OBJECTIVETo establish the human osteoblasts culture system in vitro, observe the effects of icariin on human osteoblasts proliferation and expression of OPG protein, and to explore the mechanism of promoting bone formation about human osteoblast in icariin.

METHODSThe femoral cancellous bone pieces were obtained from the operation. The enzyme digestion method was used for culturing. The third passage of human osteoblast was taken for experiments. The cells were divided into four groups, the control group was treated with 15% NCS-DMEM-F12 (1:1), the experimental groups were respectively treated with 10(-6), 10(-8), 10(-10) mol/L icariin. The MTT method was used to observe the proliferation of human osteoblast on 1, 3, 5, 7, 9 d; in 8, 10, 12 d, western blot was used to determine the expression of OPG protein on human osteoblast.

RESULTS1) Results of MTT: the icariin promoted the proliferation of human osteoblast. There was a concentration-response relation,while with the concentration of icariin increased, the ability was more obvious. There was statistically difference between 10(-6) mol/L icariin group (0.402 +/- 0.033) and the control group (0.268 +/- 0.031) (P<0.05). In the timely research, as the time prolong, the number of human osteoblast were more. At the fifth day, the human osteoblast entered rapid growth period, and access the growth platform stage; the icariin began to promote the proliferation of human osteoblast from the fifth day, which almost maintained to the 7th day and the 9th day, and most obvious in the 9th day. There was statistically difference between 10(-6) mol/L icariin group (0.402 +/- 0.033) and the control group (0.268 +/- 0.031) at the 9th day. (The results of OPG protein expression: in the control group, the expression of OPG protein was detected at the 8th day (1.01 +/- 0.08), and reached the expression peak (1.80 +/- 0.10), there was statistically different (P<0.05). In the different days and different concentration icariin groups, the expressions of OPG protein were all inferior to the control group. While the concentration decreasing, the expression was less. There were statistically difference (P<0.05). At the day 12, there was no significant difference of OPG protein expression between the 10(-6) mol/L icariin group and the 10(-8) mol/L icariin group (P>0.05).

CONCLUSIONThe effect of icariin promoting the proliferation of human osteoblast maybe is one of the mechanisms of improving the bone formation on human osteoblast.

Animals ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; Humans ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteogenesis ; drug effects ; Osteoprotegerin ; metabolism

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.
Animal ; Calcium/*metabolism ; Cells, Cultured ; Hydrogen Peroxide/*pharmacology ; Osteoblasts/*drug effects/*metabolism ; Oxidants/*pharmacology ; Rats

OBJECTIVETo evaluate the biological effects of nano-hydroxyapatite/polyamide 66(nHA-PA66) on the growth and activity of osteoblast.

METHODSMTT assay was used to determine the growth of osteoblast, enzymatic measure was used to determine the activity of ALP and quantitative RT-PCR (QRT-PCR) to evaluate the changes of osteoclacin mRNA expression in osteoblasts treated by DMEM eluate of nHA-PA66.

RESULTSOsteoblasts of different test groups demonstrated relative proliferation rate ranging from 98% - 106% without dose-dependent effect. The ALP activity and osteocalcin mRNA expression were similar in test and control groups (P > 0.05).

CONCLUSIONnHA-PA66 has no negative effects on the osteoblast and its osteoblast-compatibility is proved.

Durapatite ; pharmacology ; Nylons ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; metabolism ; RNA, Messenger ; metabolism

Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Ceramics ; pharmacology ; Gene Expression Regulation ; drug effects ; Glass ; Humans ; Osteoblasts ; drug effects ; metabolism ; Osteogenesis ; drug effects

OBJECTIVETo observe the intake of minocycline and its amount in mature rat mandibular osteoblasts (MRMOB) in vitro, and to identify the feasibility of intracellular anti-bacterial activity of minocycline.

METHODSFour groups of MRMOB were incubated in 100 mg/L minocycline for 15, 30, 45 and 60 minutes respectively, and the accumulation of minocycline within MRMOB was measured using a fluorescence spectrophotometer.

RESULTSThe intracellular accumulation amount of minocycline in the four groups of MRMOB was (17.29 +/- 1.49), (16.87 +/- 1.57), (16.96 +/- 1.67) and (17.94 +/- 1.63) mg/g respectively after osteoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained minocycline for 15, 30, 45 and 60 min. There was no significant difference in amount of minocycline among the four groups of MRMOB.

CONCLUSIONSThe mature rat mandibular osteoblasts can ingest minocycline, and the accumulation amount of minocycline in MRMOB is irrelevant with the exposure time of MRMOB to minocycline.

Animals ; Cells, Cultured ; Mandible ; cytology ; drug effects ; metabolism ; Minocycline ; pharmacokinetics ; Osteoblasts ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of zoledronic acid on proliferation and osteogenic differentiation of osteoblasts.

METHODSOsteoblasts were obtained from newly born rabbit jaw bones and cultured by the method of bone-tissue cultivation. Primary cultivated osteoblast was identified by alkaline phosphatase (ALP) and the mineralization nodes. Zoledronic acid at various concentrations was added to six groups of media with serial subcultivated cells (the final concentration: 0, 10(-5), 10(-6), 10(-7), 10(-8) and 10(-9) mol/L). At different time, ALP, osteoprotegerin and osteocalcin were observed and calculated.

RESULTSThe concentration of 10(-6), 10(-7), 10(-8) and 10(-9) mol/L zoledronic acid significantly increased ALP activity [(5.91 ± 0.35), (7.62 ± 0.33), (10.00 ± 0.38), (8.91 ± 0.29) U/L]. Protein expression of osteoprotegerin and osteocalcin was enhanced. The differences among the groups were significant (P < 0.01). Peak level was attained at a concentration of 10(-8) mol/L.

CONCLUSIONSZoledronic acid promotes osteoblast proliferation and maturation and modulates osteoprotegerin production.

Alkaline Phosphatase ; Animals ; Bone and Bones ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Diphosphonates ; pharmacology ; Imidazoles ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; drug effects ; metabolism ; Osteogenesis ; Osteoprotegerin ; drug effects ; metabolism ; Rabbits

OBJECTIVETo observe the effects of two main isoflavones, daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro.

METHODSOsteoblasts obtained from newborn Sprague-dawley rat calvaria 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.

RESULTSCompared with the control, the numbers of nodules were both increased by incubation with daidzein and genistein. 17 beta-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation that daidzein and genistein. The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein, as well as 17 beta-estradiol on day 7 and day 12 (day 12 were higher). The estrogen receptor antagonist ICI 182780 completely blocked the genistein- and 17 beta-estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts. However, the effects induced by daidzein could not be inhibited by ICI 182780.

CONCLUSIONThese findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts. The effects, like those induced by 17 beta-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.

Animals ; Cells, Cultured ; Genistein ; pharmacology ; Isoflavones ; pharmacology ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteocalcin ; biosynthesis ; Osteogenesis ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; drug effects ; metabolism

OBJECTIVETo investigate the effect of alendronate on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in mouse osteoblasts.

METHODSMouse calvarial osteoblasts cultured in vitro were identified by alkaline phosphatase (ALP) staining and immunofluorescence assay of OPG and RANKL expressions. The second passage of the osteoblasts were treated with different concentrations of alendronate (10(-4) to 10(-7) mol/L) for 48 h, and the changes in OPG and RANKL mRNA and protein expressions were examined using real-time PCR and Western blotting, respectively.

RESULTSThe isolated osteoblasts were positive for ALP and expressed OPG and RANKL. Real-time PCR and Western blotting showed that at the concentration of 1×10(-4) mol/L, alendronate caused an obvious down-regulation of OPG and RANKL expressions in the cells, whereas at lower concentrations, alendronate increased the expressions of both genes with the highest expressions occurring after treatment with 1×10(-5) mol/L.

CONCLUSIONHigh concentrations of alendronate (>1×10(-4) mol/L) decrease the expressions of OPG and RANKL, whereas low concentrations (1×10(-5) to 1×10(-7) mol/L) increase their expressions in mouse osteoblasts cultured in vitro.

Alendronate ; pharmacology ; Animals ; Cells, Cultured ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; drug effects ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism

The Wnt/beta-catenin pathway has a role in osteoblast differentiation and bone formation. We screened 100 plant extracts and identified an extract from Euodia sutchuenensis Dode (ESD) leaf and young branch as an effective activator of the Wnt/beta-catenin pathway. ESD extract increased beta-catenin levels and beta-catenin nuclear accumulation in murine primary osteoblasts. The ESD extract also increased mRNA levels of osteoblast markers, including RUNX2, BMP2 and COL1A1, and enhanced alkaline phosphatase (ALP) activity in murine primary osteoblasts. Both ESD extract-induced beta-catenin increment and ALP activation were abolished by beta-catenin knockdown, confirming that the Wnt/beta-catenin pathway functions in osteoblast differentiation. ESD extract enhanced terminal osteoblast differentiation as shown by staining with Alizarin Red S and significantly increased murine calvarial bone thickness. This study shows that ESD extract stimulates osteoblast differentiation via the Wnt/beta-catenin pathway and enhances murine calvarial bone formation ex vivo.
Animals ; Cell Differentiation/*drug effects ; Evodia/*chemistry ; HEK293 Cells ; Humans ; Mice ; Osteoblasts/cytology/*drug effects/*metabolism ; Osteogenesis/drug effects ; Plant Extracts/chemistry/*pharmacology ; Skull/anatomy & histology/drug effects/metabolism ; Wnt Signaling Pathway/*drug effects ; beta Catenin/genetics/metabolism

Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly increased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.
Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Osteoblasts ; drug effects ; metabolism ; Osteogenesis ; drug effects ; RANK Ligand ; metabolism ; Rats ; Signal Transduction ; drug effects ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism