated calcium entry via calcium-sensing receptors: a pathway contributes to the proliferation of osteoblasts.

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    Fen Hu

    Full Text Available AIMS: The local concentration of extracellular Ca(2+ ([Ca(2+]o in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca(2+]o induced store-operated calcium entry (SOCE in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca(2+]o to osteoblastic proliferation. METHODS: Cytosolic Ca(2+ concentration ([Ca(2+]c of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR as well as inhibitors of phospholipase C (PLC, SOCE and voltage-gated calcium (Cav channels were applied to study the mechanism in detail. RESULTS: Our data showed that elevating [Ca(2+]o evoked a sustained increase of [Ca(2+]c in a dose-dependent manner. This [Ca(2+]c increase was blocked by TMB-8 (Ca(2+ release inhibitor, 2-APB and BTP-2 (both SOCE blockers, respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist or U73122 (a PLC inhibitor strongly reduced the [Ca(2+]o-induced [Ca(2+]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca(2+]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca(2+]o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator, 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists. CONCLUSIONS: Elevating [Ca(2+]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca(2+ concentration.

  17. Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes

    DEFF Research Database (Denmark)

    Karsdal, M A; Levin Andersen, Thomas; Bonewald, L;

    2004-01-01

    , and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation...... in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival...... of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from...

  18. Trimethyltin (TMT) neurotoxicity in organotypic rat hippocampal slice cultures

    DEFF Research Database (Denmark)

    Noraberg, J; Gramsbergen, J B; Fonnum, F

    1998-01-01

    The neurotoxic effects of trimethyltin (TMT) on the hippocampus have been extensively studied in vivo. In this study, we examined whether the toxicity of TMT to hippocampal neurons could be reproduced in organotypic brain slice cultures in order to test the potential of this model for neurotoxico......The neurotoxic effects of trimethyltin (TMT) on the hippocampus have been extensively studied in vivo. In this study, we examined whether the toxicity of TMT to hippocampal neurons could be reproduced in organotypic brain slice cultures in order to test the potential of this model...... for neurotoxicological studies, including further studies of neurotoxic mechanisms of TMT. Four-week-old cultures, derived from 7-day-old donor rats and grown in serum-free medium, were exposed to TMT (0.5-100 microM) for 24 h followed by 24 h in normal medium. TMT-induced neurodegeneration was then monitored by (a...... of TMT neurotoxicity....

  19. The effect of plasma-nitrided titanium surfaces on osteoblastic cell adhesion, proliferation, and differentiation.

    Science.gov (United States)

    Ferraz, Emanuela P; Sa, Juliana C; de Oliveira, Paulo T; Alves, Clodomiro; Beloti, Marcio M; Rosa, Adalberto L

    2014-04-01

    In this study, we evaluated the effect of new plasma-nitrided Ti surfaces on the progression of osteoblast cultures, including cell adhesion, proliferation and differentiation. Ti surfaces were treated using two plasma-nitriding protocols, hollow cathode for 3 h (HC 3 h) and 1 h (HC 1 h) and planar for 1 h. Untreated Ti surfaces were used as control. Cells derived from human alveolar and rat calvarial bones were cultured on Ti surfaces for periods of up to 14 days and the following parameters were evaluated: cell morphology, adhesion, spreading and proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, and gene expression of key osteoblast markers. Plasma-nitriding treatments resulted in Ti surfaces with distinct physicochemical characteristics. The cell adhesion and ALP activity were higher on plasma-nitrided Ti surfaces compared with untreated one, whereas cell proliferation and extracellular matrix mineralization were not affected by the treatments. In addition, the plasma-nitrided Ti surfaces increased the ALP, reduced the osteocalcin and did not affect the Runx2 gene expression. We have shown that HC 3 h and planar Ti surfaces slightly favored the osteoblast differentiation process, and then these surfaces should be considered for further investigation using preclinical models. Copyright © 2013 Wiley Periodicals, Inc.

  20. Transformation of adult rat cardiac myocytes in primary culture.

    Science.gov (United States)

    Banyasz, Tamas; Lozinskiy, Ilya; Payne, Charles E; Edelmann, Stephanie; Norton, Byron; Chen, Biyi; Chen-Izu, Ye; Izu, Leighton T; Balke, C William

    2008-03-01

    We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

  1. Cholesterol induces fetal rat enterocyte death in culture

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    Gazzola J.

    2004-01-01

    Full Text Available The effect of cholesterol on fetal rat enterocytes and IEC-6 cells (line originated from normal rat small intestine was examined. Both cells were cultured in the presence of 20 to 80 µM cholesterol for up to 72 h. Apoptosis was determined by flow cytometric analysis and fluorescence microscopy. The expression of HMG-CoA reductase and peroxisome proliferator-activated receptor gamma (PPARgamma was measured by RT-PCR. The addition of 20 µM cholesterol reduced enterocyte proliferation as early as 6 h of culture. Reduction of enterocyte proliferation by 28 and 41% was observed after 24 h of culture in the presence and absence of 10% fetal calf serum, respectively, with the effect lasting up to 72 h. Treatment of IEC-6 cells with cholesterol for 24 h raised the proportion of cells with fragmented DNA by 9.7% at 40 µM and by 20.8% at 80 µM. When the culture period was extended to 48 h, the effect of cholesterol was still more pronounced, with the percent of cells with fragmented DNA reaching 53.5% for 40 µM and 84.3% for 80 µM. Chromatin condensation of IEC-6 cells was observed after treatment with cholesterol even at 20 µM. Cholesterol did not affect HMG-CoA reductase expression. A dose-dependent increase in PPARgamma expression in fetal rat enterocytes was observed. The expression of PPAR-gamma was raised by 7- and 40-fold, in the presence and absence of fetal calf serum, respectively, with cholesterol at 80 mM. The apoptotic effect of cholesterol on enterocytes was possibly due to an increase in PPARgamma expression.

  2. Isolating highly pure rat spermatogonial stem cells in culture.

    Science.gov (United States)

    Hamra, F Kent; Chapman, Karen M; Wu, Zhuoru; Garbers, David L

    2008-01-01

    Methods are detailed for isolating highly pure populations of spermatogonial stem cells from primary cultures of testis cells prepared from 22- to 24-day-old rats. The procedure is based on the principle that testicular somatic cells bind tightly to plastic and collagen matrices when cultured in serum-containing medium, whereas spermatogonia and spermatocytes do not bind to plastic or collagen when cultured in serum-containing medium. The collagen-non-binding testis cells obtained using these procedures are thus approx. 97% pure spermatogenic cells. Stem spermatogonia are then easily isolated from the purified spermatogenic population during a short incubation step in culture on laminin matrix. The spermatogenic cells that bind to laminin are more than 90% undifferentiated, type A spermatogonia and are greatly enriched in genetically modifiable stem cells that can develop into functional spermatozoa. This method does not require flow cytometry and can also be applied to obtain enriched cultures of mouse spermatogonial stem cells. The isolated spermatogonia provide a highly potent and effective source of stem cells that have been used to initiate in vitro and in vivo culture studies on spermatogenesis.

  3. A comparison of the cytotoxicity and proinflammatory cytokine production of EndoSequence root repair material and ProRoot mineral trioxide aggregate in human osteoblast cell culture using reverse-transcriptase polymerase chain reaction.

    Science.gov (United States)

    Ciasca, Maria; Aminoshariae, Anita; Jin, Ge; Montagnese, Thomas; Mickel, Andre

    2012-04-01

    The purpose of this study was to compare the cytotoxicity and cytokine expression profiles of EndoSequence Root Repair Material (ERRM; Brasseler, Savannah, GA) putty, ERRM flowable, and ProRoot mineral trioxide aggregate (MTA; Dentsply Tulsa Dental, Johnson City, TN) using osteoblast cells (MG-63). Four millimeters in diameter of each material was placed in the center of a 6-well culture plate, and a 2-mL suspension (10(5) cells/mL) of human osteoblasts was seeded in each well. Photomicrograph images were used to evaluate cytotoxicity as evidenced by the lack of osteoblast cell growth in relation to the materials with AH-26 (Dentsply Tulsa Dental) as the positive control. In addition, reverse-transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the expression of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Cytokine expression of MG-63 cells upon lipopolysaccharide treatment was used as controls. RT-PCR results were normalized by the expression of the housekeeping gene β-actin and were used to measure cytokine expression. Statistical analysis was performed using analysis of variance. Results showed that ERRM putty and MTA exhibited minimal levels of cytotoxicity; however, ERRM was slightly more cytotoxic although not statistically significant. The expression of IL-1β, IL-6, and IL-8 was detected in all samples with minimal TNF-α expression. We concluded that ERRM and MTA showed similar cytotoxicity and cytokine expressions. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  4. Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method.

    Science.gov (United States)

    Liu, Di; Liang, Xiao-Chun; Zhang, Hong

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(Pculture method group,and (74.50±4.23)% in the explants-culture method group(Pculture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

  5. Mycelial culture of Phellinus linteus protects primary cultured rat hepatocytes against hepatotoxins.

    Science.gov (United States)

    Kim, S H; Lee, H S; Lee, S; Cho, J; Ze, K; Sung, J; Kim, Y C

    2004-12-01

    Hepatoprotective activity of Phellinus linteus was studied using H(2)O(2)- or galactosamine-injured primary cultures of rat hepatocytes as screening systems. The methanolic extract of the mycelial culture of Phellinus linteus significantly protected against hepatotoxins-induced toxicity in primary cultured rat hepatocytes as seen from the decreased level of glutamic pyruvic transaminase released from the injured hepatocytes. The methanolic extract of the mycelial culture of Phellinus linteus was subsequently fractionated with n-hexane, ethyl acetate, n-butanol and water. Among these fractions, 100 microg/mL of the ethyl acetate fraction was the most active one. The relative protections were 68.9 +/- 5.3% in H(2)O(2)-injured hepatocytes and 46.8 +/- 3.9% in galactosamine-injured hepatocytes, respectively. The ethyl acetate fraction appeared to maintain the glutathione level which was decreased by the treatment of H(2)O(2) or galactosamine and restored the level of RNA synthesis more than two times compared to galactosamine-injured hepatocytes. These results suggest that the ethyl acetate fraction of the mycelial culture of Phellinus linteus protects hepatocytes from H(2)O(2)- or galactosamine-induced injury by maintaining hepatic glutathione level and RNA synthesis as well.

  6. Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

    Science.gov (United States)

    Parra, M.; Vercoutere, W.; Roden, C.; Banerjee, I.; Krauser, W.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense

  7. A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

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    Sui-Yi Xu

    2012-01-01

    Full Text Available The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2 onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV; cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

  8. Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function▿

    Science.gov (United States)

    Wang, Yu-Hsiung; Jiang, Jin; Zhu, Qiang; AlAnezi, Amer Z.; Clark, Robert B.; Jiang, Xi; Rowe, David W.; Nichols, Frank C.

    2010-01-01

    Porphyromonas gingivalis produces unusual sphingolipids that are known to promote inflammatory reactions in gingival fibroblasts and Toll-like receptor 2 (TLR2)-dependent secretion of interleukin-6 from dendritic cells. The aim of the present study was to examine whether P. gingivalis lipids inhibit osteoblastic function. Total lipids from P. gingivalis and two fractions, phosphoglycerol dihydroceramides and phosphoethanolamine dihydroceramides, were prepared free of lipid A. Primary calvarial osteoblast cultures derived from 5- to 7-day-old CD-1 mice were used to examine the effects of P. gingivalis lipids on mineralized nodule formation, cell viability, apoptosis, cell proliferation, and gene expression. P. gingivalis lipids inhibited osteoblast differentiation and fluorescence expression of pOBCol2.3GFP in a concentration-dependent manner. However, P. gingivalis lipids did not significantly alter osteoblast proliferation, viability, or apoptosis. When administered during specific intervals of osteoblast growth, P. gingivalis total lipids demonstrated inhibitory effects on osteoblast differentiation only after the proliferation stage of culture. Reverse transcription-PCR confirmed the downregulation of osteoblast marker genes, including Runx2, ALP, OC, BSP, OPG, and DMP-1, with concurrent upregulation of RANKL, tumor necrosis factor alpha, and MMP-3 genes. P. gingivalis total lipids and lipid fractions inhibited calvarial osteoblast gene expression and function in vivo, as determined by the loss of expression of another osteoblast differentiation reporter, pOBCol3.6GFPcyan, and reduced uptake of Alizarin complexone stain. Finally, lipid inhibition of mineral nodule formation in vitro was dependent on TLR2 expression. Our results indicate that inhibition of osteoblast function and gene expression by P. gingivalis lipids represents a novel mechanism for altering alveolar bone homeostasis at periodontal disease sites. PMID:20584977

  9. Influence of rat substrain and growth conditions on the characteristics of primary cultures of adult rat spinal cord astrocytes.

    Science.gov (United States)

    Codeluppi, Simone; Gregory, Ebba Norsted; Kjell, Jacob; Wigerblad, Gustaf; Olson, Lars; Svensson, Camilla I

    2011-04-15

    Primary astrocyte cell cultures have become a valuable tool for studies of signaling pathways that regulate astrocyte physiology, reactivity, and function; however, differences in culture preparation affect data reproducibility. The aim of this work was to define optimal conditions for obtaining primary astrocytes from adult rat spinal cord with an expression profile most similar to adult human spinal cord astrocytes. Hence, we examined whether different Sprague-Dawley substrains and culture conditions affect astrocyte culture quality. Medium supplemented with fetal bovine serum from three sources (Sigma, Gibco, Hyclone) or a medium with defined composition (AM medium) was used to culture astrocytes isolated from spinal cords of adult Harlan and Charles River Spraque-Dawley rats. Purity was significantly different between cultures established in media with different sera. No microglia were detected in AM or Hyclone cultures. Gene expression was also affected, with AM cultures expressing the highest level of glutamine synthetase, connexin-43, and glutamate transporter-1. Interestingly, cell response to starvation was substrain dependent. Charles River-derived cultures responded the least, while astrocytes derived from Harlan rats showed a greater decrease in Gfap and glutamine synthetase, suggesting a more quiescent phenotype. Human and Harlan astrocytes cultured in AM media responded similarly to starvation. Taken together, this study shows that rat substrain and growth medium composition affect purity, expression profile and response to starvation of primary astrocytes suggesting that cultures of Harlan rats in AM media have optimal astrocyte characteristics, purity, and similarity to human astrocytes.

  10. Biomimetic Multispiked Connecting Ti-Alloy Scaffold Prototype for Entirely-Cementless Resurfacing Arthroplasty Endoprostheses—Exemplary Results of Implantation of the Ca-P Surface-Modified Scaffold Prototypes in Animal Model and Osteoblast Culture Evaluation

    Directory of Open Access Journals (Sweden)

    Ryszard Uklejewski

    2016-06-01

    Full Text Available We present here—designed, manufactured, and tested by our research team—the Ti-alloy prototype of the multispiked connecting scaffold (MSC-Scaffold interfacing the components of resurfacing arthroplasty (RA endoprostheses with bone. The spikes of the MSC-Scaffold prototype mimic the interdigitations of the articular subchondral bone, which is the natural biostructure interfacing the articular cartilage with the periarticular trabecular bone. To enhance the osteoinduction/osteointegration potential of the MSC-Scaffold, the attempts to modify its bone contacting surfaces by the process of electrochemical cathodic deposition of Ca-P was performed with further immersion of the MSC-Scaffold prototypes in SBF in order to transform the amorphous calcium-phosphate coating in hydroxyapatite-like (HA-like coating. The pilot experimental study of biointegration of unmodified and Ca-P surface-modified MSC-Scaffold prototypes was conducted in an animal model (swine and in osteoblast cell culture. On the basis of a microscope-histological method the biointegration was proven by the presence of trabeculae in the interspike spaces of the MSC-Scaffold prototype on longitudinal and cross-sections of bone-implant specimens. The percentage of trabeculae in the area between the spikes of specimen containing Ca-P surface modified scaffold prototype observed in microCT reconstructions of the explanted joints was visibly higher than in the case of unmodified MSC-Scaffold prototypes. Significantly higher Alkaline Phosphatase (ALP activity and the cellular proliferation in the case of Ca-P-modified MSC-Scaffold pre-prototypes, in comparison with unmodified pre-prototypes, was found in osteoblast cell cultures. The obtained results of experimental implantation in an animal model and osteoblast cell culture evaluations of Ca-P surface-modified and non-modified biomimetic MSC-Scaffold prototypes for biomimetic entirely-cementless RA endoprostheses indicate the

  11. FGF Suppresses Poldip2 Expression in Osteoblasts.

    Science.gov (United States)

    Katsumura, Sakie; Izu, Yayoi; Yamada, Takayuki; Griendling, Kathy; Harada, Kiyoshi; Noda, Masaki; Ezura, Yoichi

    2016-12-05

    Osteoporosis is one of the most prevalent ageing-associated diseases that are soaring in the modern world. Although various aspects of the disease have been investigated to understand the bases of osteoporosis, the pathophysiological mechanisms underlying bone loss is still incompletely understood. Poldip2 is a molecule that has been shown to be involved in cell migration of vascular cells and angiogenesis. However, expression of Poldip2 and its regulation in bone cells were not known. Therefore, we examined the Poldip2 mRNA expression and the effects of bone regulators on the Poldip2 expression in osteoblasts. We found that Poldip2 mRNA is expressed in osteoblastic MC3T3-E1 cells. As FGF controls osteoblasts and angiogenesis, FGF regulation was investigated in these cells. FGF suppressed the expression of Poldip2 in MC3T3-E1 cells in a time dependent manner. Protein synthesis inhibitor but not transcription inhibitor reduced the FGF effects on Poldip2 gene expression in MC3T3-E1 cells. As for bone-related hormones, dexamethasone was found to enhance the expression of Poldip2 in osteoblastic MC3T3-E1 cells whereas FGF still suppressed such dexamethasone effects. With respect to function, knockdown of Poldip2 by siRNA suppressed the migration of MC3T3-E1 cells. Poldip2 was also expressed in the primary cultures of osteoblast-enriched cells and FGF also suppressed its expression. Finally, Poldip2 was expressed in femoral bone in vivo and its levels were increased in aged mice compared to young adult mice. These data indicate that Poldip2 is expressed in osteoblastic cells and is one of the targets of FGF. J. Cell. Biochem. 9999: 1-8, 2017. © 2016 Wiley Periodicals, Inc.

  12. Rhynchophylline Protects Cultured Rat Neurons against Methamphetamine Cytotoxicity

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    Dan Dan Xu

    2012-01-01

    Full Text Available Rhynchophylline (Rhy is an active component isolated from species of the genus Uncaria which has been used for the treatment of ailments to the central nervous system in traditional Chinese medicine. Besides acting as a calcium channel blocker, Rhy was also reported to be able to protect against glutamate-induced neuronal death. We thus hypothesize that Rhy may have neuroprotective activity against methamphetamine (MA. The primary neurons were cultured directly from the cerebral cortex of neonatal rats, acting as in vitro model in the present study. The neurotoxicity of MA and the protective effect of Rhy were evaluated by MTT assay. The effects of MA, Rhy or their combination on intracellular free calcium concentration ([Ca2+]i were determined in individual neocortical neurons by the Fluo-3/AM tracing method. The MTT assay demonstrated that MA has a dose-dependent neurotoxicity in neuronal cultures. The addition of Rhy prior to the exposure to MA prevented neuronal death. Time course studies with the Fluo-3/AM probe showed that Rhy significantly decreased neuronal [Ca2+]i which was elevated by the exposure to MA. Our results suggested that Rhy can protect the neuronal cultures against MA exposure and promptly attenuate intracellular calcium overload triggered by MA challenge. This is the first report demonstrating an inhibitory effect of Rhy against MA impairment in cultured neurons in vitro.

  13. [Metabolic characterization of rat sertoli cell in vitro culture].

    Science.gov (United States)

    Shi, Bingyang; Zhang, Shuxiang; Guo, Meijin; Wang, Yonghong; Zhang, Siliang; Shi, Xiaolin

    2009-05-01

    Sertoli cell (SC) is intrinsic to the testis and provides an appropriate growth environment for the germ cells. It was separated from rat's testis and identified by hematoxylin and eosin staining(HE) and immunocytochemical reaction, then cultivated in vitro. Culture conditions such as pH, osmotic pressure and metabolic parameters that include consumption rates of glucose, glutamine, amino acids and formation rates of lactic acid, ammonium ion were investigated. It was showed that adhesion process of SCs was accomplished within 2-4 hours after inoculation. It was also observed that the SCs entered into the decline phase when the concentration of ammonium ion and lactic acid were above 2.3 mmol/L and 14 mmol/L, respectively, which caused osmotic pressure above 326 mosm/kg and pH below 6.8 in the medium. As the changes of amino acids during culture were concerned, Glu and Ala accumulated rapidly, while Val, Leu, Ile reduced slightly and at the same time Ser, Arg, and Gly were stable. The restrict factors for SCs grown in static culture might be high osmotic pressure and low pH, which were generated when glutamine and glucose were metabolized into lactic acid. The findings could be fundamental in the process optimization of large scale Sertoli cells in vitro culture.

  14. Characterization of glucose uptake by cultured rat podocytes.

    Science.gov (United States)

    Lewko, Barbara; Bryl, Ewa; Witkowski, Jacek M; Latawiec, Elzbieta; Gołos, Magdalena; Endlich, Nicole; Hähnel, Brunhilde; Koksch, Claudia; Angielski, Stefan; Kriz, Wilhelm; Stepinski, Jan

    2005-01-01

    The nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose ((3)H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of (3)H-2DG was linear up to 20 min and was inhibited by cytochalasin B (80% inhibition) and by phlorizin (20% inhibition). Pretreatment with insulin stimulated the (3)H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a V(max) value of approximately 10 mM and S(0.5)of 7.8 mM. The value h = 1.0 for a Hill coefficient confirmed that glucose uptake exhibited a Michaelis-Menten kinetics. Transporters GLUT2 and GLUT4 were expressed in over 90% podocytes. Of the GLUT2- and GLUT4-expressing cells, approximately one-fourth expressed the membrane-bound fraction. We conclude that cultured rat podocytes possess a differentiated glucose transport system consisting chiefly of facilitative GLUT2 and GLUT4 transporters. It seems likely that a sodium-dependent glucose cotransporter may also be present in these cells.

  15. Treatment with hydrogen molecule alleviates TNFα-induced cell injury in osteoblast.

    Science.gov (United States)

    Cai, Wen-Wen; Zhang, Ming-Hua; Yu, Yong-Sheng; Cai, Jin-Hua

    2013-01-01

    Tumor necrosis factor-alpha (TNFα) plays a crucial role in inflammatory diseases such as rheumatoid arthritis and postmenopausal osteoporosis. Recently, it has been demonstrated that hydrogen gas, known as a novel antioxidant, can exert therapeutic anti-inflammatory effect in many diseases. In this study, we investigated the effect of treatment with hydrogen molecule (H(2)) on TNFα-induced cell injury in osteoblast. The osteoblasts isolated from neonatal rat calvariae were cultured. It was found that TNFα suppressed cell viability, induced cell apoptosis, suppressed Runx2 mRNA expression, and inhibited alkaline phosphatase activity, which was reversed by co-incubation with H(2). Incubation with TNFα-enhanced intracellular reactive oxygen species (ROS) formation and malondialdehyde production increased NADPH oxidase activity, impaired mitochondrial function marked by increased mitochondrial ROS formation and decreased mitochondrial membrane potential and ATP synthesis, and suppressed activities of antioxidant enzymes including SOD and catalase, which were restored by co-incubation with H(2). Treatment with H(2) inhibited TNFα-induced activation of NFκB pathway. In addition, treatment with H(2) inhibited TNFα-induced nitric oxide (NO) formation through inhibiting iNOS activity. Treatment with H(2) inhibited TNFα-induced IL-6 and ICAM-1 mRNA expression. In conclusion, treatment with H(2) alleviates TNFα-induced cell injury in osteoblast through abating oxidative stress, preserving mitochondrial function, suppressing inflammation, and enhancing NO bioavailability.

  16. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    Science.gov (United States)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  17. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    Science.gov (United States)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  18. Computational tool for morphological analysis of cultured neonatal rat cardiomyocytes.

    Science.gov (United States)

    Leite, Maria Ruth C R; Cestari, Idágene A; Cestari, Ismar N

    2015-08-01

    This study describes the development and evaluation of a semiautomatic myocyte edge-detector using digital image processing. The algorithm was developed in Matlab 6.0 using the SDC Morphology Toolbox. Its conceptual basis is the mathematical morphology theory together with the watershed and Euclidean distance transformations. The algorithm enables the user to select cells within an image for automatic detection of their borders and calculation of their surface areas; these areas are determined by adding the pixels within each myocyte's boundaries. The algorithm was applied to images of cultured ventricular myocytes from neonatal rats. The edge-detector allowed the identification and quantification of morphometric alterations in cultured isolated myocytes induced by 72 hours of exposure to a hypertrophic agent (50 μM phenylephrine). There was a significant increase in the mean surface area of the phenylephrine-treated cells compared with the control cells (p<;0.05), corresponding to cellular hypertrophy of approximately 50%. In conclusion, this edge-detector provides a rapid, repeatable and accurate measurement of cell surface areas in a standardized manner. Other possible applications include morphologic measurement of other types of cultured cells and analysis of time-related morphometric changes in adult cardiac myocytes.

  19. Culturing primary human osteoblasts on electrospun poly(lactic-co-glycolic acid) and poly(lactic-co-glycolic acid)/nanohydroxyapatite scaffolds for bone tissue engineering.

    Science.gov (United States)

    Li, Mengmeng; Liu, Wenwen; Sun, Jiashu; Xianyu, Yunlei; Wang, Jidong; Zhang, Wei; Zheng, Wenfu; Huang, Deyong; Di, Shiyu; Long, Yun-Ze; Jiang, Xingyu

    2013-07-10

    In this work, we fabricated polymeric fibrous scaffolds for bone tissue engineering using primary human osteoblasts (HOB) as the model cell. By employing one simple approach, electrospinning, we produced poly(lactic-co-glycolic acid) (PLGA) scaffolds with different topographies including microspheres, beaded fibers, and uniform fibers, as well as the PLGA/nanohydroxyapatite (nano-HA) composite scaffold. The bone-bonding ability of electrospun scaffolds was investigated by using simulated body fluid (SBF) solution, and the nano-HA in PLGA/nano-HA composite scaffold can significantly enhance the formation of the bonelike apatites. Furthermore, we carried out in vitro experiments to test the performance of electrospun scaffolds by utilizing both mouse preosteoblast cell line (MC 3T3 E1) and HOB. Results including cell viability, alkaline phosphatase (ALP) activity, and osteocalcin concentration demonstrated that the PLGA/nano-HA fibers can promote the proliferation of HOB efficiently, indicating that it is a promising scaffold for human bone repair.

  20. PPAR agonists stimulate adipogenesis at the expense of osteoblast differentiation while inhibiting osteoclast formation and activity.

    Science.gov (United States)

    Patel, Jessal J; Butters, Oliver R; Arnett, Timothy R

    2014-06-01

    Drugs used in the treatment of type 2 diabetes and cardiovascular disease, specifically peroxisome proliferator-activated receptor (PPAR) agonists, have been reported to affect bone cell function and fracture risk. In this study, we assessed the direct effects of PPAR-γ agonists (rosiglitazone and troglitazone), used in the treatment of diabetes, and a PPAR-α agonist (fenofibrate), used to treat hyperlipidaemia, on the function of primary osteoblasts and osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by up to 85% in cultures treated with rosiglitazone and by 45% in troglitazone-treated or fenofibrate-treated cultures; at the same time, lipid droplet formation was increased by 40-70%. The expression of key osteogenic markers was similarly downregulated in cultures treated with PPAR agonists, whereas adipogenesis markers were upregulated. Formation of osteoclasts in cultures derived from mouse marrow diminished with fenofibrate treatment, whereas both glitazones reduced resorptive activity without affecting osteoclast number. Metformin, although not a PPAR agonist, is also commonly used in the treatment of type 2 diabetes. Here, metformin was found to have no effect on bone cell function. Taken together, these data suggest that PPAR-γ agonists may enhance bone loss via increased adipogenesis at the expense of osteoblast formation. In contrast, PPAR-α agonists may prevent bone loss. Given that the prevalence of diabetes and cardiovascular disease is expected to rise significantly, greater attention may need to be paid to the effects of PPAR agonists on bone homeostasis. Copyright © 2014 John Wiley & Sons, Ltd.

  1. Detection of histidine decarboxylase in rat aorta and cultured rat aortic smooth muscle cells.

    Science.gov (United States)

    Tippens, A S; Davis, S V; Hayes, J R; Bryda, E C; Green, T L; Gruetter, C A

    2004-08-01

    Having previously demonstrated release of histamine from mast-cell-deficient rat aorta, the objective of this study was to determine and localize histamine synthesis capability in the aorta by detecting histidine decarboxylase (HDC), the enzyme that catalyzes histamine formation. Experiments were conducted with nested reverse transcription-polymerase chain reaction (nRT-PCR) to detect HDC mRNA and with immunofluorescence and western blot analysis to detect HDC protein in rat aorta, cultured rat aortic smooth muscle (RASMC) and endothelial cells (RAEC). Gel electrophoresis of nRT-PCR products indicated HDC mRNA in liver, aorta and RASMC but not in RAEC or kidney. Sequence analysis confirmed that the band observed in RASMC was the target HDC amplicon. Immunofluorescence indicated the presence of HDC protein in RASMC and not in RAEC. Western Blot analysis revealed HDC protein (55 kDa) in liver, aorta, RASMC but not in RAEC or kidney. The results of this study are the first to demonstrate the presence of HDC mRNA and protein in rat aorta and more specifically in RASMC, indicative of their capability to synthesize histamine. Copyright 2004 Birkhäuser Verlag, Basel

  2. Effect of magnesium ion on human osteoblast activity

    Directory of Open Access Journals (Sweden)

    L.Y. He

    2016-01-01

    Full Text Available Magnesium, a promising biodegradable metal, has been reported in several studies to increase bone formation. Although there is some information regarding the concentrations of magnesium ions that affect bone remodeling at a cellular level, little is known about the effect of magnesium ions on cell gap junctions. Therefore, this study aimed to systematically investigate the effects of different concentrations of magnesium on bone cells, and further evaluate its effect on gap junctions of osteoblasts. Cultures of normal human osteoblasts were treated with magnesium ions at concentrations of 1, 2 and 3 mM, for 24, 48 and 72 h. The effects of magnesium ions on viability and function of normal human osteoblasts and on gap junction intercellular communication (GJIC in osteoblasts were investigated. Magnesium ions induced significant (P<0.05 increases in cell viability, alkaline phosphate activity and osteocalcin levels of human osteoblasts. These stimulatory actions were positively associated with the concentration of magnesium and the time of exposure. Furthermore, the GJIC of osteoblasts was significantly promoted by magnesium ions. In conclusion, this study demonstrated that magnesium ions induced the activity of osteoblasts by enhancing GJIC between cells, and influenced bone formation. These findings may contribute to a better understanding of the influence of magnesium on bone remodeling and to the advance of its application in clinical practice.

  3. HAIR CELL-LIKE CELL GENERATION INDUCED BY NATURE CULTURE OF ADULT RAT AUDITORY EPITHELIUM

    Institute of Scientific and Technical Information of China (English)

    Liu Hui; Zhu Hongliang; Li Shengli; Yao Xiaobao; Wang Xiaoxia

    2006-01-01

    Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.

  4. Effects of electrostimulation on glycogenolysis in cultured rat myotubes

    DEFF Research Database (Denmark)

    Elsner, Peter; Grunnet, Niels; Quistorff, Bjørn

    2003-01-01

    A model for electrostimulation causing contractions of primary cultures of rat myotubes was established. The kinetics of glycogen degradation was investigated for a 2-h period to elucidate the coupling between contraction and glycogenolytic flux. Electrostimulation caused contraction and increased...... glycogenolytic flux, but had no effect on glycogen phosphorylase-a activity. Forskolin increased glycogenolytic flux more than electrostimulation, and caused a fast activation of glycogen phosphorylase, while it did not elicit contraction. The effects of electrostimulation and forskolin on glycogenolytic flux...... were partly additive. The metabolism of glucose and glycogen was almost equally anaerobic and aerobic. The ATP content remained constant during glycogenolysis, but phosphocreatine decreased with the largest decrease in electrostimulated cells. The calculated ATP turnover rate increased about 3 times...

  5. A proteome map of primary cultured rat Schwann cells

    Directory of Open Access Journals (Sweden)

    Shen Mi

    2012-03-01

    Full Text Available Abstract Background Schwann cells (SCs are the principal glial cells of the peripheral nervous system with a wide range of biological functions. SCs play a key role in peripheral nerve regeneration and are involved in several hereditary peripheral neuropathies. The objective of this study was to gain new insight into the whole protein composition of SCs. Results Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS was performed to identify the protein expressions in primary cultured SCs of rats. We identified a total of 1,232 proteins, which were categorized into 20 functional classes. We also used quantitative real time RT-PCR and Western blot analysis to validate some of proteomics-identified proteins. Conclusion We showed for the first time the proteome map of SCs. Our data could serve as a reference library to provide basic information for understanding SC biology.

  6. [Kurloff's thymic inclusion : action on rat gonads in culture].

    Science.gov (United States)

    De Graeve, P; Vincent, M F; Amiel, S; Moatti, J P; Guilhem, A; Bimes, C

    1981-12-01

    Thymic and splenic extracts rich in FOA-KURLOFF (F.K.) body cells, obtained from guinea-pigs treated with oestrogen, were added to rat testis or ovaries in culture. Controls were prepared with extracts from thymus and spleen of non treated animals and from kidneys of treated or non treated animals. After five hours the level of sexual hormones and the germinal cells were studied. The F.K. substance has no effect on germinal cells and on progesterone and testosterone secretion. The F.K. substance induces a significative decrease of oestrogen secretion. In an other paper we established that F.K. bodies induced a hyperactivity of internal theca folliculi and of ovarian interstitial cells. It is a false image of activity in connection with a hypersecretion of FSH. The F.K. substance inhibits oestrogen synthesis.

  7. Effects of Fenvalerate on Steroidogenesis in Cultured Rat Granulosa Cells

    Institute of Scientific and Technical Information of China (English)

    JIAN-FENG CHEN; GUI-DONG DAI; XIN-RU WANG; HAI-YAN CHEN; RU LIU; JUN HE; LIN SONG; QIAN BIAN; LI-CHUN XU; JIAN-WEI ZHOU; HANG XIAO

    2005-01-01

    Objective This study was designed to examine the in vitro effects of fenvalerate on steroid production and steroidogenic enzymes mRNA expression level in rat granulosa cells. Methods Using primary cultured rat granulosa cells (rGCs) as model, fenvalerate of various concentrations (0, 1, 5, 25, 125, 625 μmol/L) was added to the medium for 24 h. In some cases, optimal concentrations of 22(R)-hydroxycholesterol (25 μmol/L), Follicle stimulating hormone (FSH, 2 mg/L), or 8-Bromo-cAMP (1 mmol/L) were provided. Concentrations of 17β-estradiol(E2) and progesterone (P4) in the medium from the same culture wells were measured by RIA and the steroidogenic enzyme mRNA level was quantified by semi-quantitative RT-PCR. Results Fenvalerate decreased both P4 and E2 production in a dose-dependent manner while it could significantly stimulate rGCs proliferation. This inhibition was stronger in the presence of FSH. Furthermore, it could not be reversed by 22(R)-hydroxycholesterol or 8-Bromo-cAMP. RT-PCR revealed that fenvalerate had no significant effect on 3β-HSD, but could increase the P450scc mRNA level. In addition, 17β-HSD mRNA level was dramatically reduced with the increase of fenvalerate dose after 24 h treatment. Conclusion Fenvalerate inhibits both P4 and E2 production in rGCs. These results support the view that fenvalerate is considered as a kind of endocrine-disrupting chemicals. The mechanism of its disruption may involve the effects on steroidogenesis signaling cascades and/or steroidogenic enzyme's activity.

  8. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R., E-mail: mundy.william@epa.gov

    2011-11-15

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  9. OSTEOBLAST ADHESION OF BREAST CANCER CELLS WITH SCANNING ACOUSTIC MICROSCOPY

    Energy Technology Data Exchange (ETDEWEB)

    Chiaki Miyasaka; Robyn R. Mercer; Andrea M. Mastro; Ken L. Telschow

    2005-03-01

    Breast cancer frequently metastasizes to the bone. Upon colonizing bone tissue, the cancer cells stimulate osteoclasts (cells that break bone down), resulting in large lesions in the bone. The breast cancer cells also affect osteoblasts (cells that build new bone). Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adherer in a different way to the substrate and to each other. To characterize cell adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days, and then assayed with a mechanical scanning acoustic reflection microscope (SAM). The SAM indicated that in normal medium the MC3T3-E1 osteoblasts were firmly attached to their plastic substrate. However, MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium displayed both an abnormal shape and poor adhesion at the substrate interface. The cells were fixed and stained to visualize cytoskeletal components using optical microscopic techniques. We were not able to observe these differences until the cells were quite confluent after 7 days of culture. However, using the SAM, we were able to detect these changes within 2 days of culture with MDA-MB-231 conditioned medium

  10. Ibuprofen augments bilirubin toxicity in rat cortical neuronal culture.

    Science.gov (United States)

    Berns, Monika; Toennessen, Margit; Koehne, Petra; Altmann, Rodica; Obladen, Michael

    2009-04-01

    Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 microg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 microM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 microg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 microM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.

  11. Ethanol induces heterotopias in organotypic cultures of rat cerebral cortex.

    Science.gov (United States)

    Mooney, Sandra M; Siegenthaler, Julie A; Miller, Michael W

    2004-10-01

    Abnormalities in the migration of cortical neurons to ectopic sites can be caused by prenatal exposure to ethanol. In extreme cases, cells migrate past the pial surface and form suprapial heterotopias or 'warts'. We used organotypic slice cultures from 17-day-old rat fetuses to examine structural and molecular changes that accompany wart formation. Cultures were exposed to ethanol (0, 200, 400 or 800 mg/dl) and maintained for 2-32 h. Fixed slices were sectioned and immunolabeled with antibodies directed against calretinin, reelin, nestin, GFAP, doublecortin, MAP-2 and NeuN. Ethanol promoted the widespread infiltration of the marginal zone (MZ) with neurons and the focal formation of warts. The appearance of warts is time- and concentration-dependent. Heterotopias comprised migrating neurons and were not detected in control slices. Warts were associated with breaches in the array of Cajal-Retzius cells and with translocation of reelin-immunoexpression from the MZ to the outer limit of the wart. Ethanol also altered the morphology of the radial glia. Thus, damage to the integrity of superficial cortex allows neurons to infiltrate the MZ, and if the pial-subpial glial barrier is also compromised these ectopic neurons can move beyond the normal cerebral limit to form a wart.

  12. Effect of ETBE on reproductive steroids in male rats and rat Leydig cell cultures.

    Science.gov (United States)

    de Peyster, Ann; Stanard, Bradley; Westover, Christian

    2009-10-08

    These experiments were conducted to follow up on a report of testis seminiferous tubular degeneration in Fischer 344 rats treated with high doses of ethyl t-butyl ether (ETBE). Also, high doses of a related compound, methyl t-butyl ether (MTBE), had been shown to reduce circulating testosterone (T) in rats. Isolated rat Leydig cells were used to compare hCG-stimulated T production following exposure to ETBE, MTBE, and their common main metabolite, TBA. In addition, male Fischer 344 rats were gavaged daily with 600 mg/kg, 1200 mg/kg or 1800 mg/kg ETBE in corn oil (n=12) for 14 days, the 1200 mg/kg dose chosen for comparison with a prior 14-day MTBE gavage experiment. In cell culture experiments, TBA was more potent than either ETBE or MTBE, both of which caused similar inhibition of T production at equimolar concentrations. In the in vivo study, no significant plasma T reduction was seen 1h after the final 1200 mg/kg ETBE dose, whereas 1200 mg/kg MTBE had significantly lowered T when administered similarly to Sprague-Dawley rats. Some rats treated with 1800 mg/kg ETBE had noticeably lower T levels, and the group average T level was 66% of corn oil vehicle control (p>0.05) with high variability also evident in ETBE-treated rats. 17beta-Estradiol had been increased by 1200 mg/kg MTBE, and was elevated in the 1200 and 1800 mg/kg ETBE dose groups (p<0.05), both groups also experiencing significantly reduced body weight gain. None of these effects were seen with 600 mg/kg/day ETBE. No definitive evidence of androgen insufficiency was seen in accessory organ weights, and no testicular pathology was observed after 14 days in a small subset of 1800 mg/kg ETBE-treated animals. Like MTBE, ETBE appears to be capable of altering reproductive steroid levels in peripheral blood sampled 1h after treatment, but only with extremely high doses that inhibit body weight gain and may produce mortality.

  13. P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis.

    Science.gov (United States)

    Panupinthu, Nattapon; Rogers, Joseph T; Zhao, Lin; Solano-Flores, Luis Pastor; Possmayer, Fred; Sims, Stephen M; Dixon, S Jeffrey

    2008-06-02

    Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7-/- mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7-/- mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is suppressed in calvarial cell cultures from P2rx7-/- mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin E2. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.

  14. Three-dimensional co-culture of mesenchymal stromal cells and differentiated osteoblasts on human bio-derived bone scaffolds supports active multi-lineage hematopoiesis in vitro: Functional implication of the biomimetic HSC niche

    Science.gov (United States)

    Huang, Xiaobing; Zhu, Biao; Wang, Xiaodong; Xiao, Rong; Wang, Chunsen

    2016-01-01

    Recent studies have indicated that the hematopoietic stem/progenitor cell (HSPC) niche, consisting of two major crucial components, namely osteoblasts (OBs) and mesenchymal stromal cells (MSCs), is responsible for the fate of HSPCs. Thus, closely mimicking the HSPC niche ex vivo may be an efficient strategy with which to develop new culture strategies to specifically regulate the balance between HSPC self-renewal and proliferation. The aim of this study was to establish a novel HSPC three-dimensional culture system by co-culturing bone marrow-derived MSCs and OBs differentiated from MSCs without any cytokines as feeder cells and applying bio-derived bone from human femoral metaphyseal portion as the scaffold. Scanning electron microscopy revealed the excellent biocompatibility of bio-derived bone with bone marrow-derived MSCs and OBs differentiated from MSCs. Western blot analysis revealed that many cytokines, which play key roles in HSPC regulation, were comprehensively secreted, while ELISA revealed that extracellular matrix molecules were also highly expressed. Hoechst 33342/propidium iodide fluorescence staining proved that our system could be used to supply a long-term culture of HSPCs. Flow cytometric analysis and qPCR of p21 expression demonstrated that our system significantly promoted the self-renewal and ex vivo expansion of HSPCs. Colony-forming unit (CFU) and long-term culture-initiating cell (LTC-IC) assays confirmed that our system has the ability for both the expansion of CD34+ hematopoietic stem cells (HPCs) and the maintenance of a primitive cell subpopulation of HSCs. The severe-combined immunodeficient mouse repopulating cell assay revealed the promoting effects of our system on the expansion of long-term primitive transplantable HSCs. In conclusion, our system may be a more comprehensive and balanced system which not only promotes the self-renewal and ex vivo expansion of HSPCs, but also maintains primitive HPCs with superior phenotypic and

  15. Quantification of carbon nanotube induced adhesion of osteoblast on hydroxyapatite using nano-scratch technique.

    Science.gov (United States)

    Lahiri, Debrupa; Benaduce, Ana Paula; Kos, Lidia; Agarwal, Arvind

    2011-09-02

    This paper explores the nano-scratch technique for measuring the adhesion strength of a single osteoblast cell on a hydroxyapatite (HA) surface reinforced with carbon nanotubes (CNTs). This technique efficiently separates out the contribution of the environment (culture medium and substrate) from the measured adhesion force of the cell, which is a major limitation of the existing techniques. Nano-scratches were performed on plasma sprayed hydroxyapatite (HA) and HA-CNT coatings to quantify the adhesion of the osteoblast. The presence of CNTs in HA coating promotes an increase in the adhesion of osteoblasts. The adhesion force and energy of an osteoblast on a HA-CNT surface are 17 ± 2 µN/cell and 78 ± 14 pJ/cell respectively, as compared to 11 ± 2 µN/cell and 45 ± 10 pJ/cell on a HA surface after 1 day of incubation. The adhesion force and energy of the osteoblasts increase on both the surfaces with culture periods of up to 5 days. This increase is more pronounced for osteoblasts cultured on HA-CNT. Staining of actin filaments revealed a higher spreading and attachment of osteoblasts on a surface containing CNTs. The affinity of CNTs to conjugate with integrin and other proteins is responsible for the enhanced attachment of osteoblasts. Our results suggest that the addition of CNTs to surfaces used in medical applications may be beneficial when stronger adhesion of osteoblasts is desired.

  16. Chronaxie Measurements in Patterned Neuronal Cultures from Rat Hippocampus.

    Science.gov (United States)

    Stern, Shani; Agudelo-Toro, Andres; Rotem, Assaf; Moses, Elisha; Neef, Andreas

    2015-01-01

    Excitation of neurons by an externally induced electric field is a long standing question that has recently attracted attention due to its relevance in novel clinical intervention systems for the brain. Here we use patterned quasi one-dimensional neuronal cultures from rat hippocampus, exploiting the alignment of axons along the linear patterned culture to separate the contribution of dendrites to the excitation of the neuron from that of axons. Network disconnection by channel blockers, along with rotation of the electric field direction, allows the derivation of strength-duration (SD) curves that characterize the statistical ensemble of a population of cells. SD curves with the electric field aligned either parallel or perpendicular to the axons yield the chronaxie and rheobase of axons and dendrites respectively, and these differ considerably. Dendritic chronaxie is measured to be about 1 ms, while that of axons is on the order of 0.1 ms. Axons are thus more excitable at short time scales, but at longer time scales dendrites are more easily excited. We complement these studies with experiments on fully connected cultures. An explanation for the chronaxie of dendrites is found in the numerical simulations of passive, realistically structured dendritic trees under external stimulation. The much shorter chronaxie of axons is not captured in the passive model and may be related to active processes. The lower rheobase of dendrites at longer durations can improve brain stimulation protocols, since in the brain dendrites are less specifically oriented than axonal bundles, and the requirement for precise directional stimulation may be circumvented by using longer duration fields.

  17. Chronaxie Measurements in Patterned Neuronal Cultures from Rat Hippocampus.

    Directory of Open Access Journals (Sweden)

    Shani Stern

    Full Text Available Excitation of neurons by an externally induced electric field is a long standing question that has recently attracted attention due to its relevance in novel clinical intervention systems for the brain. Here we use patterned quasi one-dimensional neuronal cultures from rat hippocampus, exploiting the alignment of axons along the linear patterned culture to separate the contribution of dendrites to the excitation of the neuron from that of axons. Network disconnection by channel blockers, along with rotation of the electric field direction, allows the derivation of strength-duration (SD curves that characterize the statistical ensemble of a population of cells. SD curves with the electric field aligned either parallel or perpendicular to the axons yield the chronaxie and rheobase of axons and dendrites respectively, and these differ considerably. Dendritic chronaxie is measured to be about 1 ms, while that of axons is on the order of 0.1 ms. Axons are thus more excitable at short time scales, but at longer time scales dendrites are more easily excited. We complement these studies with experiments on fully connected cultures. An explanation for the chronaxie of dendrites is found in the numerical simulations of passive, realistically structured dendritic trees under external stimulation. The much shorter chronaxie of axons is not captured in the passive model and may be related to active processes. The lower rheobase of dendrites at longer durations can improve brain stimulation protocols, since in the brain dendrites are less specifically oriented than axonal bundles, and the requirement for precise directional stimulation may be circumvented by using longer duration fields.

  18. Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

    Directory of Open Access Journals (Sweden)

    Bost Kenneth L

    2003-04-01

    Full Text Available Abstract Background Staphylococcus aureus infection of normal osteoblasts induces expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL. Results Normal osteoblasts were incubated in the presence of purified bacterial products over a range of concentrations. Results demonstrate that purified surface structures and a selected superantigen present in the extracellular environment are not capable of inducing TRAIL expression by osteoblasts. Osteoblasts were co-cultured with S. aureus at various multiplicities of infection utilizing cell culture chamber inserts. Results of those experiments suggest that direct contact between bacteria and osteoblasts is necessary for optimal TRAIL induction. Finally, S. aureus infection of osteoblasts in the presence of anti-TRAIL antibody demonstrates that TRAIL mediates caspase-8 activation and apoptosis of infected cells. Conclusions Collectively, these findings suggest a mechanism whereby S. aureus mediates bone destruction via induction of osteoblast apoptosis.

  19. Osteoblast role in osteoarthritis pathogenesis.

    Science.gov (United States)

    Maruotti, Nicola; Corrado, Addolorata; Cantatore, Francesco P

    2017-11-01

    Even if osteoarthritis pathogenesis is still poorly understood, numerous evidences suggest that osteoblasts dysregulation plays a key role in osteoarthritis pathogenesis. An abnormal expression of OPG and RANKL has been described in osteoarthritis osteoblasts, which is responsible for abnormal bone remodeling and decreased mineralization. Alterations in genes expression are involved in dysregulation of osteoblast function, bone remodeling, and mineralization, leading to osteoarthritis development. Moreover, osteoblasts produce numerous transcription factors, growth factors, and other proteic molecules which are involved in osteoarthritis pathogenesis. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  20. Dietary Zinc Reduces Osteoclast Resorption Activities and Increases Markers of Osteoblast Differentiation, Matrix Maturation, and Mineralization in the Long Bones of Growing Rats

    Science.gov (United States)

    The nutritional influence of zinc (Zn) on markers of bone extracellular matrix (ECM) resorption and mineralization was investigated in growing rats. Thirty male weanling rats were randomly assigned to consume AIN-93G based diets containing 2.5, 5, 7.5, 15, or 30 µg Zn/g diet for 24 d. Femur Zn incre...

  1. Thiazide diuretics directly induce osteoblast differentiation and mineralized nodule formation by targeting a NaCl cotransporter in bone

    Science.gov (United States)

    Dvorak, Melita M; De Joussineau, Cyrille; Carter, D Howard; Pisitkun, Trairak; Knepper, Mark A; Gamba, Gerardo; Kemp, Paul J; Riccardi, Daniela

    2008-01-01

    Thiazide diuretics are used, worldwide, as the first-choice drug for patients with uncomplicated hypertension. In addition to their anti-hypertensive actions, they increase bone mineral density and reduce the prevalence of fractures, indicating that thiazides may have a role in the management of postmenopausal osteoporosis. Traditionally, the bone-protective effects of thiazides have been attributed to an increase in renal calcium reabsorption, secondary to the inhibition of the sodium chloride cotransporter, NCC, expressed in the kidney distal tubule. Whether thiazides exert a direct osteoanabolic effect independently of their renal action is controversial. Here we demonstrate that freshly frozen sections of human and rat bone express NCC, principally in bone-forming cells, the osteoblasts. In primary and established culture models of osteoblasts, fetal rat calvarial (FRC) and human MG63 cells, NCC protein is virtually absent in proliferating cells while its expression is dramatically increased during differentiation. Thiazides directly stimulate the production of osteoblast markers, runt-related transcription factor 2 (runx2) and osteopontin, in the absence of a proliferative effect. Using overexpression/knockdown studies in FRC cells, we show that thiazides, but not loop diuretics, increase mineralized nodule formation acting on NCC. Overall, our study demonstrates that thiazides stimulate osteoblast differentiation and bone mineral formation independently of their renal actions. In addition to their use as part of a therapeutic treatment plan for elderly, hypertensive individuals, our discovery opens up the possibility that bone-specific drug targeting by thiazides may be developed for the prevention and treatment of osteoporosis in the patient population as a whole. PMID:17656470

  2. The experimental study of the damage of environmental neurotoxins on the cultured rat dopaminergic neurons

    Institute of Scientific and Technical Information of China (English)

    WANG Jian; LU Chuanzhen; JIANG Yuping

    2000-01-01

    Objective To establish the culture system of rat dopaminergic neurons. and to determine whether Paraquat and Dieldrin selectively destroy cultured rat dopaminergic neurons respectively. Methods The cultured rat dopaminergic neurons were treated for 24h with Paraquat and Dieldrin(0.001 to 100 μ mol/L) respectively, Data were expressed as percentage of surviving TH-positive(TH+) cells and other cells per culture dish. Results Paraquat was not effective in selectively destroying TH+ neurons. Dieldrin (1 μ mol/L) selectively decreased the number of TH+ neurons without affecting other cells. The EC50 of Dieldrin on TH+ neurons was 27.6 l mol/L. Conclusion: Paraquat can not selectively destroy dopaminergic neurons in culture. Dieldrin (1 μ mol/L) can selectively destroy the dopaminergic neurons in culture, which make it a potential etiological agent for PD. The possible parkinsonogenic effect of Dieldrin is deserved for further investigation.

  3. MEK5 suppresses osteoblastic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kaneshiro, Shoichi [Department of Orthopaedic Surgery, Japan Community Health Care Organization Osaka Hospital, 4-2-78 Fukushima, Fukushima Ward, Osaka City, Osaka 553-0003 (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Higuchi, Chikahisa, E-mail: c-higuchi@umin.ac.jp [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-07-31

    Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts.

  4. 钛合金表面纳米化对大鼠成骨细胞的影响%The effect of nanoporous surface on titanium alloy on rat osteoblast

    Institute of Scientific and Technical Information of China (English)

    嵇伟平; 张小农; 韩培; 蒋垚; 顾新丰; 张先龙; 王琦; 沈灏; 邵俊杰; 赵常利

    2008-01-01

    目的 探讨钛合金表面纳米化对成骨细胞黏附、增殖、分化和成骨矿化能力的影响.方法 采用塑性变形和化学处理方法在Ti6Al4V合金上制得一种新型多孔纳米晶体的表面结构.将原代培养的大鼠成骨细胞与纳米表面、光滑表面及粗糙表面钛合金共培养.分别通过细胞计数、扫描电镜、MTT比色法、逆转录聚合酶链反应(reverse transcriptase-polymerase chain reaction,RT-PCR)分析和钙结节荧光染色等方法观察成骨细胞在3组钛合金表面的黏附、增殖、分化及成骨能力,所得数据进行统计学分析.结果 黏附能力实验结果显示早期纳米表面黏附的成骨细胞数多于其它两组(P<0.05).扫描电镜观察显示纳米表面上成骨细胞可以较其它两组更早的伸展黏附.增殖能力实验显示,3组细胞的生长曲线大体形态基本相同,但纳米表面成骨细胞的指数增殖期比光滑表面和粗糙表面长3~10 d,且最高峰更高.RT-PCR研究表明,共培养14 d后,纳米表面黏附的成骨细胞中碱性磷酸酶、Ⅰ型胶原和骨钙素mRNA的转录量均升高(P<0.05).另外,与光滑和粗糙表面相比,纳米表面形成钙结节面积显著增大(P<0.05).结论 纳米多孔表面技术能促进成骨细胞的体外早期黏附、增殖、分化和成骨能力,为纳米技术进一步应用到人工关节提供新的方向.%Objective To evaluate the effect of nanoporous surface of titanium alloy on adhesion,proliferation,differentiation and mineralization of osteoblasts.Methods The present study was to prepare a novel nanostructured surface of Ti6Al4V alloy by the severe plastic deformation (SPD).Osteoblasts were cultured on nanoporous, smooth and rough titanium alloy in vitro.The adhesion, proliferation,differentiation and mineralization of osteoblasts were observed by cells counting,scanning electron micrography(SEM),standard MTT assay,reverse transcriptase-polymerase chain reaction

  5. Constant Applied Force Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

    Science.gov (United States)

    Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E. A. C.

    2003-01-01

    Reduced weight-bearing caused by immobilization, bed-rest or microgravity leads to atrophy in mechanosensitive tissue such as muscle and bone. We hypothesize that bone tissue requires earth s gravity (1-g) for the maintenance of extracellular matrix, integrin, and kinase-mediated cell growth and survival pathways. We investigate the role of matrix-integrin signaling in bone cells using cell culture centrifugation to provide different levels of hypergravity mechanostimulation. The 10-50-g range we use also mimics physiological intermedullary pressure (1.2 - 5 kPa). 24 hours at 50-g increased primary rat osteoblast proliferation on collagen Type I and fibronectin, but not laminin or uncoated plastic. BrdU incorporation in primary osteoblasts over 24 h showed hypergravity increased the number of cells actively synthesizing DNA from about 60% at 1-g to over 90% at 25-g. Primary rat fibroblasts grown at 50-g (24 h) showed no proliferation increase, suggesting this is a tissue-specific phenomenon. These results suggest that the betal and alpha4 integrins may be involved. To further test this, we used osteocytic-like MLO-Y4 cells that showed increased proliferation at 1-g with stable expression of a betal integrin cytoplasmic tail and transmembrane domain construct. At 50-g, MLO-Y4/betal cells showed greater MAPK activation than MLO-Y4 vector controls, suggesting that betal integrin is involved in transducing mitogenic signals in response to hypergravity. Preliminary results also show that interfering with the alpha4 integrin in primary osteoblasts grown on fibronectin blocked the proliferation response. These results indicate that cells from mechanosensitive bone tissue can respond to gravity-generated forces, and this response involves specific matrix and integrin-dependent signaling pathways.

  6. Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response

    NARCIS (Netherlands)

    Peters, S.J.A.C.; Vanhaecke, T.; Papeleu, P.; Rogiers, V.; Haagsman, H.P.; Norren, van K.

    2010-01-01

    Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6- stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CCRLEC), a collagen type

  7. Effects of drotaverine hydrochloride on viability of rat cultured cerebellar granulocytes.

    Science.gov (United States)

    Demushkin, V P; Zhavoronkova, E V; Khaspekov, L G

    2012-02-01

    The neurocytotoxic effect of drotaverine hydrochloride was studied in culture of rat cerebellar granulocytes. Incubation of cells with 100 and 250 μM drotaverine reduced neuronal survival to 60 and 4%, respectively.

  8. Glutamate enhances the expression of vascular endothelial growth factor in cultured SD rat astrocytes

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To study the effect of glutamate on the expression of vascular endothelial growth factor (VEGF) mRNA and protein in cultured rat astrocytes. Methods Cultured rat astrocytes were randomly divided into 6 groups:control group (C),glutamate group (G),QA group (Q),DCG-IV group (D),L-AP4 group (L) and glutamate+MCPG group (G+M). Cells were cultured under nomoxic condition (95% air,5% CO2). RT-PCR and ELISA methods were used to detect the expression of VEGF mRNA and protein in cultured astrocytes,respect...

  9. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lei [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wu, Zhong [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wang, Jianguang, E-mail: jianguangwang@163.com [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Jianguo, E-mail: gehujianguo68@163.com [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China)

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  10. Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

    Science.gov (United States)

    Eleniste, Pierre P; Patel, Vruti; Posritong, Sumana; Zero, Odette; Largura, Heather; Cheng, Ying-Hua; Himes, Evan R; Hamilton, Matthew; Baughman, Jenna; Kacena, Melissa A; Bruzzaniti, Angela

    2016-06-01

    Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.

  11. RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

    Science.gov (United States)

    do Bomfim, Fernando R. C.; Sella, Valéria R. G.; Zanaga, Jéssica Q.; Pereira, Nayara S.; Nouailhetas, Viviane L. A.; Plapler, Hélio

    2014-03-01

    Purpose: Osteoblasts are capable to produce different compounds directly connected to bone mineralization process. This study aims to standardize the reverse transcriptase polymerase chain reaction (RT-PCR) for adult osteoblasts to observe the effect of low level laser therapy on bone mineralization. Methods: Five-millimeter long fragments obtained from the mead femoral region of male Wistar rats were assigned into group A (n=10, laser) and group B (n=10, no laser), submitted to mechanic and enzymatic digestion. After 7 days, cultures of group A were irradiated daily on a single spot with a GaInAs laser, λ=808nm, 200mW/cm2, 2J/cm2, bean diameter of 0,02mm, 5 seconds for 6 days. Group B was manipulated but received no laser irradiation. After 13 days the cells were trypsinized for 15 minute and stabilized with RNA later® for RNA extraction with Trizol®. cDNA synthesis used 10μg of RNA and M-MLV® enzyme. PCR was accomplished using the β-actin gene as a control. Another aliquot was fixed for Hematoxylin-Eosin and Von Kossa staining to visualize bone mineralization areas. Results: Under UV light we observed clearly the amplification of β-actin gene around 400bp. HE and Von Kossa staining showed osteoblast clusters, a higher number of bone cells and well defined mineralization areas in group A. Conclusion: The cell culture, RNA extraction and RT-PCR method for adult osteoblasts was effective, allowing to use these methods for bone mineralization studies. Laser improved bone mineralization and further studies are needed involving osteogenesis, calcium release mechanisms and calcium related channels.

  12. 1,25(OH)2D3 inhibits the deleterious effects induced by high glucose on osteoblasts through undercarboxylated osteocalcin and insulin signaling.

    Science.gov (United States)

    Wu, Ying-ying; Yu, Tao; Zhang, Xiao-hui; Liu, Yan-shan; Li, Feng; Wang, Yan-ying; Wang, Yong-yue; Gong, Ping

    2012-10-01

    Diabetes mellitus (DM) is associated with multiple skeletal disorders, and vitamin D may play a functional role in the preservation of glucose tolerance. However, the relationship between vitamin D deficiency and DM is not well known. The aim of this study was to investigate the potential molecular link between 1,25(OH)(2)D(3) regulation and glucose homeostasis. Rat primary osteoblasts were cultured in different conditioned medium: normal glucose, high glucose, high glucose and insulin, high glucose and 1,25(OH)(2)D(3), high glucose and insulin and 1,25(OH)(2)D(3). The activity of osteoblasts was measured by cell viability, alkaline phosphatase and osteocalcin assay. The potential mechanism of how 1,25(OH)(2)D(3) affect insulin sensitivity was investigated by the assay of insulin receptor (IR) and vitamin D receptor (VDR) expression, and undercarboxylated osteocalcin (ucOC) level. The combined treatment has the strongest effect of inhibiting the deleterious effects induced by high glucose on osteoblasts, and it promoted the %ucOC value to approximately 40%, which is much higher than that in high glucose without treatment. Levels of IR and VDR of osteoblasts in combined treatment culture increased significantly compared with that in high glucose without treatment. So maybe 1,25(OH)(2)D(3) promotes insulin sensitivity of osteoblasts by activating insulin signaling and simultaneously stimulating ucOC secretion, which in turn regulate insulin production and sensitivity. 1,25(OH)(2)D(3) might be beneficial not only for diabetes, but also, for osteoporosis by promoting bone formation.

  13. Plasminogen binding to rat hepatocytes in primary culture and to thin slices of rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Gonias, S.L.; Braud, L.L.; Geary, W.A.; VandenBerg, S.R. (Univ. of Virginia Health Sciences Center, Charlottesville (USA))

    1989-08-01

    Human {sup 125}I-plasminogen bound readily to rat hepatocytes in primary culture at 4 {degree}C and at 37{degree}C. Binding was inhibited by lysine and reversed by lysine, epsilon-aminocaproic acid, or nonradiolabeled plasminogen. The Kd for binding of {sup 125}I-plasminogen to hepatocytes was 0.59 +/- 0.16 mumol/L, as determined from the saturation isotherm by nonlinear regression (r2 = 0.99) and the Scatchard transformation by linear regression (r2 = 0.93). The number of sites per cell was 14.1 +/- 1.1 x 10(6). Fibrinogen synthesis and secretion by hepatocytes was insufficient to account for the major fraction of plasminogen binding, as determined by enzyme-linked immunosorbent assay (ELISA). Polyacrylamide gel electrophoresis and trichloroacetic acid precipitation studies demonstrated that plasminogen is neither activated nor degraded when bound to hepatocytes at 37{degree}C. Thin slices of whole rat liver (500 microns), isolated and prepared totally at 4{degree}C, bound {sup 125}I-plasminogen. Binding was inhibited by lysine. {sup 125}I-albumin binding to liver slices was minimal and not inhibited by lysine. Activation of plasminogen by tissue plasminogen activator (t-PA) was enhanced by hepatocytes in primary culture. When lysine was included in the media, the enhanced rate of activation was no longer observed. After activation with t-PA, much of the plasmin remained associated with hepatocyte surfaces and was partially protected from inhibition by alpha 2-antiplasmin. These studies suggest that hepatocyte plasminogen binding sites may provide important surface anticoagulant activity.

  14. Effects of trypan blue on rat and rabbit embryos cultured in vitro.

    Science.gov (United States)

    Ninomiya, H; Kishida, K; Ohno, Y; Tsurumi, K; Eto, K

    1993-11-01

    Mouse and rat whole embryo cultures are widely used in teratogenicity studies. We attempted to improve the technique of culturing rabbit embryo. Rabbit embryos of the Japanese White strain were explanted on day 9, 10 or 11 of gestation and cultured for 24 or 48 hr. Rabbit embryos on day 9 of gestation were cultured in 100% rabbit serum with a gas mixture containing 20% O(2) for the first 24 hr and 95% O(2) for the following 24 hr. Rabbit embryos on day 10 or 11 of gestation were cultured in 100, 80 or 60% rabbit serum with a gas mixture of 95% O(2) for 48 or 24 hr. The development of embryos cultured for 48 hr from day 9 or day 10 or for 24 hr from day 11 was nearly the same as that of embryos that had developed in vivo. These results indicate that rabbit embryo culture is a useful and promising technique in teratogenicity studies. We then examined the effects of trypan blue on cultured rat and rabbit embryos. Slc:SD rat embryos on day 9.5 of gestation were explanted and cultured in rat serum exposed to trypan blue (300-2700 mug/ml) for 48 hr. Rabbit embryos on day 9 or 10 of gestation were explanted and cultured in rabbit serum containing trypan blue (300-2700 mug/ml) for 48 or 24 hr. Cultured rat embryos exposed to trypan blue showed neural tube abnormalities, and all growth parameters were suppressed with increasing concentrations of trypan blue. However, trypan blue had no effect on cultured rabbit embryos. These results indicate that trypan blue has species-specific effects on embryos.

  15. IGF-1 mRNA expression of adult rat thyroid cell cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    HE Feng-ping(何凤屏); YIN Rui-xing(尹瑞兴); XUAN Su(冼苏); JEAN Joss

    2003-01-01

    Objective:To investigate the law of age-related changes of insulin-like growth factor-1(IGF-1)expression of rat thyroid cells cultured in vitro.Methods:Rat thyroid of different age(10,45,65,100,150 weeks)was isolated and thyrocytes cultured.Total RNA was extracted in different rat age group when thyroid cells had been cultured for two weeks,mRNA IGF-1 expression was measured with reverse-transcription polymerase chain reaction(RT-PCR)in each group and compared.Results:Quantity of total RNA in thyroid cells decreased with ageing when the rat thyroid cells had been cultured for 2 weeks.There is significant difference among groups(P < 0.05).Expression of IGF-1 mRNA could be detected in thyroid cells of different age cultured in vitro.Quantity of IGF-1 mRNA expression by RTPCR analysis increased from 10 to 45 weeks old,and then decreased with ageing.Conclusion:Rat thyroid cells from different age cultured in vitro can express IGF-1 mRNA.Quantity of total RNA in thyroid cells cultured in vitro decreased with aging.IGF-1 mRNA expression was correlated to age(r =0.401,P <0.05).

  16. A novel method for toxicology: in vitro culture system of a rat preantral follicle.

    Science.gov (United States)

    Wan Xuying; Zhu Jiangbo; Zhu Yuping; Xili, Ma; Liu Zhen; Wang Fei; Xu Guifeng; Zhang Tianbao

    2011-08-01

    Preantral follicle in vitro culture systems have been successfully or nearly successfully established for sheep, pig and mouse, and applied on follicle development and regulation research on reproductive biology and physiology. However, there have been few studies concerning rat preantral follicle in vitro development. The objective is to establish an in vitro culture system for rat preantral follicles which can be used for reproductive biology and toxicology research. Rat preantral follicles are mechanically separated, cultured in vitro in single follicle mode for continuous 12 days using 96-well plates, and then administrated ovulation induction. The observation on follicle development, hormone level, and ovum formation are recorded and assessed. Taking in vivo growth and in vitro maturation of oocytes group as control group, in vitro growth and maturation of oocytes group is assessed to see whether this in vitro culture method is successful. The conditions for rat follicle culture are determined based on the mouse pre-antral follicle culture. The in vitro culture system for rat preantral follicles established in this study is feasible and successful, and can serve as model for reproductive biology and toxicology research.

  17. Mechanisms regulating osteoblast response to surface microtopography and vitamin D

    Science.gov (United States)

    Bell, Bryan Frederick, Jr.

    (OH) 2D3. Silencing of the beta1 integrin in osteoblast-like MG63 cells significantly reduced osteogenic response to surface topography and 1alpha,25(OH)2D3. Silencing of the alpha 5 subunit did not alter the response of MG63 cells to changing surface roughness or chemistry, although future work must confirm these results given similar cell surface alpha5 integrin expression observed in control and alpha5-silenced cells. Multifunctional RGD, KRSR, and KSSR coated surfaces show that RGD increased osteoblast proliferation and reduced differentiation, KRSR had no affect on osteoblast phenotype, and KSSR increased osteoblast differentiation. These results suggest that titanium surfaces can be modified to manipulate proliferation and differentiation and that RGD/KSSR functionalized surfaces could be further investigated for use as osteointegrative surfaces. The results using VDR deficient osteoblasts demonstrate that 1alpha,25(OH)2D3 acts via VDR-dependent mechanisms in cells cultured on titanium surfaces that support terminal differentiation. In caveolae deficient osteoblasts, 1alpha,25(OH)2D3 affected cell number, alkaline phosphatase activity, and TGF-beta1 levels, although levels of osteocalcin and PGE2 were not affected. These results are consistent with the hypothesis that VDR is required for the actions of 1alpha,25(OH)2D3, but that caveolae-dependent membrane 1alpha,25(OH)2D3 signaling modulates traditional VDR signaling. The exact mechanisms for this interaction remain to be shown. Overall, these results are important in better understanding the role of beta 1 integrin partners in mediating osteoblast response to implant surfaces and in understanding how integrin signaling can alter osteoblast differentiation and responsiveness to 1alpha,25(OH)2D3 via genomic and non-genomic pathways.

  18. Differential sensitivity of osteoblasts and bacterial pathogens to 405-nm light highlighting potential for decontamination applications in orthopedic surgery

    Science.gov (United States)

    Ramakrishnan, Praveen; Maclean, Michelle; MacGregor, Scott J.; Anderson, John G.; Grant, M. Helen

    2014-10-01

    Healthcare associated infections pose a major threat to patients admitted to hospitals and infection rates following orthopedic arthroplasty surgery are as high as 4%. A 405-nm high-intensity narrow spectrum light has been proven to reduce environmental contamination in hospital isolation rooms, and there is potential to develop this technology for application in arthroplasty surgery. Cultured rat osteoblasts were exposed to varying light intensities and it was found that exposures of up to a dose of 36 J/cm2 had no significant effect on cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], function (alkaline phosphatase activity), and proliferation rate (BrdU cell proliferation assay). High irradiance exposures (54 J/cm2) significantly affected the cell viability indicating that the effects of 405-nm light on osteoblasts are dose dependent. Additionally, exposure of a variety of clinically related bacteria to a dose of 36 J/cm2 resulted in up to 100% kill. These results demonstrating the differential sensitivity of osteoblasts and bacteria to 405-nm light are an essential step toward developing the technique for decontamination in orthopedic surgery.

  19. Dedifferentiated fat cells differentiate into osteoblasts in titanium fiber mesh.

    Science.gov (United States)

    Kishimoto, Naotaka; Momota, Yoshihiro; Hashimoto, Yoshiya; Ando, Kayoko; Omasa, Takeshi; Kotani, Junichiro

    2013-01-01

    Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, L-ascorbic acid 2-phosphate and β-glycerophosphate for 14 days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering.

  20. How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

    Science.gov (United States)

    Globus, Ruth K.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

  1. Ototoxicity of paclitaxel in rat cochlear organotypic cultures

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yang [Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Ding, Dalian; Jiang, Haiyan [Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Shi, Jian-rong [Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Salvi, Richard [Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Roth, Jerome A., E-mail: jaroth@buffalo.edu [Department of Pharmacology and Toxicology, University at Buffalo, NY 14214 (United States)

    2014-11-01

    Paclitaxel (taxol) is a widely used antineoplastic drug employed alone or in combination to treat many forms of cancer. Paclitaxel blocks microtubule depolymerization thereby stabilizing microtubules and suppressing cell proliferation and other cellular processes. Previous reports indicate that paclitaxel can cause mild to moderate sensorineural hearing loss and some histopathologic changes in the mouse cochlea; however, damage to the neurons and the underlying cell death mechanisms are poorly understood. To evaluate the ototoxicity of paclitaxel in more detail, cochlear organotypic cultures from postnatal day 3 rats were treated with paclitaxel for 24 or 48 h with doses ranging from 1 to 30 μM. No obvious histopathologies were observed after 24 h treatment with any of the paclitaxel doses employed, but with 48 h treatment, paclitaxel damaged cochlear hair cells in a dose-dependent manner and also damaged auditory nerve fibers and spiral ganglion neurons (SGN) near the base of the cochlea. TUNEL labeling was negative in the organ of Corti, but positive in SGN with karyorrhexis 48 h after 30 μM paclitaxel treatment. In addition, caspase-6, caspase-8 and caspase-9 labeling was present in SGN treated with 30 μM paclitaxel for 48 h. These results suggest that caspase-dependent apoptotic pathways are involved in paclitaxel-induced damage of SGN, but not hair cells in cochlea. - Highlights: • Paclitaxel was toxic to cochlear hair cells and spiral ganglion neurons. • Paclitaxel-induced spiral ganglion degeneration was apoptotic. • Paclitaxel activated caspase-6, -8 and -8 in spiral ganglion neurons.

  2. Vitamin a is a negative regulator of osteoblast mineralization.

    Directory of Open Access Journals (Sweden)

    Thomas Lind

    Full Text Available An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1 with the active metabolite of vitamin A; retinoic acid (RA, a retinoic acid receptor (RAR antagonist (AGN194310, and a Cyp26 inhibitor (R115866 which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

  3. Vitamin a is a negative regulator of osteoblast mineralization.

    Science.gov (United States)

    Lind, Thomas; Sundqvist, Anders; Hu, Lijuan; Pejler, Gunnar; Andersson, Göran; Jacobson, Annica; Melhus, Håkan

    2013-01-01

    An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s) behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1) with the active metabolite of vitamin A; retinoic acid (RA), a retinoic acid receptor (RAR) antagonist (AGN194310), and a Cyp26 inhibitor (R115866) which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin) were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

  4. Three-dimensional co-culture of mesenchymal stromal cells and differentiated osteoblasts on human bio-derived bone scaffolds supports active multi-lineage hematopoiesis in vitro: Functional implication of the biomimetic HSC niche.

    Science.gov (United States)

    Huang, Xiaobing; Zhu, Biao; Wang, Xiaodong; Xiao, Rong; Wang, Chunsen

    2016-10-01

    Recent studies have indicated that the hematopoietic stem/progenitor cell (HSPC) niche, consisting of two major crucial components, namely osteoblasts (OBs) and mesenchymal stromal cells (MSCs), is responsible for the fate of HSPCs. Thus, closely mimicking the HSPC niche ex vivo may be an efficient strategy with which to develop new culture strategies to specifically regulate the balance between HSPC self-renewal and proliferation. The aim of this study was to establish a novel HSPC three-dimensional culture system by co-culturing bone marrow-derived MSCs and OBs differentiated from MSCs without any cytokines as feeder cells and applying bio-derived bone from human femoral metaphyseal portion as the scaffold. Scanning electron microscopy revealed the excellent biocompatibility of bio-derived bone with bone marrow-derived MSCs and OBs differentiated from MSCs. Western blot analysis revealed that many cytokines, which play key roles in HSPC regulation, were comprehensively secreted, while ELISA revealed that extracellular matrix molecules were also highly expressed. Hoechst 33342/propidium iodide fluorescence staining proved that our system could be used to supply a long-term culture of HSPCs. Flow cytometric analysis and qPCR of p21 expression demonstrated that our system significantly promoted the self-renewal and ex vivo expansion of HSPCs. Colony-forming unit (CFU) and long-term culture-initiating cell (LTC-IC) assays confirmed that our system has the ability for both the expansion of CD34+ hematopoietic stem cells (HPCs) and the maintenance of a primitive cell subpopulation of HSCs. The severe-combined immunodeficient mouse repopulating cell assay revealed the promoting effects of our system on the expansion of long-term primitive transplantable HSCs. In conclusion, our system may be a more comprehensive and balanced system which not only promotes the self-renewal and ex vivo expansion of HSPCs, but also maintains primitive HPCs with superior

  5. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    Energy Technology Data Exchange (ETDEWEB)

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated /sup 3/H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of /sup 3/H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.

  6. Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

    DEFF Research Database (Denmark)

    Autrup, Herman; Stoner, G.D.; Jackson, F.

    1978-01-01

    An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm2 in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, beta-retinyl acetate, and either 2.5% bo...

  7. 骨搬移技术治疗骨不连的分子机制体外实验动物研究-新型力学加载式生物反应器对雄性大鼠成骨细胞OPG/RANKL 表达的影响%Move technique in the treatment of bone nonunion vitro animal studies the molecular mechanisms-new mechanical loading bioreactor in male rat osteoblast OPG/RANKL expression

    Institute of Scientific and Technical Information of China (English)

    于大淼; 廉永云

    2013-01-01

    目的:新型力学加载式生物反应器相同振动频率和不同振动应变作用于体外三维支架条件下培养的雄性大鼠成骨细胞,探究OPG/RANKL表达的影响。方法雄性大鼠成骨细胞培养、传代后分组:振动频率为1.0 Hz ,不同振动应变(A B C三组)及对照组(无振动应变及振动频率)(D组)4组。应用 RT-PCR和Western blot检测干预后OPG和RANKL mRNA和蛋白水平的表达。结果仅振动应变2000με(B组)作用于细胞后,细胞中 OPGmRNA 蛋白水平表达升高,RANKLmRNA 及蛋白水平则降低(均P<0.05)。其余实验组未见阳性反应。结论通过新型力学加载式生物反应器(振动应变2000με振动频率1.0 Hz)的压应力作用于细胞后可以正性调节OPG/RANKL轴的表达。%Objective to model the same vibration frequency of the mechanical loading bioreactor and different vibration strain effect on the three-dimensional scaffold in vitro cultured osteoblast in male rats ,influence on OPG/RANKL expression .Methods the male rat osteoblast culture ,subculture group :vibration frequency is 1 Hz ,the different vibration strain (ABC three group) and control group (no vibration strain and vibration frequency) (group D) of 4 groups .Expression of OPG and RANKL mRNA and protein levels of RT-PCR and Western blot detection after intervention .Results only vibration strain 2000 με (group B) acting on cells ,elevated levels of OPGmRNA protein expression in RANKLmRNA cells ,and the protein level decreased (all P<0.05) .The experimental group had no positive reac-tion .Conclusion by the new type of mechanical loading bioreactor (vibration strain 2000 μεvibration frequency 1 Hz) pressure expression for the stress acting on cells can positively regulate the OPG/RANKL axis .

  8. Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

    DEFF Research Database (Denmark)

    Autrup, Herman; Stoner, G.D.; Jackson, F.

    1978-01-01

    An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm2 in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, beta-retinyl acetate, and either 2....... The explants were incubated at 30 degrees C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[alpha]pyrene, 7...

  9. Upregulation of endothelin ETB receptor-mediated vasoconstriction in rat coronary artery after organ culture

    DEFF Research Database (Denmark)

    Eskesen, Karen; Edvinsson, Lars

    2006-01-01

    The aim of this study was to examine if endothelin ET(B) receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET(B) receptors was studied by measuring the change in isometric tension in rings of left anterior...... descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction......(+)-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET(B) receptor-mediated contraction by inducing synthesis of new protein....

  10. Stimulation of Mucosal Mast Cell Growth in Normal and Nude Rat Bone Marrow Cultures

    Science.gov (United States)

    Haig, David M.; McMenamin, Christine; Gunneberg, Christian; Woodbury, Richard; Jarrett, Ellen E. E.

    1983-07-01

    Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.

  11. Comparison of coumarin-induced toxicity between sandwich-cultured primary rat hepatocytes and rats in vivo: A toxicogenomics approach

    NARCIS (Netherlands)

    Kienhuis, A.S.; Wortelboer, H.M.; Hoflack, J.C.; Moonen, E.J.; Kleinjans, J.C.S.; Ommen, B. van; Delft, J.H.M. van; Stierum, R.H.

    2006-01-01

    Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. However, loss of liver-specific functions, in particular, the decline of cytochrome P450 (P450) enzyme activity, limits the value of this model for prediction of in vivo toxicity. In this st

  12. Bilaminar co-culture of primary rat cortical neurons and glia.

    Science.gov (United States)

    Shimizu, Saori; Abt, Anna; Meucci, Olimpia

    2011-11-12

    This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions(1). At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods(1-3). Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology(4), cellular and molecular biology(5-8), biochemistry(5), imaging and microscopy(4,6,7,9,10). The primary neurons extend axons and dendrites to form functional synapses(11), a process which is not observed in neuronal cell lines, although some

  13. Effect of simvastatin on the expression ofβ-catenin and Runx2 at the early stage of the osteoblast differentiation by rat bone marrow stromal cells%辛伐他汀对 BMSCs 成骨分化早期β-catenin 及 Runx2表达的影响

    Institute of Scientific and Technical Information of China (English)

    贾忠宝; 田发明; 崔纳; 张柳

    2013-01-01

    .Methods Twenty 4-week female Sprague-Dawley rats were executed by cervical dislocation .BMSCs in the bilateral femur and the tibia were collected under sterile conditions , and the cells were cultured in vitro to differentiate into osteoblasts . The 2nd-passage cells were divided into 2 groups:experimental group and control groups .Cells in experimental group were cultured with complete medium additioned with 10 -7 mol/L SIM ( SIM was dissolved in DMSO first , then diluted with complete medium ) , while cells in control group were cultured with complete medium additioned alone with same volume of DMSO .After 12h and 36h, the protein and RNA in 2 groups were extracted , respectively.The expression of β-catenin and Runx2 was detected using Western blotting .The mRNA expression of β-catenin and Runx2 was detected using real-time RT-PCR.Results The results of Western blotting revealed that, after 12h intervention of SIM, the expression ofβ-catenin and Runx2 in experimental group was not significantly higher than that in control group (P>0.05).But after 36h intervention SIM, the expression of β-catenin and Runx2 in experimental group was significantly higher than that in control group ( P 0.05).Conclusion SIM has some effect on the osteoblast differentiation of rat BMSCs at the early stage in vitro, which may involve the activation of the Wnt signaling pathway , while the role at different time points was different .

  14. Comparison of coumarin-induced toxicity between sandwich-cultured primary rat hepatocytes and rats in vivo: a toxicogenomics approach.

    Science.gov (United States)

    Kienhuis, Anne S; Wortelboer, Heleen M; Hoflack, Jean-Christophe; Moonen, Edwin J; Kleinjans, Jos C S; van Ommen, Ben; van Delft, Joost H M; Stierum, Rob H

    2006-12-01

    Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. However, loss of liver-specific functions, in particular, the decline of cytochrome P450 (P450) enzyme activity, limits the value of this model for prediction of in vivo toxicity. In this study, we investigated whether a hepatic in vitro system with improved metabolic competence enhances the predictability for coumarin-induced in vivo toxicity by using a toxicogenomics approach. Therefore, primary rat hepatocytes were cultured in sandwich configuration in medium containing a mixture of low concentrations of P450 inducers, phenobarbital, dexamethasone, and beta-naphthoflavone. The toxicogenomics approach used enabled comparison of similar mechanistic end-points at the molecular level between in vitro and in vivo conditions, namely, compound-induced changes in multiple genes and signaling pathways. Toxicant-induced cytotoxic effects and gene expression profiles observed in hepatocytes cultured in modified medium and hepatocytes cultured in standard medium (without inducers) were compared with results from a rat in vivo study. Coumarin was used as a model compound because its toxicity depends on bioactivation by P450 enzymes. Metabolism of coumarin toward active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepatocytes cultured in modified medium compared with hepatocytes cultured in standard medium. In addition, more genes and biological pathways were similarly affected by coumarin in hepatocytes cultured in modified medium and in vivo. In conclusion, these experiments showed that for coumarin-induced toxicity, sandwich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared with hepatocytes cultured in standard medium.

  15. Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode.

    Science.gov (United States)

    Rikihisa, Y; Lin, Y C; Fukaya, T

    1985-06-01

    In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.

  16. 25-Hydroxy- and 1α,25-Dihydroxycholecalciferol Have Greater Potencies than 25-Hydroxy- and 1α,25-Dihydroxyergocalciferol in Modulating Cultured Human and Mouse Osteoblast Activities

    OpenAIRE

    Zarei, Allahdad; Hulley, Philippa A.; Sabokbar, Afsie; Javaid, M Kassim; Morovat, Alireza

    2016-01-01

    Despite differences in the phamacokinetics of 25-hydroxycholecalciferol (25(OH)D3) and 25-hydroxyergocalciferol (25(OH)D2) in man, the effects of these and their 1α-hydroxylated forms (1,25(OH)2D3 and 1,25(OH)2D2) on cellular activity of vitamin D-responsive cells have hardly been compared. We studied differences in the effects of these metabolites on cell number, gene transcription, protein expression and mineralisation of cultured human bone marrow-derived stromal cells (hBMSC) and rapidly ...

  17. Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes

    NARCIS (Netherlands)

    Treijtel, N.; Eijkeren, J.C.H.v.; Nijmeijer, S.; Greef de - Sandt, I.C.J. van der; Freidig, A.P.

    2009-01-01

    The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich

  18. Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes.

    NARCIS (Netherlands)

    Treijtel, N.; van Eijkeren, J.C.; Nijmeijer, S.; de Greef-van der Sandt, I.C.; Freidig, A.

    2009-01-01

    The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich

  19. 咀嚼压力增强对大鼠牙槽骨白细胞介素-1β表达的影响%Effect of increased bite force on the expression of IL-1β in rat alveolar bone osteoblasts

    Institute of Scientific and Technical Information of China (English)

    袁林; 周伟东; 赵云凤

    2001-01-01

    目的检测大鼠牙槽骨成骨细胞中IL-1β在正常及增强咀嚼压力状态下的动态表达,探讨IL-1β在牙槽骨改建中的分子机制。方法采用HE染色和免疫组化的方法,观察牙周形态变化以及牙槽骨成骨细胞中IL-1β蛋白表达。结果生理限度内咀嚼压力增强时,形态学显示大鼠牙周膜增宽、牙槽骨新骨形成;免疫组化观察到成骨细胞中IL-1β表达较正常咀嚼压力时明显增强。结论咀嚼压力增强促使牙周组织产生IL-1β明显增多,诱发了破骨功能,同时,还激活了成骨功能。提示IL-1β在咀嚼压力影响牙槽骨改建的过程中起着重要的调节作用。%Objective To explore the molecular mechanism of alveolar bone remodeling by studying the dynamic changes of IL- 1β expression in rat alveolar bone osteoblasts. Methods Rat models of increased bite force of the back teeth were established, and the expression of IL-1β in the alveolar bone osteoblasts were determined by HE staining and immunohistochemistry. Observation of the changes in the histological morphology of the periodontium was conducted microscopically. Rats with normal bite force served as control. Results The increase of bite force (within the physiological limit) induced the widening of the periodontal ligament and the osteogenesis in the alveolar bone. Significant enhancement of IL-1β expression was observed in the osteoblasts of rats with increased bite force, in comparison with that in the rats with normal bite force. Conclusion Increased bite force causes higher expression levels of IL-1β in the alveolar bone osteoblasts, initiating the destruction process of the bone but simultaneously the activation of the ossification, suggesting that IL-1β plays an important role in the regulation of periodontium remodeling in response to changes in the bite force

  20. Seeding Osteoblasts onto Osteocytes: An In Vitro 3D Study

    Directory of Open Access Journals (Sweden)

    Judith Green

    2006-01-01

    Full Text Available Understanding the mechanisms by which bone cells communicate is vital in exploring diseases characterized by bone degeneration, namely, osteoporosis. Cell seeding has been used in two dimensional (2D cell cultures to study how bone cells interact with one another, specifically, to prove the existence of gap junctions between osteocytes and osteoblasts. However, the natural three dimensional (3D state of bone tissue requires examining it in 3D. Accordingly, the cell seeding procedure was tested on trabecular bone core explants to ascertain whether it is useful in 3D studies as well. When the dye concentrations taken from past 2D experiments were used, Day 1 showed many osteoblasts, but by Day 2 the cells were not visible. The dye concentrations were then doubled to determine if the osteoblasts were still seeded onto the bone cores and viable but not visible, or if they had actually died. With these dye concentrations, the stained osteoblasts were still visible on the second day after seeding, indicating that the cells were seeded and living. According to these results, it is evident that with minor modifications of the 2D procedure, it is possible to seed osteoblasts onto osteocytes in 3D, making this a credible test for the presence of gap junctions in 3D bone tissue.

  1. Transplantation of primary cultured embryonic mesencephalic neural precursor cells for treating Parkinsonian rats

    Institute of Scientific and Technical Information of China (English)

    Li Fei; Chengchuan Jiang; Linyin Feng; Yaodong Ji; Zhongliang Ding

    2006-01-01

    BACKGROUND: Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy (CRT) for treating Parkinson disease (PD). Embryonic mesencephalic precursor cells (MPCs) can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far.OBJECTIVE: To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats.DESIGN: A randomized and controlled trial taking SD rats as experimental animals.SETTING: Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University.MATERIALS: This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats,with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences.METHODS: Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1out of 20 model rats after execution, and the other 19 rats were randomly divided into control group (n=5),sham transplantation group (n=5)and cell grafted group (n=9). Primary cultured E12 MPC cell suspension (1.2×1011 L-1)were used as donor cells. 4 μL primary cultured E12 MPC cell suspension prepared freshly was injected

  2. Biomimetic Versus Sintered Calcium Phosphates: The In Vitro Behavior of Osteoblasts and Mesenchymal Stem Cells.

    Science.gov (United States)

    Sadowska, Joanna-Maria; Guillem-Marti, Jordi; Montufar, Edgar Benjamin; Espanol, Montserrat; Ginebra, Maria-Pau

    2017-02-21

    The fabrication of calcium phosphates using biomimetic routes, namely, precipitation processes at body temperature, results in distinct features compared to conventional sintered calcium phosphate ceramics, such as a high specific surface area (SSA) and micro- or nanometric crystal size. The aim of this article is to analyze the effects of these parameters on cell response, focusing on two bone cell types: rat mesenchymal stem cells (rMSCs) and human osteoblastic cells (SaOS-2). Biomimetic calcium-deficient hydroxyapatite (CDHA) was obtained by a low temperature setting reaction, and α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate were subsequently obtained by sintering CDHA either at 1400°C or 1100°C. Sintered stoichiometric hydroxyapatite (HA) was also prepared using ceramic routes. The materials were characterized in terms of SSA, skeletal density, porosity, and pore size distribution. SaOS-2 cells and rMSCs were seeded either directly on the surfaces of the materials or on glass coverslips subsequently placed on top of the materials to expose the cells to the CaP-induced ionic changes in the culture medium, while avoiding any topography-related effects. CDHA produced higher ionic fluctuations in both cell culture media than sintered ceramics, with a strong decrease of calcium and a release of phosphate. Indirect contact cell cultures revealed that both cell types were sensitive to these ionic modifications, resulting in a decrease in proliferation rate, more marked for CDHA, this effect being more pronounced for rMSCs. In direct contact cultures, good cell adhesion was found on all materials, but, while cells were able to proliferate on the sintered calcium phosphates, cell number was significantly reduced with time on biomimetic CDHA, which was associated to a higher percentage of apoptotic cells. Direct contact of the cells with biomimetic CDHA resulted also in a higher alkaline phosphatase activity for both cell types compared to sintered Ca

  3. Correlation Study of Estrogen Receptor and IL-10 in Osteoblasts of Skull in Osteoporosis Rats%骨质疏松大鼠成骨细胞雌激素受体与颅骨IL-10表达的相关性研究

    Institute of Scientific and Technical Information of China (English)

    覃淑云; 蔡科军; 冯照善; 韦启后

    2011-01-01

    目的 探讨骨质疏松SD大鼠颅骨中IL-10的表达与成骨细胞内雌激素受体表达的相关性.方法 40只SD雌性大鼠用随机数字表法随机分为对照组20只和去势(去卵巢)组20只(去势第5周组、第7周组各10只),用双能X线吸收法测定各组骨密度,用免疫组织化学ABC法及图像分析技术观察各组颅骨组织中IL-10表达;用二次酶消化、反复贴壁法分离纯化培养大鼠颅骨成骨细胞,并用钙-钴法碱性磷酸酶(ALP)染色法及Ⅰ型胶原免疫组化染色法观察、鉴定大鼠成骨细胞.应用半定量Western blot法检测各组SD大鼠成骨细胞内雌激素受体.结果 去势组骨密度明显低于对照组(P<0.01);去势组骨小梁周边IL-10阳性成骨细胞数明显少于对照组(P<0.01),并且染色较对照组浅.ALP染色、Ⅰ型胶原免疫组化及形态学观察符合成骨细胞特征.去势组雌激素受体表达明显低于对照组,去势第7周低于去势第5周(P<0.01).结论 雌激素受体的表达与成骨细胞IL-10表达水平成正相关.%Objective To observe the correlation of estrogen receptor in osteoblasts and IL-10 expression of skull in osteoporosis rats. Methods 40 SD rats were randomly divided into control group( n = 20 )and ovariectomized group( n =20,5 weeks 10 rats,7 weeks 10 rats ). The bone mineral density was measured by ray absorptiometry,IL-10 expression in the skull were detected by immunohistochemical staining of SABC method and the image analysis techniques;The rat osteoblasts were purified by two-step enzymatic digestion method and successive adherence method. The osteoblasts were observed by calcium alkaline phosphatase cobalt method( ALP )and collagen type Ⅰ immunohistochemical staining method. The estrogen receptors of osteoplasts in SD rats were identified by semi-quantitative western blot. Results The ovariectomized group showed less bone mineral density( P< 0.01 );The IL-10 positive osteoblasts in the trabecula of

  4. Purmorphamine increased adhesion, proliferation and expression of osteoblast phenotype markers of human dental pulp stem cells cultured on beta-tricalcium phosphate.

    Science.gov (United States)

    Rezia Rad, Maryam; Khojaste, Moein; Hasan Shahriari, Mehrnoosh; Asgary, Saeed; Khojasteh, Arash

    2016-08-01

    Growth factors play a significant role in cell proliferation and differentiation during different stages of the bone repair. However, several limitations have been brought researchers attention to an osteoinductive small molecule including Purmorphamine. In this study, we aimed to evaluate the effect of Purmorphamine on adhesion, proliferation and differentiation of human dental pulp stem cells (hDPSCs) seaded on beta-tricalcium phosphate (β-TCP) granules. hDPSCs were established from extracted wisdom teeth of healthy volenteers. Cells at passage 3 were seeded on β-TCP in the presence or absence of Purmorphamine. Cell adhesion and proliferation were assessed using scanning electeron microscopy (SEM) and DNA counting assay, respectively, after 1, 3 and 5days. Then, hDPSCs seeded on β-TCP were subjected to osteogenic medium with or without Purmorphamine. After 7 and 14days osteogenic diffrentiation capability of hDPSCs were determined using real-time RT-PCR and alkaline phosphatase (ALP) activity assay. The significant increase in amount of DNA was observed at day 3 and 5 in the presence of Purmorphamine. SEM imaging also was confirmed the DNA counting assay; in all given time points, hDPSC attachment and growth was significantly higher in the presence of Purmorphamine. ALP activity was increased by Purmorphamine at both 7 and 14days of induction. Purmorphamine showed to effect on osteopontin expression at earlier stage of osteogenic differentiation, whereas for osteocalcin expression, this effect was more evident at later stage of differentiation. Purmorphamine had a promotive effect on adhesion, proliferation and osteogenic differentiation of hDPSCs cultured on β-TCP. The outcome of the current study would help in development of in vitro culture conditions for better osteogenic differentiation of hDPSCs prior to transplantation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Protective effects of berberine against amyloid beta-induced toxicity in cultured rat cortical neurons

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Yanjun Zhang; Shuai Du; Mixia Zhang

    2011-01-01

    Berberine, a major constituent of Coptidis rhizoma, exhibits neural protective effects. The present study analyzed the potential protective effect of berberine against amyloid G-induced cytotoxicity in rat cerebral cortical neurons. Alzheimer's disease cell models were treated with 0.5 and 2 μmol/Lberberine for 36 hours to inhibit amyloid G-induced toxicity. Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons. In addition, western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons, which coincided with significantly decreased abnormal up-regulation of activated caspase-3. These results showed that berberine exhibited a protective effect against amyloid 13-induced cytotoxicity in cultured rat cortical neurons.

  6. A novel transgenic mouse model to study the osteoblast lineage in vivo.

    Science.gov (United States)

    Maes, Christa; Kobayashi, Tatsuya; Kronenberg, Henry M

    2007-11-01

    Over the past few decades, osteoblast differentiation has been studied extensively in a variety of culture systems and findings from these experiments have shaped our understanding of the bone-forming cell lineage. However, in vitro assays are bound by intrinsic limitations and are unable to effectively mirror many aspects related to osteoblasts in vivo, including their origin, destiny, and life span. Therefore, these fundamental questions strongly advocate the need for novel models to characterize the osteoblast lineage in vivo. Here, we developed a transgenic mouse system to study stage-specific subsets of osteoblast lineage cells. We believe that this system will prove to be a helpful tool in deciphering multiple aspects of osteoblast biology in vivo.

  7. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  8. Leptin Modulates Norepinephrine-Mediated Melatonin Synthesis in Cultured Rat Pineal Gland

    OpenAIRE

    Rodrigo Antonio Peliciari-Garcia; Jéssica Andrade-Silva; José Cipolla-Neto; Carla Roberta de Oliveira Carvalho

    2013-01-01

    Pineal melatonin synthesis can be modulated by many peptides, including insulin. Because melatonin appears to alter leptin synthesis, in this work we aimed to investigate whether leptin would have a role on norepinephrine- (NE-)mediated melatonin synthesis in cultured rat pineal glands. According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb). Pineal expression of Ob-Rb mRNA was also observed in vivo. Administration of leptin (1 nM) associated with NE (1 µM)...

  9. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

    Full Text Available Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF. CF-related bone disease (CFBD is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR, the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/- mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+ littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl mRNA was detected, significantly less osteoprotegerin (Opg was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt

  10. Evaluation of PFOS-mediated neurotoxicity in rat primary neurons and astrocytes cultured separately or in co-culture.

    Science.gov (United States)

    Li, Zhenwei; Liu, Qi; Liu, Chang; Li, Chunna; Li, Yachen; Li, Shuangyue; Liu, Xiaohui; Shao, Jing

    2017-02-01

    Perfluorooctane sulfonate (PFOS) is a potential neurotoxicant reported by epidemiological investigations and experimental studies, while the underlying mechanisms are still unclear. Astrocytes not only support for the construction of neurons, but also conduct neuronal functions through glutamate-glutamine cycle in astrocyte-neuron crosstalk. In the present study, the effect of PFOS exposure on rat primary hippocampal neurons or cortex astrocytes was evaluated. Then the role of the astrocytes in PFOS-induced toxic effect on neurons was explored with astrocyte-neuron co-culture system. Exposure of rat primary hippocampal neurons to PFOS has led to oxidation-antioxidation imbalance, increased apoptosis and abnormal autophagy. The adverse effect of PFOS on rat primary cortex astrocytes manifested in the form of altered extracellular glutamate and glutamine concentrations, decreased glutamine synthase activity, as well as decreased gene expression of glutamine synthase, glutamate transporters and glutamine transporters in the glutamate-glutamine cycle. Especially, the alleviation of PFOS-inhibited neurite outgrowth in neurons could be observed in astrocyte-neuron co-culture system, though the ability of astrocytes in fostering neurite outgrowth was affected by PFOS. These results indicated that both astrocytes and neurons might be the targets of PFOS-induced neurotoxicity, and astrocytes could protect against PFOS-inhibited neurite outgrowth in primary cultured neurons. Our research might render some information in explaining the mechanisms of PFOS-induced neurotoxicity. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. TERATOGENIC EFFECTS OF TRANSPLACENTAL TRANSFUSION OF HETEROLOGOUS ANTISERA SIMULATED IN AN EXPERIMENTAL-MODEL USING INVITRO WHOLE RAT EMBRYO CULTURE

    NARCIS (Netherlands)

    VANDERZEE, DC; POELMANN, RE; ZWIERSTRA, RP; MENTINK, MMT; VERMEIJKEERS, C

    1991-01-01

    The effects of the transplacental transfusion of heterologous rabbit-anti-rat antiserum (RAR antiserum) and subsequent immunological interaction on the development of 9-10 days old rat embryos (stages 8-10 somites) were studied using an in vitro whole rat embryo culture. Transplacental transfusion w

  12. Organ explant culture of neonatal rat ventricles: a new model to study gene and cell therapy.

    Directory of Open Access Journals (Sweden)

    A Dénise den Haan

    Full Text Available Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture may be used as a model to study gene and cell therapy. We compared (immuno histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20% and freshly isolated tissue (17%, but common (82% in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, -80.5±3.5 mV in freshly isolated tissue, and -60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively. Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms, while monolayers could not. Successful lentiviral (LV transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar

  13. Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

    NARCIS (Netherlands)

    P.J.K. Kuppen (P. J K); A.M.M. Eggermont (Alexander); A.W. Marinelli (Andreas); E. de Heer (Emile); C.J.H. van de Velde (Cornelis); G.J. Fleuren (G.)

    1991-01-01

    textabstractThe role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3-5 d

  14. An Optimized Culture Method of Rat Dorsal Root Ganglion Neurons

    Institute of Scientific and Technical Information of China (English)

    LIUYin; CHENJing-Hong; GONGZe-Hui

    2004-01-01

    AIM: To establish a primary culture technique of acutely isolated dorsal root ganglion (DRG) neurons, and provide a simple & useful in vitro model for study of analgesia. Methods: Acutely isolated dorsal root ganglion (DRG) neurons were planted and cultured; the configuration and growth characters of DRG neurons were observed through inverted microscope.

  15. Effect of Microcystin-LR on Cultured Rat Endothelial Cells

    Science.gov (United States)

    1990-02-26

    mmol, New England Nuclear Corp., Boston, MA), tissue culture media and fetal bovine serum albumin (Gibco, Grand Island, NY), tissue culture ware (Becton...WAGNER, H. AND FIEBIG, M. (1984) Actions of flavonoligans from silybum marianum fruits. Planta Med. 50, 248-250. JACKSON, A. R., RUNNEGAR, M. T

  16. Human serum teratogenicity studies using in vitro cultures of rat embryos

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N.W.; Chatot, C.L.; Plenefisch, J.D.; Carey, S.W.

    1982-01-01

    Those conditions that constitute reproductive risks to man are being analyzed. Particular concern is with those conditions that cannot be or have not been identified by present methodologies. These conditions constitute the majority of factors causing fetal wastages and birth defects. The test system uses intact rat embryos that are cultured in vitro for 2 days. Findings to date suggest that this system may have a number of distinct advantages: (1) whole-embryo culture provides the test with the entire repertoire of processes involved in embryonic development; (2) whole-rat embryos can be cultured on high levels of blood serum; and (3) they can be cultured on serum from human subjects, which provides a direct and unique evaluation of the principal organism of concern. In regard to this last point, it is important to recognize that there is a large range of teratogenic responses and sensitivities to teratogens dependent upon both individual and species differences. (ERB)

  17. Selection of medium for serum-free primary culture of adult rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Miyazaki,Masahiro

    1990-02-01

    Full Text Available To select a suitable medium for serum-free primary culture of adult rat hepatocytes, ten commercially-available synthetic media were compared for their ability to maintain the cells under serum-free and serum-supplemented conditions with special reference to attachment, survival and albumin secretion. It was found that Williams' medium E and DM-160 medium were the best among the ten media for maintaining hepatocytes under serum-free conditions in primary culture.

  18. Stimulation of DNA and Collagen Synthesis by Autologous Growth Factor in Cultured Fetal Rat Calvaria

    Science.gov (United States)

    Canalis, Ernesto; Peck, William A.; Raisz, Lawrence G.

    1980-11-01

    Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline into collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

  19. Comparison of osteoblast-like cell responses to calcium silicate and tricalcium phosphate ceramics in vitro.

    Science.gov (United States)

    Ni, Siyu; Chang, Jiang; Chou, Lee; Zhai, Wanyin

    2007-01-01

    Calcium silicate ceramics have been proposed as new bone repair biomaterials, since they have proved to be bioactive, degradable, and biocompatible. Beta-tricalcium phosphate ceramic is a well-known degradable material for bone repair. This study compared the effects of CaSiO3 (alpha-, and beta-CaSiO3) and beta-Ca3(PO4)2 (beta-TCP) ceramics on the early stages of rat osteoblast-like cell attachment, proliferation, and differentiation. Osteoblast-like cells were cultured directly on CaSiO3 (alpha-, and beta-CaSiO3) and beta-TCP ceramics. Attachment of a greater number of cells was observed on CaSiO3 (alpha-, and beta-CaSiO3) ceramics compared with beta-TCP ceramics after incubation for 6 h. SEM observations showed an intimate contact between cells and the substrates, significant cells adhesion, and that the cells spread and grew on the surfaces of all the materials. In addition, the proliferation rate and alkaline phosphatase (ALP) activity of the cells on the CaSiO3 (alpha-, and beta-CaSiO3) ceramics were improved when compared with the beta-TCP ceramics. In the presence of CaSiO3, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period. In conclusion, the results of the present study revealed that CaSiO3 ceramics showed greater ability to support cell attachment, proliferation, and differentiation than beta-TCP ceramic. 2006 Wiley Periodicals, Inc.

  20. THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    刘学光; 张志刚; 张秀荣; 朱虹光; 陈琦; 郭慕依

    2002-01-01

    Objective. To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). Methods. A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. Results. A specific monoclonal antibody against AM was successfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. Conclusion. AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.

  1. THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLSA

    Institute of Scientific and Technical Information of China (English)

    刘学光; 张志刚; 等

    2002-01-01

    Objective:To observe the localization of adrenomedullin(AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC).Methods:A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry.The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC)and MsC were investigated by Northern blot assay,and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H] thymidine incorporation as an index.Results:A specific monoclonal antibody against AM was successfull developed.AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells),some cortical proximal tubules,medullary collecting duct cells,interstitial cells,vascular smooth muscle cells and endothelial cells.Northern blot assay showed the AM mRNA was expressed only on cultured GEC,but not on MsC,however,AM receptor CRLR mRNA was only expressed on MsC.GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect.Conclusion:AM produced by GEC inhibits the proliferation of MsC,which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.

  2. Osteoblastic cells: differentiation and trans-differentiation

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Abdallah, Basem; Saeed, Hamid

    2008-01-01

    The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast...... differentiation from MSC is a central topic in bone biology that can provide insight into mechanisms of bone maintenance and also novel pharmacological targets to increase osteoblast differentiation and consequently bone formation....

  3. Effects of Frequency and Acceleration Amplitude on Osteoblast Mechanical Vibration Responses: A Finite Element Study

    Directory of Open Access Journals (Sweden)

    Liping Wang

    2016-01-01

    Full Text Available Bone cells are deformed according to mechanical stimulation they receive and their mechanical characteristics. However, how osteoblasts are affected by mechanical vibration frequency and acceleration amplitude remains unclear. By developing 3D osteoblast finite element (FE models, this study investigated the effect of cell shapes on vibration characteristics and effect of acceleration (vibration intensity on vibrational responses of cultured osteoblasts. Firstly, the developed FE models predicted natural frequencies of osteoblasts within 6.85–48.69 Hz. Then, three different levels of acceleration of base excitation were selected (0.5, 1, and 2 g to simulate vibrational responses, and acceleration of base excitation was found to have no influence on natural frequencies of osteoblasts. However, vibration response values of displacement, stress, and strain increased with the increase of acceleration. Finally, stress and stress distributions of osteoblast models under 0.5 g acceleration in Z-direction were investigated further. It was revealed that resonance frequencies can be a monotonic function of cell height or bottom area when cell volumes and material properties were assumed as constants. These findings will be useful in understanding how forces are transferred and influence osteoblast mechanical responses during vibrations and in providing guidance for cell culture and external vibration loading in experimental and clinical osteogenesis studies.

  4. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Marion, Tracy L., E-mail: tracylmarion@qualyst.com [Curriculum in Toxicology, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7270 (United States); Perry, Cassandra H., E-mail: cassandraperry@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); St Claire, Robert L., E-mail: bobstclaire@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); Brouwer, Kim L.R., E-mail: kbrouwer@unc.edu [Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, CB 7569 Kerr Hall, Chapel Hill, NC 27599-7569 (United States)

    2012-05-15

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

  5. A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling

    NARCIS (Netherlands)

    Kienhuis, A.S.; Wortelboer, H.M.; Maas, W.J.; Herwijnen, M. van; Kleinjans, J.C.S.; Delft, J.H.M. van; Stierum, R.H.

    2007-01-01

    A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte

  6. Serum obtained from rats after partial hepatectomy enhances growth of cultured colon carcinoma cells

    NARCIS (Netherlands)

    de Jong, KP; Brouwers, MAM; van Veen, ML; Brinker, M; de Vries, EGE; Daemen, T; Scherphof, GL; Slooff, MJH

    1999-01-01

    Tumour-bearing rats were randomized to a 70% partial hepatectomy or a sham operation. At days 1, 3 or 14, portal and systemic serum was obtained and colon carcinoma cells were cultured in the presence of 5, 10, 20 or 50% serum. Proliferation and epidermal growth factor receptor (EGFr) expression was

  7. Embryotoxicant-specific transcriptomic responses in rat postimplantation whole-embryo culture

    NARCIS (Netherlands)

    Robinson, J.F.; van Beelen, V.A.; Verhoef, A.; Renkens, M.F.J.; Luijten, M.; van Herwijnen, M.; Westerman, A.; Pennings, J.L.; Piersma, A.H.

    2010-01-01

    Rat postimplantation whole-embryo culture (WEC) is a promising alternative test for the assessment of developmental toxicity. Toxicogenomic-based approaches may improve the predictive ability of the WEC model by providing a means to identify compound-specific mechanistic responses associated with em

  8. A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling

    NARCIS (Netherlands)

    Kienhuis, A.S.; Wortelboer, H.M.; Maas, W.J.; Herwijnen, M. van; Kleinjans, J.C.S.; Delft, J.H.M. van; Stierum, R.H.

    2007-01-01

    A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved

  9. Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin

    DEFF Research Database (Denmark)

    Pappinen, Sari; Hermansson, Martin; Kuntsche, Judith

    2008-01-01

    The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the presen...

  10. The antiapoptotic effect of insulin against anoxia/reoxygenation injury in cultured cardiomyocyte of neonatal rat

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective: To study protective effect of insulin against cardiomyocyte apoptosis in anoxia/reoxygenation (A/R)injury of neonatal rat. Methods: The model of A/R injury was finished through receiving anoxia for 2 h and reoxygenation for 4 h in cultured cardiomyocytes of neonatal rat. The cardiomyocytes were divided randomly into 3 groups: control group (CON), anoxia/reoxygenation group (A/R) and insulin-treated group (INS). At the end of reoxygenation of 4 hours, activities of lactate dehydrogenase (LDH),contents of malondialdehyde (MDA) were assessed through spectrophotometric procedures, myocyte apoptosis were detected through TUNEL and DNA Ladder. Results: MDA, LDH, and Apoptosis Index were significantly decreased in INS group compared with A/R group (P<0.01). Conclusion: Insulin has a protective effect against A/R injury in cultured cardiomyocyte of neonatal rat; the protective mechanism may contribute to antiapoptosis of insulin.

  11. The rat whole embryo culture assay using the Dysmorphology Score system.

    Science.gov (United States)

    Zhang, Cindy; Panzica-Kelly, Julie; Augustine-Rauch, Karen

    2013-01-01

    The rat whole embryo culture (WEC) system has been used extensively for characterizing teratogenic properties of test chemicals. In this chapter, we describe the methodology for culturing rat embryos as well as a new morphological score system, the Dysmorphology Score (DMS) system for assessing morphology of mid gestation (gestational day 11) rat embryos. In contrast to the developmental stage focused scoring associated with the Brown and Fabro score system, this new score system assesses the respective degree of severity of dysmorphology, which delineates normal from abnormal morphology of specific embryonic structures and organ systems. This score system generates an approach that allows rapid identification and quantification of adverse developmental findings, making it conducive for characterization of compounds for teratogenic properties and screening activities.

  12. Urea production in long-term cultures of adult rat hepatocytes.

    Science.gov (United States)

    Sierra-Santoyo, A; López, M L; Hernández, A; Mendoza-Figueroa, T

    1994-04-01

    To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cycle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with medium containing either 0.4 mm arginine or 0.4 mm ornithine and in cultures exposed to different concentrations of NH(4)Cl, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea production decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 days with medium containing ornithine or arginine. Hepatocytes exposed for 24 hr to 1, 3 and 5 mm NH(4)Cl showed an average increase in urea production of 25, 50 and 69%, respectively, above that of unexposed cultures over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hepatocytes seeded on plastic substratum, urea production decreased to 24% of the initial value after 8 days in culture. OTC and arginase activities also decreased to 13 and 10% of their initial values after 8 days in culture. These results show that 3T3-hepatocyte cultures from adult rats produce urea from ornithine and/or arginine for at least 15 days and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC and arginase activities, respectively, owing probably to an adaptative response dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals.

  13. EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON GROWTH OF ISOLATED CELLS FROM EMBRYONIC CHICKEN FRONTAL BONE CULTURED IN VITRO (A HISTOCHEMICAL STUDY) Ⅱ.THE DEVELOPMENT AND MATURATION OF OSTEOBLAST-LIKE CELLS

    Institute of Scientific and Technical Information of China (English)

    徐荣辉; 柴本甫; 朱雅萍

    1993-01-01

    The isolated osteoblast-like cells from embryonic chicken frontal bone werecultured in vitro and histochemical methods adopted to observe the effect of RadixSalviac Miltiorrhizae (RSM) on proliferation, differentiation, and osteogenic capacity ofthese cells. It was found that: 1. The mitosis and proliferation of the osteoblast-like cellscould be accelerated by RSM, resulting in increased density of the cells in RSM groupas compared with the control. 2. After 48 h, the pseudopodia stretched out and drew backactively in osteoblast-like cells in RSM group. Small particles produced in the cells weresecreted through exocytosis to the extracellular medium. However, in the control group,the capacity to form and secrete these particles was limited. These particles showed posi-tive Alcian blue staining in Alcian blue-Sirius red reaction, so they were acidmucopolysaccharide particles. 3. The osteoblast-like cells could secrete vesicular particles 3micra in diameter. These vesicular particles could be stained with Alcian blue in earlystage, then they could be stained with Sirius red, and finally by Alizarin red S. Thesevesicular particles could aggregate and fuse around the cell colonies, forming bonenodules and bone flakes. The quantity and volume of the bone nodules and flakes inRSM group were larger than in the control group. 4. The bone nodules and flakes couldbe labeled vitally with tetracycline, and show strong yellow fluorescence under thefluorescence microscope. Therefore, these substances were the newly formed bone sub-stances.

  14. Collagenlα1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Osteoblasts participate in bone formation,bone mineralization,osteoclast differentiation and many pathological processes.To study the function of genes in osteoblasts using Cre-LoxP system,we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlal (Coilal) promoter(Coilatl-Cre).Two founders were identified by genomic PCR from 16 offsprings.and the integration efficiency is 12.5%.In order tO determine the tissue distribution and the activity of Cre rccombinase in the transgenic mice,the Collal-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4co/co).Multiple tissue PCR of Collal-Cre;Smad4co/+mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon.LacZ staining in the Coilal-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5.Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice.All these data indicated that the Collal-Cre transgenic mice could Serve as a valuabletool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.

  15. Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes.

    Science.gov (United States)

    Rincón-Sánchez, A R; Hernández, A; López, M L; Mendoza-Figueroa, T

    1992-01-01

    The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from female rats and explore the potential use of this culture system to study the effect of hormones, drugs and toxic chemicals on it. Hepatocytes, seeded on a feeder layer of 3T3 cells, maintained for 2 weeks their typical morphology. The cultures were able to take up [14C]acetic and [14C]oleic acid from the culture medium and incorporate them into lipids. The synthesis and secretion of lipids by [14C]acetic acid-labeled cultures had a maximum value after 11 and 13 days in culture. Triacylglycerols were the main lipidic species synthesized and secreted by hepatocytes (up to 67% of the total lipids); they also synthesized and secreted phospholipids, cholesterol and cholesterol esters from [14C]acetic acid. Similarly, [14C]oleic acid-labeled cultures synthesized and secreted mostly triacylglycerols (up to 60-70% of the total lipids), but they were also able to incorporate the labeled precursor into both cellular and secreted phospholipids and cholesterol esters. The activity of glycerol-phosphate-dehydrogenase, marker enzyme of glycerolipid synthesis, decreased slightly during the culture time whereas the activity of malic enzyme, marker of fatty acid synthesis, increased. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially triacylglycerols, from both [14C]acetic and [14C]oleic acid for at least 2 weeks and that they maintain enzyme activities related with the synthetic pathways of glycerolipids and fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. The topographical properties of silica nanoparticle film preserve the osteoblast-like cell characteristics in vitro

    Science.gov (United States)

    Shim, Wooyoung; Lee, Seung Yun; Kim, Hyo-Sop; Kim, Jae-Ho

    2016-07-01

    The Transplantation of osteoblasts, along with an artificial implant, is experimentally considered as a therapeutics for degenerative bone diseases. However, osteoblasts have several limitations for application of transplantation in therapeutics, including a low-efficiency for bone mineralization and easy loss of characteristics in in vitro culture condition. In this study, we fabricated silica nano-particle (SNP) films using particles of different sizes to culture osteoblast-like cells for analysis the effect of topography on cellular behavior and characteristics. The physical parameters of films, such as intervals, height and roughness, were proportionally increased depending on the SNP diameter. When osteoblast-like cells were cultured on the various SNP films, the cell attachment rate on SNP-300 and SNP-700 was significantly decreased when it compared to tissue culture polystyrene (TCPS) group. In addition, the genes responsible for cell adhesion showed differential expression profiles in SNP films. The expression and activity of alkaline phosphatase were elevated in SNP-300 and SNP-700, and the extra-cellular matrix and osteoblast marker showed increased gene expression in these SNP films when compared to TCPS group. In the present study, we demonstrate that the topographical property of a nano-scale structure preserves the characteristics of osteoblast-like cells, and regulates the cellular behavior.

  17. Susceptibility of primary culture neurons from rats of different ages to encephalomyocarditis (EMC) virus infection.

    Science.gov (United States)

    Su, Weiping; Ikegami, Hisashi; Nakayama, Yumi; Suzuki, Kazuhiko; Katayama, Kei-ichi; Nakayama, Hiroyuki; Doi, Kunio

    2003-10-01

    The changes in susceptibility of neurons to the D variant of EMC virus (EMC-D) (10(6) PFU/well) were investigated in developing hippocampal primary cultures from postnatal days of 1, 7, and 56 Fischer 344 rats (P1, P7, and P56) for up to 12 h after infection (12 HAI). The virus titer of primary culture neurons increased at 1 HAI, decreased at 2 HAI, increased at 3 HAI, peaked at 8 HAI, and decreased at 12 HAI in all age groups. The titers at 1 and 8 HAI were lowest in P56 cultures. The virus titer of neurons was always higher than that of culture media, especially at 1 HAI, in P1 cultures, whereas the former was lower than the latter from 2 to 3 HAI in P7 cultures and from 2 to 4 HAI in P56 cultures, respectively. Signals of viral RNA detected by in situ hybridization were first observed in the peripheral cytoplasm of neurons at 1 HAI in P1 and P7 cultures and at 4 HAI in P56 cultures, respectively. The signals spread to a large or whole area of cytoplasm and also to processes thereafter. The number of viral RNA-positive neurons and the amount of signals decreased with age. The present results indicated that the susceptibility of primary culture neurons to EMC-D decreased with age but viral replication still occurred in P56 cultures.

  18. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Sørensen, O H;

    2004-01-01

    D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP...... enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce...

  19. Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

    DEFF Research Database (Denmark)

    Bi, Yanming; Nielsen, Karina L; Kilts, Tina M;

    2006-01-01

    the effects of Bgn on 1alpha, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate...... protease inhibitor (slpi) in bgn deficient osteoblasts treated with 1,25-(OH)(2)D(3). These findings point to a novel molecular factor made by osteoblasts that could potentially be involved in LPS-induced osteolysis....

  20. Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration

    Science.gov (United States)

    Sato, Ayako; Kajiya, Hiroshi; Mori, Nana; Sato, Hironobu; Fukushima, Tadao; Kido, Hirofumi

    2017-01-01

    The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration. PMID:28060874

  1. Organotypic explant culture of adult rat retina for in vitro investigations of neurodegeneration, neuroprotection and cell transplantation

    OpenAIRE

    sprotocols

    2015-01-01

    This protocol details a method for isolating retinal tissue from adult rats as an organotypic culture to study neurobiological processes in mature tissue. It combines the efficiency and control common to in vitro techniques with close imitation of the in vivo environment. Eyes from adult rats are enucleated and the neural retina is isolated. Tissue is cut into quarters, yielding eight retinal explants per animal, and cultured at a fluid/air interface on organotypic culture membranes. Explanta...

  2. Effect of a Bone Graft Substitute β Tricalcium Phosphate on Osteoblastic Genes mRNA Exprssion

    Institute of Scientific and Technical Information of China (English)

    QIU Tong; WANG Youfa; HAN Yinchao; JIANG Xin; LI Shipu

    2012-01-01

    To investigate the molecular aspects of osteoblastic interactions with β tricalcium phosphate (β-TCP) particles,human osteoblast-like MG-63 cells were cultured with β-TCP particles at a density of 6 mg/mL culture medium for 48 h.Then,the mRNA expression of selected genes were quantified by realtime polymerase chain reaction (PCR),including the attachment-related genes (α integrin and actin),the proliferation-related gene (c-jun),and the osteoblastic markers genes (type Ⅰ collagen,osteonectin,alkaline phosphatase,RUNX2 and osteoclain).The results showed that β-TCP particles (the average size 809 nm)significantly promote the attachment and the proliferation of MG-63 cells,and slightly enhance the osteoblastic differentiation based on the analyses of the related genes expression.This study provided scientific evidences to better reveal the underlines of functions of β-TCP in bone repair.

  3. Effects of degradable MG-ND-ZN-ZR alloy on osteoblastic cell function.

    Science.gov (United States)

    Wang, Y; Ouyang, Y; Pang, X; Mao, L; Yuan, G; Jiang, Y; He, Y

    2012-01-01

    This study aimed to investigate the effects of a novel patented Mg-3Nd-0.2Zn-0.4Zr (weight %, JDBM) alloy on osteoblastic cell function, as these cells play an important role in bone repair and remodeling. The associated effects of the JDBM alloy on osteoblastic cell function involving cell adhesion, cell proliferation, and mineralization were investigated using scanning electron microscopy (SEM), MTT assay and ambramycin staining, respectively. At the same time, the in vitro degradation behavior of the JDBM alloy in cell culture medium was evaluated by the weight-loss method and SEM. Pure magnesium was used as control. The results showed that osteoblastic cells cultured on JDBM alloy samples manifested better cell adhesion, improved cell proliferation and increased mineralization ability, compared with cells seeded on pure magnesium samples. Our data indicate that the JDBM alloy has excellent bioactivity, improving the cell function of osteoblastic cells seeded on it.

  4. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    Energy Technology Data Exchange (ETDEWEB)

    Muthukumaran, Padmalosini [Department of Bioengineering, National University of Singapore (Singapore); Lim, Chwee Teck [Department of Bioengineering, National University of Singapore (Singapore); Department of Mechanical Engineering, National University of Singapore (Singapore); Mechanobiology Institute, National University of Singapore (Singapore); Singapore-MIT Alliance for Research and Technology (SMART), National University of Singapore (Singapore); Lee, Taeyong, E-mail: bielt@nus.edu.sg [Department of Bioengineering, National University of Singapore (Singapore)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  5. Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

    DEFF Research Database (Denmark)

    Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa

    2008-01-01

    Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic...... differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co......-culture, large numbers of tyrosine hydroxylase (TH)-immunoreactive, catecholaminergic cells could be found underneath individual striatal slices. Cell counting revealed that up to 25.3% (average 16.1%) of the total number of cells in these areas were TH-positive, contrasting a few TH-positive cells (

  6. Trimethyltin (TMT) neurotoxicity in organotypic rat hippocampal slice cultures

    DEFF Research Database (Denmark)

    Noraberg, J; Gramsbergen, J B; Fonnum, F

    1998-01-01

    ) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux into the culture medium, (c) cellular cobalt uptake as an index of calcium influx, (d) ordinary Nissl cell staining, and (e) immunohistochemical staining for microtubule-associated protein 2 (MAP-2). Cellular degeneration as assessed...

  7. Enhanced expression of contractile endothelin ET(B) receptors in rat coronary artery after organ culture

    DEFF Research Database (Denmark)

    Johnsson, E.; Maddahi, A.; Wackenfors, A.;

    2008-01-01

    . In cardiovascular disease and in organ culture in vitro, endothelin ET(B) receptors are up-regulated on smooth muscle cells. The objectives of the present study were to characterise the endothelin receptor-induced vasoconstriction and quantify the endothelin receptor mRNA levels and immunoreactivity in fresh...... but produced significant vasoconstriction after organ culture. The endothelin ET(B) receptor mRNA level and the receptor protein immunoreactivity were increased, whereas the level of endothelin ET(A) receptor mRNA was down-regulated but not its receptor protein immunoreactivity after organ culture...... and cultured rat coronary arteries. We demonstrate that endothelin-1 induces strong and equal concentration-dependent contractions in fresh and cultured segments from the left anterior descending coronary artery. Sarafotoxin 6c, an endothelin ET(B) receptor agonist, had negligible effect in fresh arteries...

  8. Primary culture and identification of sinoatrial node cells from newborn rat

    Institute of Scientific and Technical Information of China (English)

    宋治远; 钟理; 仝识非; 何国祥

    2003-01-01

    Objective To establish a reliable approach to primary culture and identification of sinoatrial node (SAN) cells. Methods The SAN cells were cultured from SAN tissue removed from neonatal Wistar rats and purified with differential attachment and 5'-bromodeoxyuridine (BrdU) treatment. The obtained cells were morphologically observed with inverted microscopy and transmission electron microscopy. Its action potential was recorded using electrophysiological methods.Results Three distinctly different cells were observed in the cultured SAN cells: spindle, triangle and irregular. Of these, the spindle cells comprised the greatest proportion, with their shape, structure and electrophysiological characteristics consistent with those of the pacemaker cells of SAN. The triangle cells were similar in features to the similarly shaped myocytes located in the atrial myocardium. Conclusions The culture method of differential attachment combined with BrdU treatment is a reliable approach to growing SAN cells. Of the cells cultured from SAN, the spindle cells appear to function as pacemaker cells.

  9. Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

    Directory of Open Access Journals (Sweden)

    Yongjuan Chen

    Full Text Available Zirconium (Zr is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2 or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV oxynitrate (ZrO(NO32 at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  10. Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

    Science.gov (United States)

    Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R

    2015-01-01

    Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  11. Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

    Science.gov (United States)

    Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R.

    2015-01-01

    Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling. PMID:25602473

  12. Contrasting Nephropathic Responses to Oral Administration of Extract of Cultured Penicillium polonicum in Rat and Primate

    Science.gov (United States)

    Mantle, Peter G.; McHugh, Katharine M.; Fincham, John E.

    2010-01-01

    Liquid- or solid substrate-cultured Penicillium polonicum administered in feed to rats over several days evokes a histopathological response in kidney involving apoptosis and abnormal mitosis in proximal tubules. The amphoteric toxin is yet only partly characterized, but can be isolated from cultured sporulating biomass in a fraction that is soluble in water and ethanol, and exchangeable on either anion- or cation-exchange resins. After several weeks of treatment renal proximal tubule distortion became striking on account of karyocytomegaly, but even treatment for nearly two years remained asymptomatic. Extract from a batch of solid substrate fermentation of P. polonicum on shredded wheat was incorporated into feed for rats during four consecutive days, and also given as an aqueous solution by oral gavage to a vervet monkey daily for 10 days. Treatment was asymptomatic for both types of animal. Rat response was evident as the typical renal apoptosis and karyomegaly. In contrast there was no such response in the primate; and neither creatinine clearance nor any haematological characteristic or serum component concentration deviated from a control or from historical data for this primate. The contrast is discussed concerning other negative findings for P. polonicum in pigs and hamsters. Renal karyomegaly, as a common rat response to persistent exposure to ochratoxin A, is not known in humans suspected as being exposed to more than the usual trace amounts of dietary ochratoxin A. Therefore the present findings question assumptions that human response to ochratoxin A conforms to that in the rat. PMID:22069673

  13. Contrasting Nephropathic Responses to Oral Administration of Extract of Cultured Penicillium polonicum in Rat and Primate

    Directory of Open Access Journals (Sweden)

    John E. Fincham

    2010-08-01

    Full Text Available Liquid- or solid substrate-cultured Penicillium polonicum administered in feed to rats over several days evokes a histopathological response in kidney involving apoptosis and abnormal mitosis in proximal tubules. The amphoteric toxin is yet only partly characterized, but can be isolated from cultured sporulating biomass in a fraction that is soluble in water and ethanol, and exchangeable on either anion- or cation-exchange resins. After several weeks of treatment renal proximal tubule distortion became striking on account of karyocytomegaly, but even treatment for nearly two years remained asymptomatic. Extract from a batch of solid substrate fermentation of P. polonicum on shredded wheat was incorporated into feed for rats during four consecutive days, and also given as an aqueous solution by oral gavage to a vervet monkey daily for 10 days. Treatment was asymptomatic for both types of animal. Rat response was evident as the typical renal apoptosis and karyomegaly. In contrast there was no such response in the primate; and neither creatinine clearance nor any haematological characteristic or serum component concentration deviated from a control or from historical data for this primate. The contrast is discussed concerning other negative findings for P. polonicum in pigs and hamsters. Renal karyomegaly, as a common rat response to persistent exposure to ochratoxin A, is not known in humans suspected as being exposed to more than the usual trace amounts of dietary ochratoxin A. Therefore the present findings question assumptions that human response to ochratoxin A conforms to that in the rat.

  14. Amygdalin inhibits angiogenesis in the cultured endothelial cells of diabetic rats

    Directory of Open Access Journals (Sweden)

    Hossein Mirmiranpour

    2012-01-01

    Full Text Available Background: Angiogenesis contributes to different physiological and pathological conditions. The aim of this study was to investigate for the first time the antiangiogenic effects of amygdalin on the cultured endothelial cells of diabetic rats. Materials and Methods: A total of 20 streptozotocin-induced diabetic rats were divided into two equal groups of control and amygdalin-treated animals. Eight weeks after the induction of diabetes, amygdalin was injected intraperitoneally (3 mg/kg to the rats of the treatment group. One day later, rats were sacrificed; the aortic arteries were excised and cut as 2 mm rings. Each aortic ring was incubated in a cell-culture well for 7 days. The process of angiogenesis was monitored by counting the number of microvessels and primary microtubules in each well. Results: Optic microscopy showed proliferation and migration of new endothelial cells to the fibrin gels. The endothelial cells produced primary microtubules which gradually made several branches and finally made a vascular matrix. The number of the primary microtubules and microvessels were significantly lower in the amygdalin-treated vs. control group (P < 0.01. Conclusion: Therefore, amygdalin exerts inhibitory effects on angiogenesis in aortic rings of diabetic rats and may pave a new way for treatment of unfavorable angiogenic conditions.

  15. Amygdalin inhibits angiogenesis in the cultured endothelial cells of diabetic rats.

    Science.gov (United States)

    Mirmiranpour, Hossein; Khaghani, Shahnaz; Zandieh, Ali; Khalilzadeh, O Omid; Gerayesh-Nejad, Siavash; Morteza, Afsaneh; Esteghamati, Alireza

    2012-01-01

    Angiogenesis contributes to different physiological and pathological conditions. The aim of this study was to investigate for the first time the antiangiogenic effects of amygdalin on the cultured endothelial cells of diabetic rats. A total of 20 streptozotocin-induced diabetic rats were divided into two equal groups of control and amygdalin-treated animals. Eight weeks after the induction of diabetes, amygdalin was injected intraperitoneally (3 mg/kg) to the rats of the treatment group. One day later, rats were sacrificed; the aortic arteries were excised and cut as 2 mm rings. Each aortic ring was incubated in a cell-culture well for 7 days. The process of angiogenesis was monitored by counting the number of microvessels and primary microtubules in each well. Optic microscopy showed proliferation and migration of new endothelial cells to the fibrin gels. The endothelial cells produced primary microtubules which gradually made several branches and finally made a vascular matrix. The number of the primary microtubules and microvessels were significantly lower in the amygdalin-treated vs. control group (P amygdalin exerts inhibitory effects on angiogenesis in aortic rings of diabetic rats and may pave a new way for treatment of unfavorable angiogenic conditions.

  16. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    Science.gov (United States)

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B

    1993-08-01

    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

  17. Differential feedback regulation of cholesterol 7α-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Lehmann, E.M.; Princen, H.M.G.

    1993-01-01

    We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7α-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most

  18. Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

    DEFF Research Database (Denmark)

    Meyer, Morten; Widmer, H R; Wagner, B

    1998-01-01

    improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...... days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls...... numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after...

  19. Differentiation of osteoblasts on pectin-coated titanium.

    Science.gov (United States)

    Kokkonen, H; Cassinelli, C; Verhoef, R; Morra, M; Schols, H A; Tuukkanen, J

    2008-09-01

    The gold standard for implant metals is titanium, and coatings such as collagen-I, RGD-peptide, chondroitin sulfate, and calcium phosphate have been used to modify its biocompatibility. We investigated how titanium coated with pectins, adaptable bioactive plant polysaccharides with anti-inflammatory effects, supports osteoblast differentiation. MC3T3-E1 cells, primary murine osteoblasts, and human mesenchymal cells (hMC) were cultured on titanium coated with rhamnogalacturonan-rich modified hairy regions (MHR-A and MHR-B) of apple pectin. Alkaline phosphatase (ALP) expression and activity, calcium deposition, and cell spreading were investigated. MHR-B, but not MHR-A, supports osteoblast differentiation. The MHR-A surface was not mineralized, but on MHR-B, the average mineralized area was 14.0% with MC3T3-E1 cells and 26.6% with primary osteoblasts. The ALP activity of hMCs on MHR-A was 58.3% at day 7 and 9.3% from that of MHR-B at day 10. These data indicate that modified pectin nanocoatings may enhance the biocompatibility of bone and dental implants.

  20. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E

    1981-01-01

    The effect of partially purified extracts from adult pig brains containing a glia maturation protein factor (BE) has been investigated on neural cells during carcinogenesis. Pregnant BD IX-rats were given a single transplacental dose of the carcinogen ethylnitrosourea (EtNU) on the 18th day of ge...... on GFA-content was seen any longer, although some few weakly GFA positive cells could be observed in all permanent cell lines. Fetal rat brain cells therefore seem to become less responsive to this differentiation inducer during neoplastic transformation in cell culture....

  1. Effects of Nitric Oxide Production Inhibitor Named, NG-Nitro-L-Arginine Methyl Ester (L-NAME, on Rat Mesenchymal Stem Cells Differentiation

    Directory of Open Access Journals (Sweden)

    E Arfaei

    2010-04-01

    Full Text Available Introduction & Objectives: Recently, the findings of some studies have shown that, nitric oxide (NO probably has an important role in differentiation of mesenchymal stem cells to osteoblasts. The aim of the present investigation was to study the effects of nitric oxide production inhibitor named, NG-nitro-L-arginine methyl ester (L-NAME, on rat mesenchymal stem cells differentiation to osteoblasts in vitro. Materials & Methods: This was an experimental study conducted at Hamedan University of Medical Sciences in 2009, in which rat bone marrow stem cells were isolated in an aseptic condition and cultured in vitro. After third passage, the cells were cultured in osteogenic differentiation medium. To study the effects of L-NAME on osteogenic differentiation, the L-NAME was added to the culture medium at a concentration of 125, 250, and 500 μM in some culture plates. During the culture procedure, the media were replaced with fresh ones, with a three days interval. After 28 days of culturing the mineralized matrix was stained using Alizarian red staining method. The gathered data were analyzed by SPSS software version 12 using one way ANOVA. Results: The findings of this study showed that in the presence of L-NAME, differentiation of bone marrow mesenchymal stem cells to osteoblasts was disordered and matrix mineralization significantly decreased in a dose dependent manner. Conclusion: This study revealed that, inhibition of nitric oxide production using L-NAME can prevent the differentiation of rat bone marrow mesenchymal stem cells to osteoblast. The results imply that NO is an important constituent in differentiation of mesenchymal stem cell to osteoblasts.

  2. Alterations of mineral elements in osteoblast during differentiation under hypo, moderate and high static magnetic fields.

    Science.gov (United States)

    Zhang, Jian; Ding, Chong; Shang, Peng

    2014-12-01

    Static magnetic fields (SMFs) can enhance the ability of bone formation by osteoblast and is a potential physical therapy to bone disorders and the maintenance of bone health. But, the mechanism is not clear yet. Certain mineral elements including macro and trace elements are essential for normal bone metabolism. Deficiency of these elements can cause severe bone disorders including osteoporosis. However, there are few reports regarding the role of mineral elements in the regulation of bone formation under SMFs. In this study, hypomagnetic field (HyMF) of 500 nT, moderate SMF (MMF) of 0.2 T, and high SMF (HiMF) of 16 T were used to investigate the effects of SMFs on mineral element (calcium, copper, iron, magnesium, manganese, and zinc) alteration of MC3T3-E1 cells during osteoblast mineralization. The results showed that osteoblasts in differentiation accumulated more mineral elements than non-differentiated cell cultures. Furthermore, HyMF reduced osteoblast differentiation but did not affect mineral elements levels compared with control of geomagnetic field. MMF decreased osteoblast differentiation with elevated iron content. HiMF enhanced osteoblast differentiation and increased all the mineral contents except copper. It is suggested that the altered potential of osteoblast differentiation under SMFs may partially due to the involvement of different mineral elements.

  3. Studies on responsiveness of hepatoma cells to catecholamines. II. Comparison of beta-adrenergic responsiveness of rat ascites hepatoma cells with cultured normal rat liver cells.

    Science.gov (United States)

    Miyamoto, K; Matsunaga, T; Takemoto, N; Sanae, F; Koshiura, R

    1985-05-01

    The pharmacological properties of beta-adrenoceptors in rat ascites hepatoma cells were compared with those in normal rat liver cells which were cultured for 24 hr after collagenase digestion. Adenylate cyclases in the homogenates of cultured normal rat liver cells and rat ascites hepatoma cells, AH44, AH66, AH109A, AH130 and AH7974, were all activated by isoproterenol or NaF to different degrees. The enzyme in rat liver cells was activated by several beta 2-agonists but those in all hepatoma cells hardly responded. Furthermore, salbutamol, a beta 2-partial agonist, antagonized the cyclase activation by isoproterenol in AH130 cells. The Kact value of isoproterenol for the activation of adenylate cyclase in AH130 cells was smaller than that in rat liver cells. A comparison of the Ki values of beta-antagonists for the inhibition of isoproterenol-stimulated cyclase activity shows that while the Ki values of propranolol and butoxamine in AH130 cells were similar to those in rat liver cells, a significant difference was observed in the values for beta 1-selective antagonists between AH130 cells and rat liver cells. The Ki values of metoprolol and atenolol for AH130 cells were 137- and 90-fold lower, respectively, than for normal rat liver cells. From these findings, it is strongly suggested that beta-adrenoceptors in rat ascites hepatoma cells including AH130 cells have similar properties to the mammalian beta 1-receptor.

  4. Identification of differentially expressed genes in aflatoxin B1-treated cultured primary rat hepatocytes and Fischer 344 rats.

    Science.gov (United States)

    Harris, A J; Shaddock, J G; Manjanatha, M G; Lisenbey, J A; Casciano, D A

    1998-08-01

    Aflatoxin B1 (AFB1), a mutagen and hepatocarcinogen in rats and humans, is a contaminant of the human food supply, particularly in parts of Africa and Asia. AFB1-induced changes in gene expression may play a part in the development of the toxic, immunosuppressive and carcinogenic properties of this fungal metabolite. An understanding of the-role of AFB1 in modulating gene regulation should provide insight regarding mechanisms of AFB1-induced carcinogenesis. We used three PCR-based subtractive techniques to identify AFB1-responsive genes in cultured primary rat hepatocyte RNA: differential display PCR (DD-PCR), representational difference analysis (RDA) and suppression subtractive hybridization (SSH). Each of the three techniques identified AFB1-responsive genes, although no individual cDNA was isolated by more than one technique. Nine cDNAs isolated using DD-PCR, RDA or SSH were found to represent eight genes that are differentially expressed as a result of AFB1 exposure. Genes whose mRNA levels were increased in cultured primary rat hepatocytes after AFB1 treatment were corticosteroid binding globulin (CBG), cytochrome P450 4F1 (CYP4F1), alpha-2 microglobulin, C4b-binding protein (C4BP), serum amyloid A-2 and glutathione S-transferase Yb2 (GST). Transferrin and a small CYP3A-like cDNA had reduced mRNA levels after AFB1 exposure. Full-length CYP3A mRNA levels were increased. When liver RNA from AFB1-treated male F344 rats was evaluated for transferrin, CBG, GST, CYP3A and CYP4F1 expression, a decrease in transferrin mRNA and an increase in CBG, GST, CYP3A and CYP4F1 mRNA levels was also seen. Analysis of the potential function of these genes in maintaining cellular homeostasis suggests that their differential expression could contribute to the toxicity associated with AFB1 exposure.

  5. In vitro culture of osteoblasts: A bibliometric analysis based on Science Citation Index, National Institutes of Health and Derwent Innovations Index from 2008 to 2010%成骨细胞的体外培养:来自SCI及NIH基金和德温特专利数据库2008/2010相关文献及数据分析

    Institute of Scientific and Technical Information of China (English)

    芮钢; 金旭红; 郭元利; 胡宝山

    2012-01-01

    BACKGROUND: Osteoblasts play an important role in the regulation of bone metabolism. The bone formation and loss are affected by osteoblast differentiation and apoptosis. OBJECTIVE: To identify the global research trends in in vitro culture of osteoblasts from 2008 to 2010 via a bibliometric analysis of Science Citation Index (SCI), National Institutes of Health (NIH) and Derwent Innovations Index. DESIGN: A bibliometric study. DATA RETRIEVAL: We performed a bibliometric analysis based on the SCI, NIH and Derwent Innovations Index for data regarding in vitro culture of osteoblasts from 2008 to 2010. The data were downloaded on October 18, 2011. SELECTION CRITERIA: Articles closely related to ①in vitro culture of osteoblasts, ②with funding item for in vitro culture of osteoblasts, and ③with patents related to in vitro culture of osteoblasts were included. Exclusive criteria: document types such ①as meeting abstracts, reviews, proceedings papers, notes, and letters; non②-published articles. MAIN OUTCOME MEASUREMENTS: ①Types of documents; number of author outputs②; distribution of output in subject ③categories; publication distribution of countries and institutes④; distribution of output in journals⑤; dis⑥tribution of citations; ⑦funding items; patents. ⑧ RESULTS: During 2008 to 2010, 2 612 papers focus①ing on in vitro culture of osteoblasts were added to the SCI. Articles were the most frequently used document type. The result showed that② the amount of literature was annually increased, and there were 22 core institutes, 12 core journals and 13 classical references. The Journal of Biomedical Materials Research Part A published most articles addressing in vitro culture of osteoblasts, followed by Biomaterials and Bone. Totally 1 443 articles③ (60.3%) obtained funding supports from 2 090 funding agencies. A total of 398 international patents were searched. All results ④demonstrated that the topics in osteoblast research are being

  6. Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis

    Directory of Open Access Journals (Sweden)

    Lin Shin-Jen

    2012-06-01

    Full Text Available Abstract Background Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. Methods We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP assays, and electrophoretic mobility shift assays (EMSA were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. Results We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Conclusions Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

  7. Isolation, culture and differentiation of rabbit peripheral blood mesenchymal stem cells into osteoblasts in vitro%兔外周血间充质干细胞的体外分离培养及诱导成骨

    Institute of Scientific and Technical Information of China (English)

    李中华

    2009-01-01

    BACKGROUND: A few mesenchymal stem cells (MSCs) can be found in peripheral blood.OBJECTIVE: To isolate rabbit peripheral blood MSCs and induce its differentiation into osteoblasts.DESIGN, TIME AND SE'I-I'ING: The cytological in vitro study was performed at the Central Laboratory of Shangdong Provincial Hospital from June to December 2008.MATERIALS: A total of 6 New Zealand rabbits were purchased from Shandong Academy of Agricultural Sciences. α-MEM and L-DiEM were bought from Hyclone.METHODS: Full-thickness skin (3 cm×3 cm) (dorsal muscular layer was left) was incised at various sites of rabbit back, every 3days. Incised skin was dressed following orthotopic transplantation. Each rabbit received four consecutive wounds. Peripheral blood was collected from femoral vein before injury and 1 week after injury. MSCs were harvested from peripheral blood by density gradient cantrifugation. MSCs were divided into 2 groups, which were respectively incubated in α-MEM supplemented with 10% fetal bovine serum and L-DiEM. Cells at passage 2 and 1 ×105/cm2 were incubated in a 12-well plate and induced with H-DiEM containing osteogenic inductor.MAIN OUTCOME MEASURES: The following parameters were measured: cell morphology before and after injury; colony forming efficiency of MSCs; outcome of osteogenic induction.RESULTS: Following primary medium change and before injury, obtained cells were normal. Twenty-four hours following incubation and after injury, MSCs were spindle or polygonal, and adhered to the wall. 5-6 days later, cell colonies appeared.Compared with the L-DiEM group, the number of primary culture colony formation in α-MEM group was significantly greater (P < 0.05). Peripheral blood MSCs were spindle, tdangle or polygonal, with the presence of processes, 2-3 nuclei and cell division phase, and slowly proliferated following osteogenic induction. At day 7 after differentiation of MSCs into osteoblasts, positive rate of alkaline phosphates was above 80%. At day 21

  8. In vitro study of vancomycin release and osteoblast-like cell growth on structured calcium phosphate-collagen

    Energy Technology Data Exchange (ETDEWEB)

    Pon-On, Weeraphat, E-mail: wponun@yahoo.com [Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip [Center of Calcium and Bone Research, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand); Tang, I-Ming [ThEP Center, Commission of Higher Education. 328 Si Ayuthaya Rd., Bangkok 10400 (Thailand); Department of Physics, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand)

    2013-04-01

    A drug delivery vehicle consisting of spherical calcium phosphate-collagen particles covered by flower-like (SFCaPCol) blossoms composed of nanorod building blocks and their cellular response is studied. The spherical structure was achieved by a combination of sonication and freeze-drying. The SFCaPCol blossoms have a high surface area of approximately 280 m{sup 2}g{sup −1}. The blossom-like formation having a high surface area allows a drug loading efficiency of 77.82%. The release profile for one drug, vancomycin (VCM), shows long term sustained release in simulated body fluid (SBF), in a phosphate buffer saline (PBS, pH 7.4) solution and in culture media over 2 weeks with a cumulative release ∼ 53%, 75% and 50%, respectively, over the first 7 days. The biocompatibility of the VCM-loaded SFCaPCol scaffold was determined by in vitro cell adhesion and proliferation tests of rat osteoblast-like UMR-106 cells. MTT tests indicated that UMR-106 cells were viable after exposure to the VCM loaded SFCaPCol, meaning that the scaffold (the flower-like blossoms) did not impair the cell's viability. The density of cells on the substrate was seen to increase with increasing cultured time. - Graphical abstract: A spherical calcium phosphate-collagen with flower-like blossoms consisting of nanorod building blocks (SFCaPCol) particles was achieved by a combination of sonication and freeze-drying. In vitro drug release profile and the biocompatibility of the VCM-loaded SFCaPCol composite cell adhesion and proliferation in rat osteoblast-like UMR-106 cells were determined for biomaterial applications. Highlights: ► SFCaPCol and VCM-loaded SFCaPCol composite were synthesized by a combination of ultra sonication and freeze-drying. ► VCM drug-loaded SFCaPCol composite was used as substrate for the growth of rat osteoblast-like UMR-106 cells. ► Controlled release of VCM from the composite is critically medium dependent. ► The VCM-loaded SFCaPCol composite is also

  9. Estradiol Synthesis and Release in Cultured Female Rat Bone Marrow Stem Cells

    Directory of Open Access Journals (Sweden)

    Dalei Zhang

    2013-01-01

    Full Text Available Bone marrow stem cells (BMSCs have the capacity to differentiate into mature cell types of multiple tissues. Thus, they represent an alternative source for organ-specific cell replacement therapy in degenerative diseases. In this study, we demonstrated that female rat BMSCs could differentiate into steroidogenic cells with the capacity for de novo synthesis of Estradiol-17β (E2 under high glucose culture conditions with or without retinoic acid (RA. The cultured BMSCs could express the mRNA and protein for P450arom, the enzyme responsible for estrogen biosynthesis. Moreover, radioimmunoassay revealed that BMSCs cultured in the present culture system produced and secreted significant amounts of testosterone, androstenedione, and E2. In addition, RA promoted E2 secretion but did not affect the levels of androgen. These results indicate that BMSCs can synthesize and release E2 and may contribute to autologous transplantation therapy for estrogen deficiency.

  10. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures.

    Science.gov (United States)

    Lepsch, Lucilia B; Planeta, Cleopatra S; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  11. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    Directory of Open Access Journals (Sweden)

    Lucilia B. Lepsch

    2015-01-01

    Full Text Available To study cocaine’s toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2 and/or neuronal nucleus protein (NeuN staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  12. Differentiation of rat embryonic neural stem cells promoted by co-cultured Schwann cells

    Institute of Scientific and Technical Information of China (English)

    万虹; 安沂华; 张泽舜; 张亚卓; 王忠诚

    2003-01-01

    Objective To explore the factors which induce differentiation of embryonic neural stem cells. Methods Rat embryonic neural stem cells were co-cultured with newborn rat Schwann cells in serum-free medium. The phenotype and specific-markers including tubulin-β, glial fibrillary acidic protein (GFAP) and galactorcerebroside (GalC), were domonstrated by phase contrast microscopy and double immunofluorescence staining. Results Overall, 80%±5% of neural stem cells protruded several elongated processes and expressed tubulin-β antigen at high levels, while 20±3% of them protruded several short processes and were GalC or GFAP positive. Conclusion The factors secreted by Schwann cells could induce rat embryonic neural stem cell to differentiate.

  13. Regulation of period 1 expression in cultured rat pineal

    Science.gov (United States)

    Fukuhara, Chiaki; Dirden, James C.; Tosini, Gianluca

    2002-01-01

    The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.

  14. Fabrication of poly(ethylene glycol) hydrogel micropatterns with osteoinductive growth factors and evaluation of the effects on osteoblast activity and function

    Science.gov (United States)

    Subramani, K.; Birch, M. A.

    2006-09-01

    The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of protein trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 µm were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as 10 µm and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that protein could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the morphogenetic properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (bone) regeneration in dental and

  15. Fabrication of poly(ethylene glycol) hydrogel micropatterns with osteoinductive growth factors and evaluation of the effects on osteoblast activity and function

    Energy Technology Data Exchange (ETDEWEB)

    Subramani, K [Institute for Nanoscale Science and Technology (INSAT), University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7AR (United Kingdom); Birch, M A [Institute for Nanoscale Science and Technology (INSAT), University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7AR (United Kingdom)

    2006-09-15

    The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of protein trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 {mu}m were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as 10 {mu}m and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that protein could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the morphogenetic properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (bone) regeneration in dental and

  16. IL-1β and IL-6 modulate apolipoprotein E gene expression in rat hepatocyte primary culture

    Directory of Open Access Journals (Sweden)

    Agnes Ribeiro

    1992-01-01

    Full Text Available Incubation of rat hepatocytes in primary culture with IL-1β at a concentration of 2.5 units/ml resulted in an increase (+80% in the amount of apoE mRNA without any effect upon apoE synthesis. IL-6 at a low concentration (10 units/ml induced a decrease (−35% in the amount of apoE mRNA, but increased apoE synthesis (+28%. No effect was observed with higher concentrations of IL-1β (10 units/ml or IL-6 (100 units/ml. These results suggest that inflammatory cytokines IL-1β and IL-6 modulate the expression of apoE gene in cultured rat hepatocytes, at a concentration that does not induce the acute phase response.

  17. Although it is rapidly metabolized in cultured rat hepatocytes, lauric acid is used for protein acylation

    OpenAIRE

    Rioux, Vincent; Daval, Stéphanie; Guillou, Hervé; Jan, Sophie; Legrand, Philippe

    2003-01-01

    International audience; This study was designed to examine the metabolic fate of exogenous lauric acid in cultured rat hepatocytes, in terms of both lipid metabolism and acylation of proteins. Radiolabeled [ 1-$^{14}$C] -lauric acid at 0.1 mM in the culture medium was rapidly taken up by the cells ($94.8 \\pm 2.2\\%$ of the initial radioactivity was cleared from the medium after a 4 h incubation) but its incorporation into cellular lipids was low ($24.6 \\pm 4.2\\%$ of initial radioactivity after...

  18. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-brain...... barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  19. Toxic effect of the glycoalkaloids solanine and tomatine on cultured neonatal rat heart cells.

    Science.gov (United States)

    Bergers, W W; Alink, G M

    1980-06-01

    The toxic effects of the glycoalkaloids, alpha-solanine and tomatine, were studied in beating heart cell cultures from 1--2-day-old rats. After addition of alpha-solanine (80 microgram/ml) and tomatine (40 microgram/ml) to the culture medium, the cells ceased beating within a few minutes. At a concentration of 40 microgram/ml alpha-solanine and 20 microgram/ml tomatine, both compounds caused a pronounced increase of the contraction frequency, lasting for at least 2h. K-strophantin, a reference heart glycoside, caused arrhythmic beating at 20 microgram/ml and complete cessation of contractions at 160 microgram/ml.

  20. Vasoactive intestinal peptide can promote the development of neonatal rat primordial follicles during in vitro culture.

    Science.gov (United States)

    Chen, Niannian; Li, Yu; Wang, Wenjun; Ma, Yun; Yang, Dongzi; Zhang, Qingxue

    2013-01-01

    Recruitment of primordial follicles is essential for female fertility. Some of the intraovarian growth factors involved in the initiation of primordial follicle growth have been identified, but the exact mechanisms regulating follicle activation are poorly understood. Strong evidence indicates that vasoactive intestinal peptide (VIP), a neuropeptide found in ovarian nerves, plays a role in the physiology of follicle development and function. The aim of the present study was to determine whether VIP might regulate the activation and growth of neonatal rat primordial follicles in an in vitro culture system. Ovaries from 4-day-old rats were cultured for 14 days in medium containing 10(-7) M VIP. At the end of the culture, the developmental stages and viability of the follicles were evaluated using histological sections. Immunohistochemistry studies for proliferating cell nuclear antigen (PCNA) were performed to assess the mitotic activity of granulosa cells. In addition, the expression level of kit ligand (KL) mRNA was examined after culture. Histology showed that primordial follicles could survive and start to grow in vitro. The proportion of primordial follicles was decreased and the proportion of early primary follicles increased after in vitro culture with VIP. Immunolocalization of PCNA showed that follicle growth was initiated after VIP treatment. The expression level of KL mRNA was increased in the VIP treatment group. Thus, VIP can promote primordial follicle development, possibly mediated in part through upregulating the expression of KL.

  1. Surface proteins in normal and transformed rat liver epithelial cells in culture.

    Science.gov (United States)

    Bannikov, G. A.; Saint Vincent, L.; Montesano, R.

    1980-01-01

    The pattern of surface proteins of different types of normal and transformed rat liver cells have been studied in culture by means of lactoperoxidase-catalysed iodination procedures, followed by SDS-gel electrophoresis. The cells examined were primary cultures of epithelial liver cells, long-term cultures of epithelial liver cells, in vitro transformed epithelial liver cell lines and liver tumour-cell lines; mesenchymal cells from liver and skin were also examined. The principal surface proteins of primary cultures of epithelial cells from adult or neonatal rats had components with mol. wts of 140,000-160,000, 100,000 and 40,000-70,000. A band that had the same position as fibronectin from mesenchymal cells was also present and this band, as well as other iodinated components, were less sensitive to trypsin than fibroblastic fibronectin. A similar pattern of iodinated proteins was seen in long-term cultures of epithelial liver cells, with a great reduction in the number and intensity of the bands in the mol. wt region below 100,000. Almost all the in vitro transformed and tumour epithelial cell lines contain a protein with a mol. wt 135,000 as one of the major iodinated bands, and in contrast to the observation in transformed fibroblasts, the fibronectin was retained by most of these transformed cell lines. Images Fig. 1 Fig. 2 Fig. 3 PMID:7053205

  2. Glutamate enhances the expression of vascular endothelial growth factor in cultured SD rat astrocytes

    Institute of Scientific and Technical Information of China (English)

    Chong-xiao Liu; Yong Liu; Wei Shi; Xin-lin Chen; Xin-li Xiao; Ling-yu Zhao; Yu-mei Tian; Jun-feng Zhang

    2009-01-01

    Objective To study the effect of glutamate on the expression of vascular endothelial growth factor (VEGF) mRNA and protein in cultured rat astrocytes. Methods Cultured rat astrocytes were randomly divided into 6 groups: control group (C), glutamate group (G), QA group (Q), DCG-IV group (D), L-AP4 group (L) and glutanmte-FMCPG gronp (G+M). Cells were cultured under nomoxic condition (95% air, 5% CO2). RT-PCR and ELISA methods were used to detect the expression of VEGF mRNA and protein in cultured astrocytes, respectively. G+ M group was preincubated with lmM MCPG for 30 min prior to the stimulation with glutamate. There were 7 time points at 0,4,8,12,16,24 and 48 h in each group except G+M group. Results The expression of VEGF mRNA and protein did not differ significantly among D group, L group and C group. Different from that in C group, the expression of VEGF mRNA and protein could be enhanced both in a dose-dependent and time-dependent manner in G group and Q group. Meanwhile, the enhanced expression of VEGF mRNA and protein in G group was completely suppressed by MCPG after 24 h. Conclusion Glutamate can increase the expression of VEGF mRNA and protein in cultured astrocytes, which may be due to the activation of group I metabotropic glutamate receptors in astrocytes.

  3. Identification and pharmacological characterization of the histamine H3 receptor in cultured rat astrocytes.

    Science.gov (United States)

    Mele, Tina; Jurič, Damijana Mojca

    2013-11-15

    Recently we reported that cultured rat cortical astrocytes express histamine H3 receptor that is functionally coupled to Gi/o proteins and participates to the stimulatory effect of histamine. Due to the lack of data on the distribution of histamine H3 receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions. Real-time PCR was performed to examine the expression of native histamine H3 receptor in cultured rat astrocytes from cortex,cerebellum, hippocampus and striatum.Double-antigen immunofluorescence staining and[3H]N-α-methylhistamine([3H]NαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes. Histamine H3 receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes.Double-antigen immunofluorescence confirmed the presence of histamine H3 receptors on the membrane of all examined astroglial populations.[3H]NαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes(KD¼4.5570.46 nM; Bmax¼5.6370.21 fmol/mg protein)and competition assays with selective agonists and antagonists were consistent with labeling of histamine H3 receptor(range of pKi values 7.50–8.87). Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H3 receptors.The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.

  4. Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

    OpenAIRE

    Sato, K.; Robbins, J

    1981-01-01

    Primary cultured adult rat hepatocytes were used to study regulation of thyroid hormone deiodination. Control studies showed that these cells survived for at leas 4 d, during which time they actively deiodinated both the phenolic (5'-) and non-phenolic (5-) rings of L-thyroxine (T4),3,5,3'-triiodo-L-thyronine, and 3,3',5'-triiodothyronine. Increasing the substate concentration caused a decrease in fractional iodide release and a corresponding increase in conjugation with sulfate and glucuroni...

  5. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    Science.gov (United States)

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  6. Mechanical strain promotes osteoblast ECM formation and improves its osteoinductive potential

    Directory of Open Access Journals (Sweden)

    Guo Yong

    2012-10-01

    Full Text Available Abstract Background The extracellular matrix (ECM provides a supportive microenvironment for cells, which is suitable as a tissue engineering scaffold. Mechanical stimulus plays a significant role in the fate of osteoblast, suggesting that it regulates ECM formation. Therefore, we investigated the influence of mechanical stimulus on ECM formation and bioactivity. Methods Mouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with mechanical tensile strain. After removing the cells, the ECMs coated on dishes were prepared. The ECM protein and calcium were assayed and MC3T3-E1 cells were re-seeded on the ECM-coated dishes to assess osteoinductive potential of the ECM. Results The cyclic tensile strain increased collagen, bone morphogenetic protein 2 (BMP-2, BMP-4, and calcium levels in the ECM. Compared with the ECM produced by unstrained osteoblasts, those of mechanically stimulated osteoblasts promoted alkaline phosphatase activity, elevated BMP-2 and osteopontin levels and mRNA levels of runt-related transcriptional factor 2 (Runx2 and osteocalcin (OCN, and increased secreted calcium of the re-seeded MC3T3-E1 cells. Conclusion Mechanical strain promoted ECM production of osteoblasts in vitro, increased BMP-2/4 levels, and improved osteoinductive potential of the ECM. This study provided a novel method to enhance bioactivity of bone ECM in vitro via mechanical strain to osteoblasts.

  7. Cultured human embryonic neocortical cells survive and grow in infarcted cavities of adult rat brains and interconnect with host brain

    Institute of Scientific and Technical Information of China (English)

    ZENG Jin-sheng; YU Jian; CUI Chun-mei; ZHAO Zhan; HONG Hua; SHENG Wen-li; TAO Yu-qian; LI Ling; HUANG Ru-xun

    2005-01-01

    Background There are no reports on exnografting cultured human fetal neocortical cells in this infracted cavities of adult rat brains. This study was undertaken to observe whether cultured human cortical neurons and astrocytes can survive and grow in the infarcted cavities of adult rat brains and whether they interconnect with host brains.Methods The right middle cerebral artery was ligated distal to the striatal branches in 16 adult stroke-prone renovascular hypertensive rats. One week later, cultured cells from human embryonic cerebral cortexes were stereotaxically transferred to the infarcted cavity of 11 rats. The other 5 rats receiving sham transplants served as controls. For immunosuppression, all transplanted rats received intraperitoneal injection of cyclosporine A daily starting on the day of grafting. Immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament, and microtubule associated protein-2 (MAP-2) was performed on brain sections perfused in situ 8 weeks after transplantation.Results Grafts in the infarcted cavities of 6 of 10 surviving rats consisted of bands of neurons with an immature appearance, bundles of fibers, and GFAP-immunopositive astrocytes, which were unevenly distributed. The grafts were rich in synaptophysin, neurofilament, and MAP2-positive neurons with long processes. The graft/host border was diffuse with dendrites apparently bridging over to the host brain, into which neurofilament immunopositive fibers protruded. Conclusion Cultured human fetal brain cells can survive and grow in the infarcted cavities of immunodepressed rats and integrate with the host brain.

  8. The metabolism of malate by cultured rat brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. (Department of Pediatrics, University of Maryland School of Medicine, Baltimore (USA))

    1990-12-01

    Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.

  9. Therapy of Chronic Cardiosclerosis in WAG Rats Using Cultures of Cardiovascular Cells Enriched with Cardiac Stem Cell.

    Science.gov (United States)

    Chepeleva, E V; Pavlova, S V; Malakhova, A A; Milevskaya, E A; Rusakova, Ya L; Podkhvatilina, N A; Sergeevichev, D S; Pokushalov, E A; Karaskov, A M; Sukhikh, G T; Zakiyan, S M

    2015-11-01

    We developed a protocol for preparing cardiac cell culture from rat heart enriched with regional stem cells based on clonogenic properties and proliferation in culture in a medium with low serum content. Experiments on WAG rats with experimental ischemic myocardial damage showed that implantation of autologous regional stem cells into the left ventricle reduced the volume of cicatricial tissue, promoted angiogenesis in the damaged zone, and prevented the risk of heart failure development.

  10. Regulation of Osteoblast Differentiation by Acid-Etched and/or Grit-Blasted Titanium Substrate Topography Is Enhanced by 1,25(OH)2D3 in a Sex-Dependent Manner.

    Science.gov (United States)

    Olivares-Navarrete, Rene; Hyzy, Sharon L; Boyan, Barbara D; Schwartz, Zvi

    2015-01-01

    This study assessed contributions of micron-scale topography on clinically relevant titanium (Ti) to differentiation of osteoprogenitor cells and osteoblasts; the interaction of this effect with 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3); and if the effects are sex-dependent. Male and female rat bone marrow cells (BMCs) were cultured on acid-etched (A, R a = 0.87 μm), grit-blasted (GB, R a = 3.90 μm), or grit-blasted/acid-etched (SLA, R a = 3.22 μm) Ti. BMCs were sensitive to surface topography and underwent osteoblast differentiation. This was greatest on SLA; acid etching and grit blasting contributed additively. Primary osteoblasts were also sensitive to SLA, with less effect from individual structural components, demonstrated by enhanced local factor production. Sex-dependent responses of BMCs to topography varied with parameter whereas male and female osteoblasts responded similarly to surface treatment. 1α,25(OH)2D3 enhanced cell responses on all surfaces similarly. Effects were sex-dependent and male cells grown on a complex microstructured surface were much more sensitive than female cells. These results indicate that effects of the complex SLA topography are greater than acid etching or grit blasting alone on multipotent BMCs and committed osteoblasts and that individual parameters are sex-specific. The effect of 1α,25(OH)2D3 was sex dependent. The results also suggest that levels of 1α,25(OH)2D3 in the patient may be important in osseointegration.

  11. Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays.

    Science.gov (United States)

    Ito, D; Tamate, H; Nagayama, M; Uchida, T; Kudoh, S N; Gohara, K

    2010-11-24

    To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

  12. Simvastatin inhibits leptin-induced hypertrophy in cultured neonatal rat cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Tai-ping HU; Fang-ping XU; Yuan-jian LI; Jian-dong LUO

    2006-01-01

    Aim:To test the hypothesis that statins inhibit leptin-induced hypertrophy in cultured neonatal rat cardiomyocytes.Methods:Cultured neonatal rat cardiomyocytes were used to evaluate the effects of simvastatin on leptininduced hypertrophy.Intracellular reactive oxygen species (ROS) levels were determined by using 2',7'-dichlorofluorescein diacetate (DCF-DA) fluorescence.Total intracellular RNA and cell protein content,which serve as cell proliferative markers,were assayed by using propidium iodide (PI) fluorescence and the Bio-Rad DC protein assay.respectively.The cell surface area,an indicator of cell hypertrophy,was quantified by using Leica image analysis software.Results:After 72 h treatment,1eptin markedly increased RNA 1evels,cell surface area,and total cell protein levels in cardiomyocytes,which were significantly inhibited by simvastatin or catalase treatment.ROS levels were significantly elevated in cardiomyocytes treated with leptin for 4 h compared with those cells without leptin treatment.The increase in ROS levels in cardiomyocytes induced by leptin was reversed by treatment with simvastatin and catalase.Conclusion:Simvastatin inhibits leptin-induced ROS-mediated hyperophy in cultured neonatal rat cardiac myocytes.Statin therapy may provide an effective means of improving cardiac dysfunction in obese humans.

  13. Characterization of primary rat nasal epithelial cultures in CFTR knockout rats as a model for CF sinus disease.

    Science.gov (United States)

    Tipirneni, Kiranya E; Cho, Do-Yeon; Skinner, Daniel F; Zhang, Shaoyan; Mackey, Calvin; Lim, Dong-Jin; Woodworth, Bradford A

    2017-08-03

    The objectives of the current experiments were to develop and characterize primary rat nasal epithelial cultures and evaluate their usefulness as a model of cystic fibrosis (CF) sinonasal transepithelial transport and CF transmembrane conductance regulator (CFTR) function. Laboratory in vitro and animal studies. CFTR(+/+) and CFTR(-/-) rat nasal septal epithelia (RNSE) were cultured on semipermeable supports at an air-liquid interface to confluence and full differentiation. Monolayers were mounted in Ussing chambers for pharmacologic manipulation of ion transport and compared to similar filters containing murine (MNSE) and human (HSNE) epithelia. Histology and scanning electron microscopy (SEM) were completed. Real-time polymerase chain reaction of CFTR(+/+) RNSE, MNSE, and HSNE was performed to evaluate relative CFTR gene expression. Forskolin-stimulated anion transport (ΔIsc in μA/cm(2) ) was significantly greater in epithelia derived from CFTR(+/+) when compared to CFTR(-/-) animals (100.9 ± 3.7 vs. 10.5 ± 0.9; P < 0.0001). Amiloride-sensitive ISC was equivalent (-42.3 ± 2.8 vs. -46.1 ± 2.3; P = 0.524). No inhibition of CFTR-mediated chloride (Cl(-) ) secretion was exhibited in CFTR(-/-) epithelia with the addition of the specific CFTR inhibitor, CFTRInh -172. However, calcium-activated Cl(-) secretion (UTP) was significantly increased in CFTR(-/-) RNSE (CFTR(-/-) -106.8 ± 1.6 vs. CFTR(+/+) -32.2 ± 3.1; P < 0.0001). All responses were larger in RNSE when compared to CFTR(+/+) and CFTR(-/-) (or F508del/F508del) murine and human cells (P < 0.0001). Scanning electron microscopy demonstrated 80% to 90% ciliation in all RNSE cultures. There was no evidence of infection in CFTR(-/-) rats at 4 months. CFTR expression was similar among species. The successful development of the CFTR(-/-) rat enables improved evaluation of CF sinus disease based on characteristic abnormalities of ion transport. NA. Laryngoscope, 2017. © 2017 The American Laryngological

  14. Decreased oxygen tension lowers reactive oxygen species and apoptosis and inhibits osteoblast matrix mineralization through changes in early osteoblast differentiation.

    Science.gov (United States)

    Nicolaije, Claudia; Koedam, Marijke; van Leeuwen, Johannes P T M

    2012-04-01

    Accumulating data show that oxygen tension can have an important effect on cell function and fate. We used the human pre-osteoblastic cell line SV-HFO, which forms a mineralizing extracellular matrix, to study the effect of low oxygen tension (2%) on osteoblast differentiation and mineralization. Mineralization was significantly reduced by 60-70% under 2% oxygen, which was paralleled by lower intracellular levels of reactive oxygen species (ROS) and apoptosis. Following this reduction in ROS the cells switched to a lower level of protection by down-regulating their antioxidant enzyme expression. The downside of this is that it left the cells more vulnerable to a subsequent oxidative challenge. Total collagen content was reduced in the 2% oxygen cultures and expression of matrix genes and matrix-metabolizing enzymes was significantly affected. Alkaline phosphatase activity and RNA expression as well as RUNX2 expression were significantly reduced under 2% oxygen. Time phase studies showed that high oxygen in the first phase of osteoblast differentiation and prior to mineralization is crucial for optimal differentiation and mineralization. Switching to 2% or 20% oxygen only during mineralization phase did not change the eventual level of mineralization. In conclusion, this study shows the significance of oxygen tension for proper osteoblast differentiation, extra cellular matrix (ECM) formation, and eventual mineralization. We demonstrated that the major impact of oxygen tension is in the early phase of osteoblast differentiation. Low oxygen in this phase leaves the cells in a premature differentiation state that cannot provide the correct signals for matrix maturation and mineralization.

  15. Responses of human normal osteoblast cells and osteoblast-like cell line, MG-63 cells, to pulse electromagnetic field (PEMF

    Directory of Open Access Journals (Sweden)

    Suttatip Kamolmatyakul

    2008-01-01

    Full Text Available The objective of this in vitro study is to investigate the effect of pulsed electromagnetic field (PEMF on cellular proliferation and osteocalcin production of osteoblast-like cell line, MG-63 cells, and human normal osteoblast cells (NHOC obtained from surgical bone specimens. The cells were placed in 24-well culture plates in the amount of 3x104 cell/wells with 2 ml αMEM media supplemented with 10% FBS. The experimental plates were placed between a pair of Helmoltz coils powered by a pulse generator (PEMF, 50 Hz, 1.5 mV/cm in the upper compartment of a dual incubator (Forma. The control plates were placed in the lower compartment of the incubator without Helmotz coils. After three days, the cell proliferation was measured by the method modified from Mossman (J. Immunol Methods 1983; 65: 55-63. Other sets of plates were used for osteocalcin production assessment. Media from these sets were collected after 6 days and assessed for osteocalcin production using ELISA kits. The data were analyzed using a one-way analysis of variance (ANOVA. The results showed that MG-63 cells from the experimental group proliferated significantly more than those from the control group (20% increase, p<0.05. No significant difference in osteocalcin production was detected between the two groups. On the other hand, NHOC from the experimental group produced larger amount of osteocalcin (25% increase, p<0.05 and proliferated significantly more than those from the control group (100% increase, p<0.05. In conclusion, PEMF effect on osteoblasts might depend on their cell type of origin. For osteoblast-like cell line, MG-63 cells, PEMF increased proliferation rate but not osteocalcin production of the cells. However, PEMF stimulation effect on human normal osteoblast cells was most likely associated with enhancement of both osteocalcin production and cell proliferation.

  16. Experimental Study of Rat Beta Islet Cells Cultured under Simulated Microgravity Conditions

    Institute of Scientific and Technical Information of China (English)

    ChunSONG; Xiu-QingDUAN; XiLI; Li-OuHAN; PingXU; Chun-FangSONG:; Lian-HongJIN

    2004-01-01

    To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3,7 and 14. The morphology of the cells was observed by electron microscopy.Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured underthe microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient transport tubules. The microgravity cultured group also has higher insulin levels in the media as compared with the control group (P<0.01). Our results indicate that microgravity cultivation of islet cells has advantages over the routine culture methods.

  17. Experimental Study of Rat Beta Islet Cells Cultured under Simulated Microgravity Conditions

    Institute of Scientific and Technical Information of China (English)

    Chun SONG; Xiu-Qing DUAN; Xi LI; Li-Ou HAN; Ping XU; Chun-Fang SONG; Lian-Hong JIN

    2004-01-01

    To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3, 7 and 14. The morphology of the cells was observed by electron microscopy.Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured under the microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient transport tubules. The microgravity cultured group also has higher insulin levels in the media as compared with the control group(P<0.01). Our results indicate that microgravity cultivation of islet cells has advantages over the routine culture methods.

  18. Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

    Science.gov (United States)

    Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

  19. Preparation and Primary Culture of Liver Cells Isolated from Adult Rats by Dispase Perfusion

    Directory of Open Access Journals (Sweden)

    Wahid,Syarifuddin

    1984-06-01

    Full Text Available The dispase perfusion technique was used to isolate liver cells from adult rats. The optimum conditions for obtaining many isolated liver cells with high viability were an enzyme concentration of 2000 U/ml, a pH of 7.5 and a perfusion time of 20 min. The population of isolated liver cells prepared with dispase consisted of 43.6% cells with diameters less than 20 micron and 56.4% cells with diameters above 20 micron. The isolated liver cells were cultured in basal culture medium either supplemented with or without dexamethasone (1 X 10(-5M and insulin (10 micrograms/ml. The addition of hormones to the culture medium improved the attachment efficiency of the isolated liver cells and delayed the disappearance of mature hepatocytes. Epithelial-like clear cells proliferated early in primary culture even in the presence of hormones. Therefore, functioning mature hepatocytes and proliferating epithelial-like clear cells coexisted well in the hormone-containing medium. Furthermore, the number of cultured cells reached a maximal level earlier in the presence of hormones than in the absence of hormones. The level of TAT activity in primary cultured cells was higher up to 3 days after inoculation in the presence of hormones than in their absence. No difference between G6Pase activity in primary cultured cells in the presence of hormones and that in the absence of hormones was found.

  20. Identification of novel regulators of osteoblast matrix mineralization by time series transcriptional profiling.

    Science.gov (United States)

    Staines, Katherine Ann; Zhu, Dongxing; Farquharson, Colin; MacRae, Vicky Elizabeth

    2014-05-01

    Bone mineralization is a carefully orchestrated process, regulated by a number of promoters and inhibitors that function to ensure effective hydroxyapatite formation. Here we sought to identify new regulators of this process through a time series microarray analysis of mineralising primary osteoblast cultures over a 27 day culture period. To our knowledge this is the first microarray study investigating murine calvarial osteoblasts cultured under conditions that permit both physiological extracellular matrix mineralization through the formation of discrete nodules and the terminal differentiation of osteoblasts into osteocytes. RT-qPCR was used to validate and expand the microarray findings. We demonstrate the significant up-regulation of >6,000 genes during the osteoblast mineralization process, the highest-ranked differentially expressed genes of which were those dominated by members of the PPAR-γ signalling pathway, namely Adipoq, Cd36 and Fabp4. Furthermore, we show that the inhibition of this signalling pathway promotes matrix mineralisation in these primary osteoblast cultures. We also identify Cilp, Phex, Trb3, Sox11, and Psat1 as novel regulators of matrix mineralization. Further studies examining the precise function of the identified genes and their interactions will advance our understanding of the mechanisms underpinning biomineralization.

  1. 二甲双胍对骨向分化骨髓基质细胞增殖和分化的影响%Effects of Metformin on Proliferation and Differentiation of Bone Marrow Stromal Cells in Osteoblastic Medium.

    Institute of Scientific and Technical Information of China (English)

    张海波; 粱伟之; 高莺; 胡静

    2011-01-01

    Objective To investigate the possible effects of metformin, a commonly used drug for the patients with type 2 diabetes, on the proliferation and differentiation of rat bone marrow stromal cells ( BMSCs) cultured in osteoblatic medium. Methods The BMSCs used in this study were isolated from the femurs and tibiae of Sprague - Dawley rats and then cultured in the osteoblatic medium with or without 100 μmol/L metforrain. MTT assay was performed to evaluate the proliferation of the cells cultured in osteoblastic medium with or without metformin, while alkaline phosphate (ALP) activities analysis and mineralization nodules assessment were performed to evaluate the osteoblastic differentiation of these cells. At last, real time RT - PCR was further performed to quantify the mRNA expression levels of osteoblastic marker genes in osteoblastic differentiation cells treated with or without metformin. Results The number of BMSCs in two groups both increased over time, and metformin induced more cells. By contrast, the cells affected by metformin showed higher ALP activities, more nodules in the culture and more calcium deposition in mineralized nodules, as examined by ARS staining. The optical density of ARS destaining solution in metformin group was significantly higher than that of control group. Moreover, the promotive effects of metformin on osteoblastic differentiation were further examined by testing the mRNA levels of osteoblastic marker genes using real - time RT - PCR. The metformin - containing medium yielded higher levels of mRNA levels for osteoblastic markers. Conclusion Metformin may promote the proliferation and differentiation of BMSCs cultured in osteoblastic medium.%目的 探讨二甲双胍对骨向分化的大鼠骨髓基质细胞增殖和分化的影响.方法 体外分离培养大鼠骨髓基质细胞,对其进行骨向诱导分化,并于实验组添加100μ mol/L二甲双胍,应用四甲基偶氮唑盐法(MTT)检测细胞增

  2. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

    Science.gov (United States)

    Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

    2016-09-01

    Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in

  3. Osteoblast Behavior on Hierarchical Micro-/Nano-Structured Titanium Surface

    Institute of Scientific and Technical Information of China (English)

    Weiyan Meng; Yanmin Zhou; Yanjing Zhang; Qing Cai; Liming Yang; Jinghui Zhao; Chnnyan Li

    2011-01-01

    In the present work, osteoblast behavior on a hierarchical micro-/nano-structured titanium surface was investigated. A hierarchical hybrid micro-/nano-structured titanium surface topography was produced via Electrolytic Etching (EE). MG-63 cells were cultured on disks for 2 h to 7 days. The osteoblast response to the hierarchical hybrid micro-/nano-structured titanium surface was evaluated through the osteoblast cell morphology, attachment and proliferation. For comparison, MG-63 cells were also cultured on Sandblasted and Acid-etched (SLA) as well as Machined (M) surfaces respectively. The results show significant differences in the adhesion rates and proliferation levels of MG-63 cells on EE, SLA, and M surfaces. Both adhesion rate and proliferation level on EE surface are higher than those on SLA and M surfaces. Therefore, we may expect that, comparing with SLA and M surfaces, bone growth on EE surface could be accelerated and bone formation could be promoted at an early stage, which could be applied in the clinical practices for immediate and early-stage loadings.

  4. Myocyte Enhancer Factor 2C, an Osteoblast Transcription Factor Identified by Dimethyl Sulfoxide (DMSO)-enhanced Mineralization*

    Science.gov (United States)

    Stephens, Alexandre S.; Stephens, Sebastien R.; Hobbs, Carl; Hutmacher, Deitmar W.; Bacic-Welsh, Desa; Woodruff, Maria Ann; Morrison, Nigel A.

    2011-01-01

    Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts. PMID:21652706

  5. Myocyte enhancer factor 2c, an osteoblast transcription factor identified by dimethyl sulfoxide (DMSO)-enhanced mineralization.

    Science.gov (United States)

    Stephens, Alexandre S; Stephens, Sebastien R; Hobbs, Carl; Hutmacher, Deitmar W; Bacic-Welsh, Desa; Woodruff, Maria Ann; Morrison, Nigel A

    2011-08-26

    Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

  6. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Michinao [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Ariyoshi, Wataru [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Iwanaga, Kenjiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Okinaga, Toshinori [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Habu, Manabu [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Yoshioka, Izumi [Division of Oral and Maxillofacial Surgery, Department of Medicine of Sensory and Motor Organs, University of Miyazaki, Kiyotake, Miyazaki 889-1692 (Japan); Tominaga, Kazuhiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580 (Japan)

    2011-02-25

    Research highlights: {yields} In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. {yields} MG63 cells were incubated with BMP-2 and HA for various time periods. {yields} Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. {yields} HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and

  7. The PPARgamma-selective ligand BRL-49653 differentially regulates the fate choices of rat calvaria versus rat bone marrow stromal cell populations

    Directory of Open Access Journals (Sweden)

    Yoshiko Yuji

    2008-07-01

    Full Text Available Abstract Background Osteoblasts and adipocytes are derived from a common mesenchymal progenitor and an inverse relationship between expression of the two lineages is seen with certain experimental manipulations and in certain diseases, i.e., osteoporosis, but the cellular pathway(s and developmental stages underlying the inverse relationship is still under active investigation. To determine which precursor mesenchymal cell types can differentiate into adipocytes, we compared the effects of BRL-49653 (BRL, a selective ligand for peroxisome proliferators-activated receptor (PPARγ, a master transcription factor of adipogenesis, on osteo/adipogeneis in two different osteoblast culture models: the rat bone marrow (RBM versus the fetal rat calvaria (RC cell system. Results BRL increased the number of adipocytes and corresponding marker expression, such as lipoprotein lipase, fatty acid-binding protein (aP2, and adipsin, in both culture models, but affected osteoblastogenesis only in RBM cultures, where a reciprocal decrease in bone nodule formation and osteoblast markers, e.g., osteopontin, alkaline phosphatase (ALP, bone sialoprotein, and osteocalcin was seen, and not in RC cell cultures. Even though adipocytes were histologically undetectable in RC cultures not treated with BRL, RC cells expressed PPAR and CCAAT/enhancer binding protein (C/EBP mRNAs throughout osteoblast development and their expression was increased by BRL. Some single cell-derived BRL-treated osteogenic RC colonies were stained not only with ALP/von Kossa but also with oil red O and co-expressed the mature adipocyte marker adipsin and the mature osteoblast marker OCN, as well as PPAR and C/EBP mRNAs. Conclusion The data show that there are clear differences in the capacity of BRL to alter the fate choices of precursor cells in stromal (RBM versus calvarial (RC cell populations and that recruitment of adipocytes can occur from multiple precursor cell pools (committed preadipocyte

  8. Growth characteristics and functional changes in rat chondrocytes cultured in porous tantalum in vitro

    Directory of Open Access Journals (Sweden)

    Ling ZHANG

    2014-08-01

    Full Text Available Objective To evaluate the growth characteristics and functional changes in rat chondrocytes cultured in porous tantalum in vitro. Methods The chondrocytes isolated from cartilage of 3-week old SD rats were cultured in vitro, then the 2nd passage cells were identified and implanted in porous tantalum scaffolds with a density of 1×106 cells/ml. The morphological characteristics of the chondrocytes cultured in porous tantalum were observed under inverted microscope, scanning electron microscope (SEM and transmission electron microscope (TEM, and the content of glycosaminoglycan (GAG in the chondrocytes was measured by chromatometry. Results The harvested cells were identified as chondrocytes by type Ⅱ collagen immunocytochemical staining, toluidine blue staining and safranin-O staining. Many chondrocytes adhering to the edge of porous tantalum were found by inverted microscope. Observation under SEM showed that chondrocytes spread well on the surface and distributed in the holes of porous tantalum, and they proliferated and secreted some extracellular matrixes. TEM observation showed that the ultrastructure of chondrocytes cultured in porous tantalum was similar to that of normal chondrocytes. Chromatometry determination showed that the chondrocytes in porous tantalum could secrete GAG continuously. Conclusion Porous tantalum is shown to have a satisfactory biocompatibility with chondrocytes in vitro, and may be used as a scaffold for cartilage tissue engineering. DOI: 10.11855/j.issn.0577-7402.2014.06.08

  9. Electrophysiology of embryonic, adult and aged rat hippocampal neurons in serum-free culture.

    Science.gov (United States)

    Evans, M S; Collings, M A; Brewer, G J

    1998-01-31

    Methods were recently developed for culturing neurons from adult rat hippocampus using the serum-free medium Neurobasal with B27 supplement. To determine whether adult cultured neurons have normal electrical properties, we studied cultures from rats of three age groups: (1) embryonic; (2) 10-11 months old and (3) 35-36 months old. Neurons had a polarized morphology with a large branching apical dendrite and small basal dendrites. Mean resting potentials were similar in the three age groups. All neurons had nonlinear current-voltage relationships, indicating the presence of voltage-sensitive ion channels. Most neurons had a voltage-sensitive inward current followed by a sustained voltage-sensitive outward current. Tetrodotoxin blocked the inward current, which is likely to be a sodium current. The sustained outward current, which is likely to be a potassium current, reversed at -71 mV. Most neurons exhibited anomalous rectification. Calcium currents were present in both embryonic and adult neurons. Embryonic neurons would sometimes fire multiple action potentials but adult neurons fired only single action potentials. Our results indicate that both embryonic and adult cultured neurons retain a clearly neuronal electrophysiological phenotype in Neurobasal/B27 serum-free medium.

  10. Leptin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland.

    Science.gov (United States)

    Peliciari-Garcia, Rodrigo Antonio; Andrade-Silva, Jéssica; Cipolla-Neto, José; Carvalho, Carla Roberta de Oliveira

    2013-01-01

    Pineal melatonin synthesis can be modulated by many peptides, including insulin. Because melatonin appears to alter leptin synthesis, in this work we aimed to investigate whether leptin would have a role on norepinephrine- (NE-)mediated melatonin synthesis in cultured rat pineal glands. According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb). Pineal expression of Ob-Rb mRNA was also observed in vivo. Administration of leptin (1 nM) associated with NE ( 1 µM) reduced melatonin content as well as arylalkylamine-N-acetyl transferase (AANAT) activity and expression in cultured pineal glands. Leptin treatment per se induced the expression of STAT3 in cultured pineal glands, but STAT3 does not participate in the leptin modulation of NE-mediated pineal melatonin synthesis. In addition, the expression of inducible cAMP early repressor (ICER) was further induced by leptin challenge when associated with NE. In conclusion, leptin inhibition of pineal melatonin synthesis appears to be mediated by a reduction in AANAT activity and expression as well as by increased expression of Icer mRNA. Peptidergic signaling within the pineal gland appears to be one of the most important signals which modulates melatonin synthesis; leptin, as a member of this system, is not an exception.

  11. Leptin Modulates Norepinephrine-Mediated Melatonin Synthesis in Cultured Rat Pineal Gland

    Directory of Open Access Journals (Sweden)

    Rodrigo Antonio Peliciari-Garcia

    2013-01-01

    Full Text Available Pineal melatonin synthesis can be modulated by many peptides, including insulin. Because melatonin appears to alter leptin synthesis, in this work we aimed to investigate whether leptin would have a role on norepinephrine- (NE-mediated melatonin synthesis in cultured rat pineal glands. According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb. Pineal expression of Ob-Rb mRNA was also observed in vivo. Administration of leptin (1 nM associated with NE (1 µM reduced melatonin content as well as arylalkylamine-N-acetyl transferase (AANAT activity and expression in cultured pineal glands. Leptin treatment per se induced the expression of STAT3 in cultured pineal glands, but STAT3 does not participate in the leptin modulation of NE-mediated pineal melatonin synthesis. In addition, the expression of inducible cAMP early repressor (ICER was further induced by leptin challenge when associated with NE. In conclusion, leptin inhibition of pineal melatonin synthesis appears to be mediated by a reduction in AANAT activity and expression as well as by increased expression of Icer mRNA. Peptidergic signaling within the pineal gland appears to be one of the most important signals which modulates melatonin synthesis; leptin, as a member of this system, is not an exception.

  12. Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway.

    Science.gov (United States)

    Yang, Yue-Hua; Chen, Ke; Li, Bo; Chen, Jiang-Wei; Zheng, Xin-Feng; Wang, Yu-Ren; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2013-11-01

    Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through

  13. Comparative effects of sodium channel blockers in short term rat whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, Mats F, E-mail: Mats.Nilsson@farmbio.uu.se [Department of Pharmaceutical Biosciences, Uppsala University (Sweden); Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma [AstraZeneca R and D Södertälje (Sweden); Cebers, Gvido [AstraZeneca R and D, iMed, 141 Portland Street, Cambridge, MA 02139 (United States); Hellmold, Heike; Gustafson, Anne-Lee [AstraZeneca R and D Södertälje (Sweden); Webster, William S [Department of Anatomy and Histology, University of Sydney (Australia)

    2013-10-15

    This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development.

  14. Morphological and functional behaviors of rat hepatocytes cultured on single-walled carbon nanotubes.

    Science.gov (United States)

    Koga, Haruka; Fujigaya, Tsuyohiko; Nakashima, Naotoshi; Nakazawa, Kohji

    2011-09-01

    This study describes the morphological and functional behaviors of rat hepatocytes on single-walled carbon nanotube (CNT)-coated surfaces. Although the hydrophobic characteristics of CNT-coated surfaces increased with increasing CNT density, hepatocyte adhesion decreased, indicating that the interaction between hepatocytes and CNTs is weak. We found that hepatocytes on a CNT-coated surface gradually gather together and form spheroids (spherical multicellular aggregates). These spheroids exhibit compact spherical morphology with a smooth surface and express connexin-32, an intracellular communication molecule. In contrast, collagen treatment in conjunction with the CNT-coated surface improved hepatocyte adhesion, and the cells maintained a monolayer configuration throughout the culture period. The albumin secretion and ammonia removal activities of hepatocyte spheroids were maintained at elevated levels for at least 15 days of culturing as compared with hepatocyte monolayers. These results indicate that CNTs can be used for the formation and long-term culture of hepatocyte spheroids.

  15. Expressions of integrin subunits in osteoblasts during weightlessness simulation using clionstat

    Science.gov (United States)

    Zhang, S.; Wang, B.; Zhao, D.; Nie, J.; Li, Y.

    Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity to simulate weightlessness environment. We then observed the responses of rat calvarial osteoblasts subsequent to rotation at 30 revolutions per minute (rpm) for 72 h. We found that the gene expressions of three integrin subunits started to change from 24 h of rotation in clinostat but not in stationary cultures. The decreased percent changes of integrin a5 mRNA at 24, 48 and 72 h were 11.3 +/- 2.6%, 18.7 +/- 4.2% and 9.8 +/ - 2.1%, respectively. The same trend was saw in the expression of integrin av mRNA as 23.0 +/- 4.7%, 12.3 +/- 1.6% and 16.7 +/- 3.2%, respectively. Moreover, the expressions of integrin ß1 mRNA in different periods were also declined with the percent changes of 15.3 +/- 1.3%, 11.4 +/- 1.2% and 26.4 +/- 5.5%, respectively. All cells contain membrane-anchored attachment proteins able to recognize specific chemical motifs in the extracellular macromolecules forming the supporting scaffold, made of various types of collagen, adhesive glycoproteins, elastin, proteoglycans, etc. These cell-matrix interactions are mainly mediated by receptors of the integrins family, heterodimeric molecules made of an extracellular domain connected through a transmembrane sequence to an intracytoplasmic tail. Our results suggest that vector-averaged gravity causes alterations of signal transduction and integrin-mediated cell adhesion in osteoblasts by altering the gene expressions of several crucial integrin subunits. These alterations might contribute to the pathogenesis of osteoporotic bone loss as observed in actual space flights.

  16. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells.

    Science.gov (United States)

    Parkinson, Fiona E; Hacking, Cindy

    2005-07-05

    The blood-brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood-brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood-brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2-14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of alpha-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood-brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood-brain barrier properties in in vitro models that utilize primary cultures of RBMECs.

  17. Failure of zinc to prevent dysmorphogenesis of cultured rat conceptuses by anti-yolk sac antiserum

    Energy Technology Data Exchange (ETDEWEB)

    Marlow, R.; Freeman, S.J.

    1989-01-01

    Day 10 rat conceptuses were cultured for 48h in the presence of either cadmium or anti-vesceral yolk sac antiserum (AVYS). Cadmium was embryotoxic at concentrations exceeding 0.25 ug/ml while AVYS caused embryonic dysmorphogenesis, particularly affecting the optic vesicles, at concentrations of 2 ul/ml and above. The effect of pretreatment with zinc on embryotoxicity caused by cadmium or AVYS was studied. Zinc ameliorated the effects of cadmium but had no effect on AVYS-induced embryonic abnormalities. In a second set of experiments inhibition of /sup 125/I-labelled PVP uptake by the yolk sac of cultured whole conceptuses was studied. Cadmium and AVYS both inhibited uptake compared to control cultures. Zinc again ameliorated the effect of cadmium but had no action against AVYS-induced inhibition. These results are in contrast to their previous findings using isolated cultured yolk sacs in which zinc ameliorated the inhibitory effects on /sup 125/I-labelled PVP uptake of both cadmium and AVYS. These data show that in experiments using the isolated cultured yolk sac and the intact cultured conceptus, a qualitatively different response in yolk sac behavior is observed under similar experimental conditions.

  18. Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH).

    Science.gov (United States)

    Naruto, Hirosuke; Huang, Hongyun; Nishikawa, Masaki; Kojima, Nobuhiko; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

    2007-10-01

    We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

  19. Time window characteristics of cultured rat hippocampal neurons subjected to ischemia and reperfusion

    Institute of Scientific and Technical Information of China (English)

    XU Zhong; XU Ru-xiang; LIU Bao-song; JIANG Xiao-dan; HUANG Tao; DING Lian-shu; YUAN Jun

    2005-01-01

    Objective: To explore cell death and apoptosis in rat hippocampal neurons at different time points after ischemia, hypoxia and reperfusion injury and to elucidate time window characteristics in ischemia neuronal injury.Methods: Hippocampal neurons were obtained from rat embryo and were cultured in vitro. The ischemia and reperfusion of cultured rat hippocampal neurons were simulated by oxygen-glucose deprivation (OGD) and recovery. OGD at different time points (0.25 h to 3.0 h) and then the same recovery (24 h) were prepared. Annexin V-PI staining and flow cytometry examined neuron death and apoptosis at different time after injury. Results: After OGD and recovery, both necrosis and apoptosis were observed. At different times after OGD, there were statistically significant differences in neuron necrosis rate (P0.05). At recovery, survival rate of hippocampal neurons further decreased while apoptosis rate increased. Furthermore, apoptosis rates of different time differed greatly (P<0.05). Apoptosis rate gradually increased with significant difference among those of different time points (P<0.05). However, 2 h after ischemia, apoptosis rate decreased markedly.Conclusions: Apoptosis is an important pathway of delayed neuron death. The therapeutic time window should be within 2 h after cerebral ischemia and hypoxia.

  20. Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture.

    Science.gov (United States)

    Montagne, Kevin; Huang, Hongyun; Ohara, Keikou; Matsumoto, Kunio; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

    2011-11-01

    Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.

  1. Nitroglycerin enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via nitric oxide pathway

    Institute of Scientific and Technical Information of China (English)

    Li HUANG; Ni QIU; Che ZHANG; Hong-yan WEI; Ya-lin LI; Hong-hao ZHOU; Zhou-sheng XIAO

    2008-01-01

    Aim: To investigate the effect of nitroglycerin (NTG) on cell proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSC) and its mechanisms. Methods: Primary HBMSC were cultured in osteogenic differentiation medium consisting of phenol red-free or-minimum es-sential media plus 10% fetal bovine serum (dextran-coated charcoal stripped)supplemented with 10 nmol/L dexamethasone, 50 mg/L ascorbic acid, and l0 mmol/Lβ-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1-10 μmol/L) alone or concurrent incubation with different nitric oxide synthase (NOS) inhibitors. Nitric oxide (NO) production was measured by using a commercial NO kit. Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. The osteoblastic differentiation of HBMSC culture was evaluated by measuring cellular alkaline phosphatase (ALP) activity and calcium deposition, as well as osteoblastic markers by real-time RT-PCR. Results: The treatment of HBMSC with NTG (0.1-10 μmol/L) led to a dose-dependent increase of NO production in the conditional medium. The release of NO by NTG resulted in increased cell proliferation and osteoblastic differentiation of HBMSC, as evi-denced by the increment of the BrdU incorporation, the induction of ALP activity in the early stage, and the calcium deposition in the latter stage. The increment of NO production was also correlated with the upregulation of osteoblastic markers in HBMSC cultures. However, the stimulatory effect of NTG (10 μmol/L) could not be abolished by either NG-nitro-L-arginine methyl ester, an antagonist of endothe-lial NOS, or 1400W, a selective blocker of inducible NOS activity. Conclusion: NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity.

  2. Plant-derived pectin nanocoatings to prevent inflammatory cellular response of osteoblasts following Porphyromonas gingivalis infection

    Science.gov (United States)

    Meresta, Anna; Folkert, Justyna; Gaber, Timo; Miksch, Korneliusz; Buttgereit, Frank; Detert, Jacqueline; Pischon, Nicole; Gurzawska, Katarzyna

    2017-01-01

    Background Bioengineered plant-derived Rhamnogalacturonan-Is (RG-Is) from pectins are potential candidates for surface nanocoating of medical devices. It has recently been reported that RG-I nanocoatings may prevent bacterial infection and improve the biocompatibility of implants. The aim of the study was to evaluate in vitro impact of bioengineered RG-I nanocoatings on osteogenic capacity and proinflammatory cytokine response of murine osteoblasts following Porphyromonas gingivalis infection. Methods Murine MC3T3-E1 osteoblasts and isolated primary calvarial osteoblasts from C57BL/6J (B6J osteoblasts) mice were infected with P. gingivalis and incubated on tissue culture polystyrene plates with or without nanocoatings of unmodified RG-Is isolated from potato pulps (PU) or dearabinanated RG-Is (PA). To investigate a behavior of infected osteoblasts cultured on RG-Is cell morphology, proliferation, metabolic activity, mineralization and osteogenic and pro-inflammatory gene expression were examined. Results Following P. gingivalis infection, PA, but not PU, significantly promoted MC3T3-E1 and BJ6 osteoblasts proliferation, metabolic activity, and calcium deposition. Moreover, Il-1b, Il-6, TNF-α, and Rankl gene expressions were downregulated in cells cultured on PU and to a higher extent on PA as compared to the corresponding control, whereas Runx, Alpl, Col1a1, and Bglap gene expressions were upregulated vice versa. Conclusion Our data clearly showed that pectin RG-Is nanocoating with high content of galactan (PA) reduces the osteoblastic response to P. gingivalis infection in vitro and may, therefore, reduce a risk of inflammation especially in immunocompromised patients with rheumatoid or periodontal disorders. PMID:28138240

  3. 二甲双胍减轻糖基化终末产物对成骨细胞功能损害的观察%Metformin ameliorating the deleterious effects of advanced glycation end products on osteoblastic cells

    Institute of Scientific and Technical Information of China (English)

    甄东户; 汤旭磊; 成建国; 韩婕; 刘丽娟; 傅松波

    2012-01-01

    Objective To explore the effect of metformin on proliferation, differentiation and mineralization in the advanced glycation end products(AGEs)-induced rat cranium osteoblasts. Methods Rat cranium osteoblasts were isolated and cultured. The proliferation of osteoblasts was assayed by MTT method, the activity of alkaline phosphatase(ALP) was measured by biochemical method, the number of mineralized nodules was assessed by Alizarin red S staining, and the calcium deposition in the mineralized nodules was detected by hydrochloric acid decalcification method. Results With 500 fig/ml AGEs, the cellular proliferation, ALP activity, number of mineralized nodules, and calcium deposition were reduced significantly. Metformine (100-500 μmol/L) increased the cellular proliferation and ALP activity, and promoted the formation of mineralized nodules and calcium deposition in both control and AGEs groups. Conclusion AGEs inhibit the proliferation, differentiation and mineralization of the primary osteoblasts. Metformine strengthens the osteogenesis of osteoblasts and relieves the deleterious effect of AGEs on osteoblasts.%目的 观察二甲双胍对晚期糖基化终末产物(AGEs)诱导的大鼠颅骨成骨细胞增殖、分化、矿化的影响.方法 分离培养大鼠颅骨成骨细胞,四氮唑蓝(MTT)比色分析法测定细胞增殖,生化法测定碱性磷酸酶(ALP)活性、茜素红S钙染法检测矿化结节形成,盐酸脱钙法检测矿化结节中钙含量.结果 500μg/ml AGEs抑制成骨细胞增殖、ALP活性、钙化结节形成、钙沉积;给予二甲双胍(100~500 μmol/L)可不同程度上提高成骨细胞数量和ALP活性,促进矿化结节形成及钙沉积,减轻AGEs对成骨细胞增殖、ALP活性、钙化结节形成及钙沉积的抑制.结论 AGEs对原代成骨细胞增殖、分化与矿化产生抑制作用,二甲双胍提高成骨细胞的成骨能力,减轻AGEs对成骨细胞功能的损害.

  4. Neuropeptides stimulate human osteoblast activity and promote gap junctional intercellular communication.

    Science.gov (United States)

    Ma, Wenhui; Zhang, Xuemin; Shi, Shushan; Zhang, Yingze

    2013-06-01

    Neuropeptides released from the skeletal nerve fibers have neurotransmitter and immunoregulatory roles; they exert paracrine biological effects on bone cells present close to the nerve endings expressing these signaling molecules. The aims of this study were a systematic investigation of the effects of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), Neuropeptide Y (NPY) and tyrosine hydroxylase (TH) on the cell viability and function of the human osteoblasts, and comparing their difference in the role of regulating bone formation. Cultures of normal human osteoblasts were treated with SP, CGRP, VIP, NPY or TH at three concentrations. We found that each of the five neuropeptides induced increases in cell viability of human osteoblasts. The stimulatory action of NPY was the highest, followed by VIP, SP and TH, while CGRP had the lowest stimulatory effect. The viability index of osteoblasts was inversely associated with the concentration of neuropeptides, and positively with the time of exposure. Moreover, the five neuropeptides increased the ALP activity and osteocalcin to different extents in a dose-dependent manner. The GJIC of osteoblasts was significantly promoted by neuropeptides. The results demonstrated that neuropeptides released from skeletal nerve endings after a stimulus appeared to be able to induce the proliferation and activity of osteoblasts via enhancing GJIC between cells, and further influence the bone formation. These findings may contribute toward a better understanding of the neural influence on bone remodeling and improving treatments related to bone diseases.

  5. Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Shiguang [Department of Intensive Care Unit, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Mao, Li [Department of Endocrinology, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Ji, Feng, E-mail: huaiaifengjidr@163.com [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Shouguo; Xie, Yue; Fei, Haodong [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Xiao-dong, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children' s Bone Diseases, The Children' s Hospital Affiliated to Soochow University, Suzhou (China)

    2016-03-18

    Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the other hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.

  6. Chronic lithium treatment increased intracellular S100ß levels in rat primary neuronal culture.

    Directory of Open Access Journals (Sweden)

    Masoumeh Emamghoreishi

    2015-02-01

    Full Text Available S100ß a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100ß. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100β in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day's embryos. Cell cultures were exposed to lithium (1mM or vehicle for 1day (acute or 7 days (chronic. RT-PCR and ELISA determined S100β mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100β in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05. Acute and chronic lithium treatments exerted no significant effects on intracellular S100β protein levels in astrocytes, and extracellular S100β protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100β mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100ß protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100ß level, with no effect on astrocytes or interaction between neurons and astrocytes.

  7. Batroxobin Against Anoxic Damage of Rat Hippocampal Neurons in Culture: Morphological Changes and Hsp70 Expression

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Batroxobin,the thrombin-like enzyme,is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

  8. Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity.

    Science.gov (United States)

    Chang, Shih-Yu; Voellinger, Jenna L; Van Ness, Kirk P; Chapron, Brian; Shaffer, Rachel M; Neumann, Thomas; White, Collin C; Kavanagh, Terrance J; Kelly, Edward J; Eaton, David L

    2017-04-01

    The liver is the main site for drug and xenobiotics metabolism, including inactivation or bioactivation. In order to improve the predictability of drug safety and efficacy in clinical development, and to facilitate the evaluation of the potential human health effects from exposure to environmental contaminants, there is a critical need to accurately model human organ systems such as the liver in vitro. We are developing a microphysiological system (MPS) based on a new commercial microfluidic platform (Nortis, Inc.) that can utilize primary liver cells from multiple species (e.g., rat and human). Compared to conventional monolayer cell culture, which typically survives for 5-7days or less, primary rat or human hepatocytes in an MPS exhibited higher viability and improved hepatic functions, such as albumin production, expression of hepatocyte marker HNF4α and canaliculi structure, for up to 14days. Additionally, induction of Cytochrome P450 (CYP) 1A and 3A4 in cryopreserved human hepatocytes was observed in the MPS. The acute cytotoxicity of the potent hepatotoxic and hepatocarcinogen, aflatoxin B1, was evaluated in human hepatocytes cultured in an MPS, demonstrating the utility of this model for acute hepatotoxicity assessment. These results indicate that MPS-cultured hepatocytes provide a promising approach for evaluating chemical toxicity in vitro. Copyright © 2017. Published by Elsevier Ltd.

  9. Growth enhancement effect of BzATP on primary cultured astrocytes from rat brain

    Institute of Scientific and Technical Information of China (English)

    Hua-Zheng LIANG; Ying LIU; Zhu-Rong YE

    2006-01-01

    Objective To explore whether BzATP could promote the growth of primary cultured astrocytes (AS) of rat and its possible mechanism, and whether TGF-β1 was involved in the event. Methods The primary cultured AS were derived from new born Sprague-Dawley rats.Glial fibrillary acidic protein (GFAP) immunofluorescent stain was used to check the purity of cultured AS. Morphometry was used to detect the changes of AS. The proliferation index of AS was detected by BrdU incorporation assay. Western blot was used to detect the changes of GFAP under different conditions. Changes of TGF-β1 gene transcription were detected by RT-PCR. ELISA was utilized to detect the variation of TGF-β1 protein in the supernate. Results The purity of primary cultured AS reached to 99%. BzATP promoted the hypertrophy of AS including the elongation of AS processes and the enlargement of cell bodies, BzATP also promoted the expression of GFAP in existence of Ca2+, but had no effect on cell proliferation. BzATP increased the transcription of TGF-β1 mRNA and the release of TGF-β1 protein in existence of Ca2+. TGF-β1 neutralizing antibody partially inhibited the expression of GFAP induced by BzATP, but had no effect on AS proliferation and cell morphology. Conclusion BzATP enhanced the hypertrophy of primary cultured AS, increased the expression of GFAP partially through TGF-β1. Mechanisms of the enhancement of AS growth induced by BzATP other than TGF-51 pathway remains to be elucidated.

  10. Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina.

    Science.gov (United States)

    Rickman, D W

    1999-11-15

    The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.

  11. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    Directory of Open Access Journals (Sweden)

    Junko Kimura-Kuroda

    2016-10-01

    Full Text Available Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of longterm (14 days and low dose (1 μM exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  12. Rapid oriented fibril formation of fish scale collagen facilitates early osteoblastic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Matsumoto, Rena; Uemura, Toshimasa; Xu, Zhefeng; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

    2015-08-01

    We studied the effect of fibril formation of fish scale collagen on the osteoblastic differentiation of human mesenchymal stem cells (hMSCs). We found that hMSCs adhered easily to tilapia scale collagen, which remarkably accelerated the early stage of osteoblastic differentiation in hMSCs during in vitro cell culture. Osteoblastic markers such as ALP activity, osteopontin, and bone morphogenetic protein 2 were markedly upregulated when the hMSCs were cultured on a tilapia collagen surface, especially in the early osteoblastic differentiation stage. We hypothesized that this phenomenon occurs due to specific fibril formation of tilapia collagen. Thus, we examined the time course of collagen fibril formation using high-speed atomic force microscopy. Moreover, to elucidate the effect of the orientation of fibril formation on the differentiation of hMSCs, we measured ALP activity of hMSCs cultured on two types of tilapia scale collagen membranes with different degrees of fibril formation. The ALP activity in hMSCs cultured on a fibrous collagen membrane was significantly higher than on a non-fibrous collagen membrane even before adding osteoblastic differentiation medium. These results showed that the degree of the fibril formation of tilapia collagen was essential for the osteoblastic differentiation of hMSCs.

  13. Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (-)-baclofen.

    OpenAIRE

    Dolphin, A C; Scott, R.H.

    1986-01-01

    Voltage-dependent inward calcium currents (ICa) activated in cultured rat dorsal root ganglion neurones were reversibly reduced in a dose-dependent manner by (-)-baclofen (10 microM to 100 microM). Baclofen (100 microM) reduced the calcium-dependent slow outward potassium current (IK(Ca)). This current was abolished in calcium-free medium and by 300 microM cadmium chloride. The action of baclofen on IK(Ca) was reduced when the calcium concentration in the medium was increased from 5 mM to 30 ...

  14. Modulations of prolactin and growth hormone gene expression and chromatin structure in cultured rat pituitary cells.

    OpenAIRE

    Levy-Wilson, B

    1983-01-01

    I have measured the effect of hormones and other regulatory factors present in the serum component of the culture medium on the levels of growth hormone and prolactin mRNAs in rat pituitary (GH4) cells. Hybridization of cytoplasmic RNA with growth hormone or prolactin cDNA clones indicate that serum depletion reduces significantly the amount of these two mRNAs. The localization of these two genes in chromatin was also analysed using micrococcal nuclease as a probe. At intermediate levels of d...

  15. Green tea polyphenol (-)-epigallocatechin gallate suppressed the differentiation of murine osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Kamon, Masayoshi; Zhao, Ran; Sakamoto, Kazuichi

    2009-12-16

    Recently, various physiological effects of the tea polyphenol catechin for alleviating diseases such as cancer, arteriosclerosis, hyperlipidaemia and osteoporosis have been reported. However, the physiological effect of catechin on bone metabolism remains unclear. We examined the physiological effect of EGCG [(-)-epigallocatechin-3-gallate], which is the main component of green tea catechin, on osteoblast development using the precursor cell line of osteoblasts, MC3T3-E1, and co-culture of the osteoblasts from mouse newborn calvaria and mouse bone marrow cells. Although EGCG did not affect the viability and proliferation of MC3T3-E1 cells, EGCG inhibited the osteoblast differentiation. Furthermore, EGCG did not affect the mineralization of differentiated MC3T3-E1 cells, and reduced osteoclast formation in co-culture. These results suggest that EGCG can effectively suppress bone resorption, and can be used as an effective medicine in the treatment of the symptoms of osteoporosis.

  16. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    Science.gov (United States)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  17. 人骨膜来源细胞在聚乳酸三维支架体外成骨的超微结构研究%Biological activity and ultrastructure of human periosteum-derived osteoblasts in vitro cultured in poly lactic acid scaffolds

    Institute of Scientific and Technical Information of China (English)

    贝抗胜; 刘延晓; 熊英辉; 吴礼杨; 卢林俊

    2015-01-01

    目的 探讨人骨膜来源细胞在聚乳酸(PLA)三维支架体外培养成骨的可行性及其超微结构特征. 方法 取材于成人胫骨骨膜,组织块法分离出骨膜细胞并体外培养传至第3代后进行定向诱导分化为骨膜来源成骨细胞,相差显微镜观察细胞形态特征,分为诱导组和未诱导组,分别在5、10、15、20 d取3个样本,采用碱性磷酸酶(ALP)试剂盒法及钙结节Von Kossa染色法检测ALP和钙结节的表达.经诱导分化的成骨细胞随机分为实验组和对照组,分别接种至经含骨形态发生蛋白-7成骨诱导剂预处理的PLA三维支架和普通二维细胞培养板,1、3、5、7、9、11d采用CCK-8法测量两组细胞的增殖能力,并绘制生长曲线;3、6、9、12d电镜下观察细胞在PLA的生长情况和超微形态特征. 结果 骨膜来源细胞经诱导分化其形态与成骨细胞相似,诱导组的ALP和钙结节染色阳性率明显高于未诱导组,差异均有统计学意义(P<0.05).实验组和对照组细胞生长曲线均呈"S"形,实验组对数生长期增殖较快,平稳期细胞数目明显下降,对照组平稳期数目则有上升,差异有统计学意义(P<0.05).电子显微镜观察体外骨膜来源成骨细胞具有良好的超微形态学结构. 结论 体外骨膜来源成骨细胞与PLA三维支架复合有良好的生物相容性、细胞增殖力和超微形态学结构;体外复合成活后应在第8~9天及时转入体内生长以发挥骨修复作用.%Objective To explore the feasibility of osteogenesis by human periosteum-derived osteoblasts in vitro cultured in poly lactic acid (PLA) three-dimensional scaffolds and the ultrastructural features of the human periosteal osteoblasts.Methods The cells derived from adult tibial periosteum were separated through tissue explants, cultured in vitro to the third generation, and directionally differentiated into the human periosteum-derived osteoblasts.Their morphological features were

  18. Cherubism Gene Sh3bp2 is Important for Optimal Bone Formation, Osteoblast Differentiation and Function

    Science.gov (United States)

    Mukherjee, Padma M.; Wang, Chiachien J.; Chen, I-Ping; Jafarov, Toghrul; Olsen, Bjorn R.; Ueki, Yasuyoshi; Reichenberger, Ernst J.

    2012-01-01

    Introduction Cherubism is a human genetic disorder that causes bilateral symmetrical enlargement of the maxilla and mandible in children. It is caused by mutations in SH3BP2. The exact pathogenesis of the disorder is an area of active research. Sh3bp2 knock-in mice were developed by introducing a Pro416Arg mutation (Pro418Arg in humans) in the mouse genome. The osteoclast phenotype of this mouse model was recently described. Methods We examined the bone phenotype of the cherubism mouse model, the role of Sh3bp2 during bone formation, osteoblast differentiation and osteoblast function. Results We observed delays in early postnatal development of homozygous Sh3bp2KI/KI mice. Sh3bp2KI/KI mice exhibit increased growth plate thickness and significantly decreased trabecular bone thickness and reduced bone mineral density. Histomorphometric and μ-CT analyses reveal bone loss in cranial and appendicular skeleton. Sh3bp2KI/KI mice also exhibit a significant decrease in osteoid formation that indicates a defect in osteoblast function. Calvarial osteoblast cell cultures exhibit a decrease in alkaline phosphatase expression and mineralization suggesting reduced differentiation potential. Gene expression of osteoblast differentiation markers like collagen type-I, alkaline phosphatase and osteocalcin are decreased in osteoblast cultures from Sh3bp2KI/KI mice. Conclusions These data suggest that Sh3bp2 function regulates bone homeostasis not only through osteoclast-specific effects but also through effects on osteoblast differentiation and function. PMID:20691350

  19. STUDY ON DIFFERENTIATION OF RATS EMBRYONIC STEM CELLS CULTURED IN BRL-CM INTO NEURAL PRECURSOR CELLS

    Institute of Scientific and Technical Information of China (English)

    张晓智; 李旭; 徐海伟; 陈葳

    2003-01-01

    Objective To investigate whether buffalo rat liver cell-conditioned medium (BRL-CM) can be used as the culture medium of embryonic stem (ES) cells, and to get relatively pure neural precursor cells (NPCs) for treatment aim. Methods Mouse ES cells were cultured in BRL-CM and medium contain leukemia inhibitory factor (LIF), respectively. NPCs were selectively cultured in serum-free medium. Alkaline phosphatase activity was visualized with NBT/BCIP and nestin antigen was detected with immunocytochemical methods. Results BRL-CM could be used as an efficiency culture condition instead of LIF in ES cells culture. About 86% of cells derived from ES cells in the serum-free culture were NPCs. Conclusion BRL-CM can replace LIF to use in ES cell culture. High purity of NPC can be induced from ES cells with serum-free culture method.

  20. [3H]ouabain binding to cultured rat vascular smooth muscle cells.

    Science.gov (United States)

    Khalil, F; Hopp, L; Searle, B M; Tokushige, A; Tamura, H; Kino, M; Aviv, A

    1984-05-01

    The number of Na+ pump units (Bmax) and the equilibrium dissociation constant (Kd) for ouabain as well as parameters of K+ binding to the Na+ pump were examined in in vitro-grown vascular smooth muscle cells ( VSMC ) derived from Sprague-Dawley rats. The technique to measure these variables utilizes analyses of [3H]ouabain displacement from its VSMC receptors by nonlabeled ouabain and K+. The mean values for Bmax and Kd in the cultured VSMCs were 1.95 X 10(5) receptor sites per single VSMC and 2.68 X 10(-6) M, respectively. The equilibrium dissociation constant for K+ (Ki) was 0.92 mM. K+ binding to the cultured VSMCs demonstrated positive cooperativity with a Hill coefficient (n) of 1.78.

  1. Quantitative analysis of the toxicity of human amniotic fluid to cultured rat spinal cord.

    Science.gov (United States)

    Drewek, M J; Bruner, J P; Whetsell, W O; Tulipan, N

    1997-10-01

    It has been proposed that the myelodysplastic components of a myelomeningocele are secondarily damaged as the result of exposure to amniotic fluid, the so-called 'two-hit' hypothesis. The critical time at which this secondary insult might occur has not been clearly defined. The present study addresses this issue by quantitatively assessing the toxic effects of human amniotic fluid of various gestational ages upon organotypic cultures of rat spinal cord. Using an assay for lactate dehydrogenase efflux to evaluate toxicity in such spinal cord cultures, we found that the amniotic fluid became toxic at approximately 34 weeks' gestation. This toxic effect of amniotic fluid appears to emerge rather suddenly. Accordingly, it seems reasonable to suggest that prevention of exposure of vulnerable spinal cord tissue to this toxicity by surgical closure of a myelomeningocele defect prior to the emergence of toxicity in amniotic fluid may prevent injury to vulnerable myelodysplastic spinal cord tissue.

  2. [POLYPEPTIDES INFLUENCE ON TISSUE CELL CULTURES REGENERATION OF VARIOUS AGE RATS].

    Science.gov (United States)

    Ryzhak, A P; Chalisova, N I; Lin'kova, N S; Khalimov, R I; Ryzhak, G A; Zhekalov, A N

    2015-01-01

    A comparative study of polypeptides extracted from the tissues of calves: Cortexin (from brain cortex), Epinorm (from pineal gland), Ventvil (from liver), Prostatilen (from prostate), Thymalin (from thymus), Chelohart (from heart), Chondrolux (from cartilage) on the relevant organotypic tissue cultures of young and old rats, in concentration 0,01-100 ng/ml was performed. Polypeptides specifically stimulated "young" and "old" cell cultures growth in concentration 20-50 ng/ml. This effect correlates with increasing of PCNA and decreasing of p53 expression in brain cortex, pineal gland, liver, prostate, heart, cartilage. Moreover, Thymalin activated CD5, CD20 expression--markers of B-cells differentiation. These data show that polypeptides isolated from different tissues have selective molecular activity on the regeneration of suitable tissues in aging.

  3. Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1998-01-01

    Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [3H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response...... of Schwann cells. Removal of extracellular Ca2+ by addition of EGTA to the culture medium suppressed [3H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca2+ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [3H] thymidine...... together with morphological evaluation of myelin association showed that proliferation occurred in Schwann cells. The results are consistent with a model in which Schwann cell proliferation is enhanced by Ca2+ through activation of calmodulin-dependent and/or PKCdependent mechanisms. Inhibition is achieved...

  4. Ethanol affects network activity in cultured rat hippocampus: mediation by potassium channels.

    Directory of Open Access Journals (Sweden)

    Eduard Korkotian

    Full Text Available The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25-0.5% ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.

  5. Real-time observations of mechanical stimulus-induced enhancements of mechanical properties in osteoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Xu; Liu Xiaoli; Sun Jialun [State Key Laboratory of Bioactive Materials, School of Physics, Nankai University, Tianjin 300073 (China); He Shuojie [State Key Laboratory of Bioactive Materials, School of Physics, Nankai University, Tianjin 300073 (China); Department of Physics, Pusan National University, Pusan (Korea, Republic of); Lee, Imshik [State Key Laboratory of Bioactive Materials, School of Physics, Nankai University, Tianjin 300073 (China)], E-mail: ilee@nankai.edu.cn2; Pak, Hyuk Kyu [Department of Physics, Pusan National University, Pusan (Korea, Republic of)

    2008-09-15

    Osteoblast, playing a key role in the pathophysiology of osteoporosis, is one of the mechanical stress sensitive cells. The effects of mechanical load-induced changes of mechanical properties in osteoblast cells were studied at real-time. Osteoblasts obtained from young Wister rats were exposed to mechanical loads in different frequencies and resting intervals generated by atomic force microscopy (AFM) probe tip and simultaneously measured the changes of the mechanical properties by AFM. The enhancement of the mechanical properties was observed and quantified by the increment of the apparent Young's modulus, E{sup *}. The observed mechanical property depended on the frequency of applied tapping loads. For the resting interval is 50 s, the mechanical load-induced enhancement of E{sup *}-values disappears. It seems that the enhanced mechanical property was recover able under no additional mechanical stimulus.

  6. Premature osteoblast clustering by enamel matrix proteins induces osteoblast differentiation through up-regulation of connexin 43 and N-cadherin.

    Directory of Open Access Journals (Sweden)

    Richard J Miron

    Full Text Available In recent years, enamel matrix derivative (EMD has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43 and N-cadherin (N-cad as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

  7. Effect of arsenite on urea production by long-term cultures of adult rat hepatocytes.

    Science.gov (United States)

    Sierra-Santoyo, A; Hernández, A; López, M L; Mendoza-Figueroa, T

    1996-01-01

    Urea cycle is a hepatic metabolic pathway involving five enzymes and several intermediary metabolites and can be altered by different chemicals. To investigate the effect of arsenic, an ubiquitous hepatotoxic agent, on urea production we exposed long-term cultures of adult rat hepatocytes, which produce urea, to 1.33 and 6.67 microM arsenite for 2 weeks. In cultures exposed to 6.67 microM, urea production decreased 60-70% and cellular arginase activity decreased 30, 70 and 85% after 4, 7 and 14 days of exposure, respectively. The arginase activity released to the medium increased significantly after 4, 7 and 14 days, with a maximum value after 7 days of exposure that was 27-fold higher than that of the untreated controls. The total arginase activity also decreased 35, 52 and 82% after 4, 7 and 14 days of exposure and protein content decreased 57 and 65% after 7 and 14 days of exposure, respectively. Exposure to 6.67 microM arsenite also produced accumulation of intracytoplasmic lipid droplets, vacuolizations and enlargement of the intercellular spaces. On the other hand, exposure of hepatocytes to 1.33 microM arsenite caused an initial decrease of 20% in urea production, did not change cellular, released and total arginase activity and cellular protein content and produced accumulation of intracytoplasmic lipid droplets. These results show that long-term exposure of cultured rat hepatocytes to 6.67 microM arsenite decreases urea production, cellular and total arginase activity and protein content and increases the release of arginase into the culture medium. These alterations could be useful markers of hepatotoxicity in in vitro assays.

  8. TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

    Science.gov (United States)

    Lu, L.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle

  9. Human osteoblast damage after antiseptic treatment.

    Science.gov (United States)

    Vörös, Pauline; Dobrindt, Oliver; Perka, Carsten; Windisch, Christoph; Matziolis, Georg; Röhner, Eric

    2014-01-01

    Antiseptics are powerful medical agents used for wound treatment and decontamination and have a high potential for defeating joint infections in septic surgery. Both chlorhexidine and polyhexanide are frequently used in clinical practice and have a broad antimicrobial range, but their effect on human osteoblasts has not been sufficiently studied. Our objective was to investigate the toxic effects of polyhexanide and chlorhexidine on human osteoblasts in vitro to evaluate their clinical applicability in septic surgery. We isolated and cultivated human osteoblasts in vitro and assayed the toxic effects of chlorhexidine 0.1% and polyhexanide 0.04%, concentrations commonly applied in clinical practice. Toxicity analysis was performed by visualisation of cell structure, lactate dehydrogenase (LDH) activity and evaluation of vital cells. Toxicity was evaluated by microscopic inspection of cell morphology, trypan blue staining and determination of LDH release. Damaged cell structure could be shown by microscopy. Both antiseptics promoted LDH activity after incubation with osteoblasts. The evaluation of vital osteoblasts showed a significant decrease of vital cells. Both antiseptics induced significant cell death of osteoblasts at optimum exposure. We therefore recommend cautious use of polyhexanide and chlorhexidine in septic surgery to avoid severe osteoblast toxicity.

  10. Dimethyl sulfoxide enhances lipid synthesis and secretion by long-term cultures of adult rat hepatocytes.

    Science.gov (United States)

    De La Vega, F M; Mendoza-Figueroa, T

    1991-05-01

    Dimethyl sulfoxide (DMSO) was tested for its effects on lipid metabolism of long-term cultures of adult rat hepatocytes. The addition of 1% DMSO to 3T3-hepatocyte cultures was not toxic to cells and in fact treated cultures maintained better their characteristic morphology for up to 14 days of exposure. DMSO treatment increased 2-3 fold the de novo synthesis of total lipids from[14C]acetate. The analysis by thin layer chromatography of cellular and secreted lipids revealed that DMSO increased the levels of cellular triglycerides, phospholipides and free and sterified cholesterol at 7 days of exposure while at 14 days there was also a 2-3-fold increase in medium secreted lipids. Additionally, DMSO increased the activity of glycerol-phosphate dehydrogenase, a marker enzyme of glycerolipid synthesis, by greater than 50% at either 7 or 14 days of exposure. These results show that 1% DMSO not only is not detrimental to cultured hepatocytes but also enhances lipid synthesis and secretion, both hepatic-differentiated functions.

  11. Dose-dependent DNA ruptures induced by the procarcinogen dimethylnitrosamine on primary rat liver cultures.

    Science.gov (United States)

    Mendoza-Figueroa, T; López-Revilla, R; Villa-Treviño, S

    1979-08-01

    The effect of certain procarcinogens, among which demethylnitrosamine (DMN) is included, has been difficult to detect in several short-term assays. An alternative system, in which DMN effects could be easily quantitated, might be useful in studies of chemical carcinogenesis and environmental contamination. To develop such a system, we tested the possibility of measuring the amount of breakage produced by DMN on radiolabeled DNA of primary liver cultures. Rat liver cells were isolated 20 to 24 hr after partial hepatectomy, cultured, and pulse labeled in vitro with [3H]thymidine. Radioactively labeled cultures were treated with DMN or with the direct carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and then lysed directly onto alkaline sucrose gradients. DMN and N-methyl-N'-nitro-N-nitrosoguanidine caused a dose-dependent reduction in the molecular weight of DNA, N-methyl-N'-nitro-N-nitrosoguanidine being approximately 1000 times more potent than DMN. DNA breaks appeared to be carcinogen specific and not due to cell death since treatment with high doses of cycloheximide, a noncarcinogenic hepatotoxic, was without significant effect. Our data indicate that detection of DNA breaks constitutes a more sensitive assay of DMN effects than does unscheduled DNA synthesis in primary liver cultures. Therefore, it could be useful to extend our work to determine the general applicability of quantitation of DNA breaks in liver cells as a short-term assay for the identification of possible carcinogens and procarcinogens.

  12. Comparison of two methods used to culture and purify rat retinal Müller cells.

    Science.gov (United States)

    Song, Wei-Tao; Zhang, Xue-Yong; Xiong, Si-Qi; Wen, Dan; Jiang, Jian; Xia, Xiao-Bo

    2013-01-01

    To study two methods for culturing and purifying Sprague-Dawley (SD) rat retinal Müller cells and determine which one is better. The passage culture method of Müller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method. After culturing retinal cells for one month through these two methods, fluorescence-activated cell sorter (FACS), RT-PCR, and immunohistochemistry technology were performed to examine the enrichment and purity of Müller glial cells, and carried out two-sample approximate t test using SSPS 13.0 to further compare the Müller cell positive rate in both methods. The statistical results showed that the purity of Müller cells was 83.2%±5.16% in group A, and the purity was 98.5%±1.08% in group B. The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant (t=-9.178, Pcells cultured by the complete pancreatic enzyme digestion method (group A) and the repeated incomplete pancreatic enzyme digestion method (group B). Compared with the complete pancreatic enzyme digestion method, this novel method was more efficient and a higher purity of Müller cells could be obtained using this approach.

  13. Physiological responses of osteoblasts to cyclic stretching and the change of intracellular calcium concentration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The development of bone tissues is regulated by mechanical stimulation. Cyclic stretching was applied to the osteoblasts that were delivered from rat calvarie. The results showed that stretching at 500 με increased the cell proliferation while loading at 1000 με and 1500 με inhabited cell growth. Loading alsoincreased the adhesive force between cells and substrate as well as spreading areas of osteobalsts. Furthermore, the fluorescence probe Fluo-3/AM was used to investigate the effect of stretching stimulation on the intracellular calcium concentration of osteoblasts. The intracellular calcium concentration of osteoblasts that were stretched at 500 με for 5 min was 92.9% higher than the control. After being treated with the panax ontoginseng saponins, the stretched osteoblasts still expressed 28.6% higher intracellular calcium concentration than that of the control, which proved that both the influx of extracellular calcium and the release of intracellular calcium store were involved in the increase of intracellular calcium concentration when osteoblasts responded to the cyclic stretching. And the influx of extracellular calcium through transmembrance channel played a main role.

  14. 改良体外培养方法及诱导剂配比条件下骨髓间充质干细胞向成骨细胞的分化%Differentiation of bone marrow mesenchymal stem cells into osteoblasts by modified in vitro culture methods and inductor matching

    Institute of Scientific and Technical Information of China (English)

    郭立达; 夏冰; 王捷

    2008-01-01

    状沉淀,碱性磷酸酶活性部位呈棕黑色.②骨钙素分泌情况:细胞经诱导后骨钙素阳性较明显,胞核呈蓝色,胞浆呈棕色.③细胞矿化作用:细胞经诱导培养后呈复层生长,并出现不透明结节,Von kossa染色可见黑色的矿化结节颗粒,颗粒大小不均一,提示有矿化基质沉积.结论:改良法可成功培养及诱导大鼠骨髓间充质干细胞成骨.%BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are in advantages of easy collection and amplification in vitro, but the culture and induction in vitro still need to be modified. OBJECTIVE: To investigate the differentiated potency of BMSCs into ostcoblasts by modified in vitro culture methods and inductor matching. DESIGN: Observational study. SETTING: Department of Medical Laboratory, General Hospital of Guangzhou Military Area Command of Chinese PLA. MATERIALS: This study was performed in the Stem Cell Tissue Engineering Laboratory, Department of Medical Laboratory, Genera Hospital of Guangzhou Military Area Command of Chinese PLA from July 2004 to June 2006. Twenty adult SD rats of clean grade, irrespective of gender, weighing 140-180 g were provided by Animal Experimental Center, General Hospital of Guangzhou Military Area Command of Chinese PLA. The experimental animals were disposed according to ethical criteria. Β-sodium glycerophosphate, dexamethasone, and vitamin C were provided by Sigma Company, USA; goat-anti-rat osteocalcin antibody by DSL Company, USA; S-P immunohistochemical kit by Maixin Biotechnology Developing Co., Ltd., Fujian. METHODS: Cells were cultured and induced into osteoblasts by modified methods. Separation and culture of BMSCs: By anesthesia, bone marrow of bilateral femur and tibia was separated to prepare single cell suspension; subsequently, the suspension was inoculated in culture bottle, and the culture media was semi-quantitatively changed after 48 and 96 hours in order to get rid of non-adherent hematopoietic cells

  15. Mechanism underlying blockade of voltage-gated calcium channels by agmatine in cultured rat hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Jian-quan ZHENG; Xie-chuan WENG; Xiao-dan GAI; Jin LI; Wen-bin XIAO

    2004-01-01

    AIM: To investigate whether agmatine could selectively block a given type of the voltage-gated calcium channels (VGCC) and whether related receptors are involved in the blocking effect of agmatine on VGCC. METHODS: The whole-cell patch recording technique was performed to record VGCC currents in the cultured neonatal rat hippocampal neurons. RESULTS: Verapamil (100 μmol/L), a selective blocker of L-type calcium channel, significantly inhibited VGCC current by 80 %± 7 %. Agmatine (100 μmol/L) could further depress the remained currents by 25 %±6 %. The α2-adrenoceptor antagonist yohimbine (10 μmol/L) and the I2 imidazoline receptor antagonist idazoxon (10 and 40 μmol/L) had no significant effect on VGCC currents when used respectively. When the mixture of yohimbine and agmatine was applied, VGCC currents were still depressed remarkably. However, the blocking effect of agmatine was decreased by 29 %± 8 % in the presence of idazoxon (10 μmol/L). The effect of idazoxon did not increase at a higher concentration (40 μmol/L). CONCLUSION: Agmatine could block the L- and other types of VGCC currents in the cultured rat hippocampal neurons. Blocking effect of agmatine on VGCC was partially related to I2 imidazoline receptor and had no relationship with α2-adrenoceptors.

  16. Evaluation of the rat embryo culture system as a predictive test for human teratogens.

    Science.gov (United States)

    Guest, I; Buttar, H S; Smith, S; Varma, D R

    1994-01-01

    Ingestion of the anticonvulsant drug valproic acid and of the angiotensin converting enzyme inhibitor captopril during pregnancy has been associated with abnormal fetal outcome in humans. In contrast, the use of the antiinflammatory drug ibuprofen and the antihistamine diphenhydramine has not been documented to be embryotoxic in humans. We evaluated the rat embryo culture system as a predictive model of teratogenesis, using these four drugs as test agents. Valproic acid, ibuprofen, and diphenhydramine were embryotoxic, inducing concentration-dependent decreases in growth and a significant increase in anomalies. Valproic acid caused an increase in neural tube defects, ibuprofen increased the incidence of abnormal maxillary processes, and diphenhydramine increased the number of embryos with distorted body morphology. These abnormalities were induced at concentrations of valproic acid and diphenhydramine that are used clinically, but ibuprofen only induced toxicity at concentrations greatly exceeding the therapeutic range. Captopril was not embryotoxic up to 5 mM, the highest concentration tested. These results suggest that the rat embryo culture system produces both false positive and false negative data on the teratogenic potential of drugs. Although such an in vitro assay may be suitable to determine the mechanism of teratogenesis, it is not a sensitive indicator of potential human teratogens on its own. These data support the view that in vitro systems can only supplement clinical and epidemiological observations in humans, possibly as a method to determine mechanisms of actions of teratogens.

  17. The role of adrenergic agonists on glycogenolysis in rat hepatocyte cultures and possible involvement of NO.

    Science.gov (United States)

    Hodis, J; Kutinová-Canová, N; Potmesil, P; Kameníková, L; Kmonícková, E; Zídek, Z; Farghali, H

    2007-01-01

    Certain liver metabolic diseases point to the presence of disturbances in glycogen deposition. Epinephrine raises the cAMP level that activates protein kinase A leading to the activation of phosphorylase and glycogen breakdown. In the present report, we sought to investigate whether NO is produced during adrenoceptor agonist-induced glycogenolysis in rat hepatocytes in cultures. Isolated glycogen rich rat hepatocytes in cultures were used. NO production (NO(2)(-)) was assessed under the effect of adrenergic agonists and adrenergic agonist/antagonist pairs, dibutyryl cyclic AMP sodium-potassium salt (db-cAMP), NO synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP). The inducible NO synthase (iNOS) mRNA was examined by the reverse transcription-polymerase chain reaction (RT-PCR). Glycogenolysis was quantified by glucose levels released into medium. The amount of glucose and NO(2)(-) released by hepatocytes was increased as a result of epinephrine, phenylephrine or db-cAMP treatments. The increase in glucose and NO(2)(-) released by epinephrine or phenylephrine was blocked or reduced by prazosin pretreatment and by NOS inhibitors aminoguanidine and L-NAME. iNOS gene expression was up-regulated by epinephrine. It can be concluded that glycogenolysis occurs through -adrenoceptor stimulation and a signaling cascade may involve NO production.

  18. Developmental features of rat cerebellar neural cells cultured in a chemically defined medium

    Energy Technology Data Exchange (ETDEWEB)

    Gallo, V.; Ciotti, M.T.; Aloisi, F.; Levi, G.

    1986-01-01

    We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8-day postnatal animals and cultured in a serum-free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L-glutamate analogue /sup 3/H-D-aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization-induced release of /sup 3/H-D-aspartate previously observed in this type of culture. In contrast, /sup 3/H-(GABA) was scarcely accumulated by glial-fibrillary-acidic-protein (GFAP)-positive astrocytes, but taken up by glutamate-decarboxylase-positive inhibitory interneurons and was released in a Ca2+-dependent way upon depolarization: /sup 3/H-GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle-associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. /sup 3/H-GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule-associated protein 2. These cells have been characterized as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high-affinity /sup 3/H-GABA uptake.

  19. alpha-Asarone toxicity in long-term cultures of adult rat hepatocytes.

    Science.gov (United States)

    López, M L; Hernández, A; Chamorro, G; Mendoza-Figueroa, T

    1993-04-01

    In this work we studied the toxic effects of alpha-asarone, a hypolipidemic active principle of Guatteria gaumeri Greenman, on long-term cultures of adult rat hepatocytes cultivated on a feeder layer of 3T3 cells. The exposure for one and two weeks to alpha-asarone (1-50 micrograms/ml) produced intracytoplasmic lipid droplets and at higher concentrations (25-50 micrograms/ml) retraction of the hepatocyte cords and cell detachment. Ultrastructurally, the treated cultures (10 micrograms/ml) showed enlargement and vacuolization of the mitochondria in addition to lipid droplets. The triacylglycerol content increased up to 2.3-fold in the cultures treated for one week with 50 micrograms/ml, whereas the protein content per culture, a rough estimate of cell number and viability, decreased by up to 53% in the cultures treated for two weeks with 50 micrograms/ml. The synthesis and secretion of proteins, measured by the incorporation of [3H]-leucine into cellular and secreted macromolecules, decreased also in the cultures exposed. After one and two week exposure to 50 micrograms/ml of alpha-asarone, the secretion of labeled proteins decreased by 53 and 67%, respectively, whereas the synthesis of cellular and total proteins decreased by 48-67%, respectively. The secretion of proteins was the most sensitive parameter of alpha-asarone toxicity. The mean inhibitory dose (ID50), i.e, that producing 50% inhibition in the incorporation of the labeled precursor, was 22.12 and 5.04 micrograms/ml after one and two weeks exposure, respectively. Our results show that long-term exposure to micromolar concentrations of alpha-asarone produces morphologic and ultrastructural alterations, triacylglycerol accumulation (fatty liver), and inhibition of protein synthesis and secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Collagen advanced glycation inhibits its Discoidin Domain Receptor 2 (DDR2)-mediated induction of lysyl oxidase in osteoblasts.

    Science.gov (United States)

    Khosravi, Roozbeh; Sodek, Katharine L; Faibish, Michael; Trackman, Philip C

    2014-01-01

    Diabetes increases the risk of bone fracture. Organic and inorganic bone extracellular matrix components determine bone strength. Previous studies indicate that in diabetes, glycation of collagen causes abnormal arrangements of collagen molecules and fragile bones. Diabetic bone fragility is additionally attributed to reduced levels of lysyl oxidase enzyme-dependent collagen cross-links. The mechanism underlying the presence of lower enzymatic collagen cross-links in diabetic bone has not been directly investigated. Here we determine in primary osteoblast cultures the regulation of lysyl oxidase protein by type I collagen and collagen modified by carboxymethylation (CML-collagen), a form of advanced glycation endproducts. Data indicate that non-glycated collagen up-regulates lysyl oxidase levels both in primary non-differentiated and in differentiating mouse and rat osteoblast cultures, while CML-collagen fails to regulate lysyl oxidase in these cells. Collagen binding to Discoidin Domain Receptor-2 (DDR2) mediates lysyl oxidase increases, determined in DDR2 shRNA knockdown studies. DDR2 binding and activation were disrupted by collagen glycation, pointing to a mechanism for the diminished levels of lysyl oxidase and consequently low lysyl oxidase-derived cross-links in diabetic bone. Our studies indicate that collagen-integrin interactions may not play a major role in up-regulating lysyl oxidase. Furthermore, non-collagenous ligands for the receptor for advanced glycation end products (RAGE) failed to alter lysyl oxidase levels. Taken together with published studies a new understanding emerges in which diabetes- and age-dependent inhibition of normal collagen-stimulated DDR2- and integrin-signaling, and independent advanced glycation-stimulated RAGE-signaling, each contributes to different aspects of diabetic osteopenia.

  1. Integrin α5β1-fimbriae binding and actin rearrangement are essential for Porphyromonas gingivalis invasion of osteoblasts and subsequent activation of the JNK pathway

    Directory of Open Access Journals (Sweden)

    Zhang Wenjian

    2013-01-01

    Full Text Available Abstract Background Chronic periodontitis is an infectious disease of the periodontium, which includes the gingival epithelium, periodontal ligament and alveolar bone. The signature clinical feature of periodontitis is resorption of alveolar bone and subsequent tooth loss. The Gram-negative oral anaerobe, Porphyromonas gingivalis, is strongly associated with periodontitis, and it has been shown previously that P. gingivalis is capable of invading osteoblasts in a dose- and time-dependent manner resulting in inhibition of osteoblast differentiation and mineralization in vitro. It is not yet clear which receptors and cytoskeletal components mediate the invasive process, nor how the signaling pathways and viability of osteoblasts are affected by bacterial internalization. This study aimed to investigate these issues using an in vitro model system involving the inoculation of P. gingivalis ATCC 33277 into primary osteoblast cultures. Results It was found that binding between P. gingivalis fimbriae and integrin α5β1 on osteoblasts, and subsequent peripheral condensation of actin, are essential for entry of P. gingivalis into osteoblasts. The JNK pathway was activated in invaded osteoblasts, and apoptosis was induced by repeated infections. Conclusions These observations indicate that P. gingivalis manipulates osteoblast function to promote its initial intracellular persistence by prolonging the host cell life span prior to its intercellular dissemination via host cell lysis. The identification of molecules critical to the interaction between P. gingivalis and osteoblasts will facilitate the development of new therapeutic strategies for the prevention of periodontal bone loss.

  2. Pharmacological assessment of ARTCEREB irrigation and perfusion solution for cerebrospinal surgery using primary cultures of rat brain cells.

    Science.gov (United States)

    Nishimura, Masuhiro; Doi, Kazuhisa; Kishimoto, Sanae; Koshitani, Osamu; Naito, Shinsaku; Yamauchi, Aiko

    2010-08-01

    ARTCEREB irrigation and perfusion solution (Artcereb), an ethical pharmaceutical, is typically applied inside the skull and spinal cavity as artificial fluid. Artcereb is composed of glucose and electrolytes (Na+, K+, Mg2+, Ca2+, Cl-, HCO3- and P) and has a pH of 7.3. An in vitro assessment of the effects of Artcereb on cell culture of rat fetal astrocytes or rat fetal brain cells was performed in comparison with normal saline and lactated Ringer's solutions. Furthermore, the effects of Artcereb on cell culture of rat fetal brain cells were also assessed in comparison with Krebs bicarbonate solution. Cell function after exposure to Artcereb was assessed based on 3H-thymidine incorporation activity. Cell function after exposure to Artcereb and lactated Ringer's solution in primary cultures of rat fetal astrocytes remained unaffected when compared to that after exposure to normal saline. Cell function after exposure to Artcereb in a primary culture of rat brain cells remained unaffected as compared to that after exposure to normal saline and lactated Ringer's solution. However, function decreased after exposure to a modified Artcereb formulation lacking bicarbonate, thus confirming that the presence of bicarbonate is essential for the Artcereb formulation.

  3. Osteoblast differentiation and migration are regulated by Dynamin GTPase activity

    OpenAIRE

    2013-01-01

    Bone formation is controlled by osteoblasts but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0–21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased...

  4. Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

    DEFF Research Database (Denmark)

    Jensen, T; Aliouat, E M; Lundgren, B

    1998-01-01

    The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

  5. Effects of pyridoxine on rat testes by means of Sertoli-germ cell co-culture system in vitro

    Institute of Scientific and Technical Information of China (English)

    Huang Houjin

    2001-01-01

    Objective To investigate the effects of pyridoxine on rat testis in vitro. Method an in vitro systen of Sertoligem cell co-culture was applied, the toxic effects of pyridoxine at different concentrations an exposed duration were olserved. Results The detachment of germ cells from sertoli cells showed marked dose-response and time response relafionships with the exposure of pyridoxine. Meanwhile, the characteristic of loosing and ratracting skeletun in the Sertoli cells was found. Conclusions The effects induced by pyridoxine in vitro may reflect damage to Sertoli cells, and testicular cells co-culture could be of value for the study of underlying mechanisms of toxic effects of pyridoxine on rat testis.

  6. Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75- and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum.

    Science.gov (United States)

    Gorski, J P; Griffin, D; Dudley, G; Stanford, C; Thomas, R; Huang, C; Lai, E; Karr, B; Solursh, M

    1990-09-05

    Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.

  7. Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopographies

    Directory of Open Access Journals (Sweden)

    Boyan B. D.

    2003-10-01

    Full Text Available Osteoblasts respond to microarchitectural features of their substrate. On smooth surfaces (tissue culture plastic, tissue culture glass, and titanium, the cells attach and proliferate but they exhibit relatively low expression of differentiation markers in monolayer cultures, even when confluent. When grown on microrough Ti surfaces with an average roughness (Ra of 4-7 µm, proliferation is reduced but differentiation is enhanced and in some cases, is synergistic with the effects of surface microtopography. In addition, cells on microrough Ti substrates form hydroxyapatite in a manner that is more typical of bone than do cells cultured on smooth surfaces. Osteoblasts also respond to growth factors and cytokines in a surface-dependent manner. On rougher surfaces, the effects of regulatory factors like 1alpha,25(OH2D3 or 17beta-estradiol are enhanced. The response to the surface is mediated by integrins, which signal to the cell through many of the same mechanisms used by growth factors and hormones. Studies using PEG-modified surfaces indicate that increased differentiation may be related to altered attachment to the surface. When osteoblasts are grown on surfaces with chemistries or microarchitectures that reduce cell attachment and proliferation, and enhance differentiation, the cells tend to increase production of factors like TGF-beta1 that promote osteogenesis while decreasing osteoclastic activity. Thus, on microrough Ti surface, osteoblasts create a microenvironment conducive to new bone formation.

  8. In Vitro Osteoblast Model for Bone Wound Infections and Antimicrobial Therapy (Addendum)

    Science.gov (United States)

    2012-01-01

    of multidrug resistant Acinetobacter baumannii , Klebsiella pneumoniae and Pseudomonas aeruginosa. Infection of osteoblasts revealed that all 3 species...cultures will be infected with clinical isolates of multidrug resistant Acinetobacter baumannii (AB), Klebsiella pneumonia (KP) and Pseudomonas...the model, and infections were performed with clinical isolates of multidrug resistant Acinetobacter baumannii , Klebsiella pneumoniae and

  9. Identification of differentiation-stage specific molecular markers for the osteoblastic phenotype

    DEFF Research Database (Denmark)

    Twine, Natalie; Chen, Li; Wilkins, Marc;

    The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity which belong mostly to extracellular matrix proteins. Also, for clinical use of human skeletal (mesenchymal) stem cells (hMSC) in bone regeneration, there ...

  10. Effects of chronic renal failure rat serum on histone acetyltransferase p300 and activation of activating transcription factor 4 of arterial smooth muscle cells cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    张耀全

    2014-01-01

    Objective To investigate the effects of the rat serum with chronic renal failure(CRF)on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4)of rat arterial vascular smooth muscle cells(VSMCs)cultured in vitro,and explore the possible mechanism.Methods Objective To establish the rat model of

  11. Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro.

    Science.gov (United States)

    Gianinazzi, C; Schild, M; Müller, N; Leib, S L; Simon, F; Nuñez, S; Joss, P; Gottstein, B

    2005-12-01

    The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

  12. Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells cultured in vitro.

    Science.gov (United States)

    Rato, Luís; Alves, Marco G; Socorro, Sílvia; Carvalho, Rui A; Cavaco, José E; Oliveira, Pedro F

    2012-02-01

    Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50 h. Glucose and pyruvate consumption, and lactate and alanine secretion w