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 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

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

This study was aimed to explore the influence of brucine on the early differentiation of osteoblasts and the metabolic pathway of osteoclast in multiple myeloma (MM) and to compare the effects of brucine and bortezomib on MM. The half inhibitory concentration (IC(50)) of brucine and bortezomib on MM cell line U266 was determined by MTT method; the mRNA levels of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and osteoprotegerin ligand (RANKL) were detected by RT-PCR after the supernatant of cultured U266 cells was added into the culture system for inducing the differentiation of osteoblast line MC3T3-E1 and culturing. The results showed that the IC(50)of bortezomib and brucine on U266 cells for 48 hours were 22.4 nmol/L and 0.16 mg/ml respectively. As compared with osteoblasts treated by supernatant of cultured MM cells alone, the mRNA levels of ALP, OC and OPG in osteoblasts treated by brucine combined with supernatant of cultured MM cells were enhanced (p < 0.05), while the RANKL mRNA level was lowered (p < 0.05), moreover the enhanced and lowered degree also was large (p < 0.05). It is concluded that the influence of brucine on metabolism of osteoblasts and osteoclasts in MM may be realized through the regulation of osteoclasts by osteoblasts. The therapeutic efficacy of brucine on MM is superior to bortezomib.
Animals ; Cell Line ; Cell Line, Tumor ; Humans ; Inhibitory Concentration 50 ; Mice ; Multiple Myeloma ; metabolism ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteoclasts ; cytology ; drug effects ; metabolism ; Strychnine ; analogs & derivatives ; pharmacology

OBJECTIVETo investigate the growth activity of osteoblast on a novel strontium incorporated calcium sulfate and make comparison with normal calcium sulfate material.

METHODSOsteoblast was inoculated on samples and cell proliferation was measured on the 1st, 3rd, 5th days, and the activities of ALP and osteocalcin were observed on the 5th day. And microcosmic morphology of osteoblast was observed by scanning electron microscopy(SEM).

RESULTSOsteoblast grows robustly on tested material. Cell quantity on the surface of novel material was obviously higher than normal calcium sulfate material (P < 0.05). The activity of ALP and osteocalcin on novel material was 57.8% and 40.2% higher than on normal calcium sulfate material respectively (P < 0.05). On strontium incorporated surface, osteoblast spread well. Cells were polygonal with abundant cytoplasm and the morphology was active.

CONCLUSIONStrontium incorporated calcium sulfate can sustain robust growth activity of osteoblast, which is promising to be used for bone substitute materials.

3T3 Cells ; Alkaline Phosphatase ; metabolism ; Animals ; Bone Substitutes ; chemistry ; pharmacology ; Calcium Sulfate ; chemistry ; pharmacology ; Cell Proliferation ; drug effects ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteocalcin ; metabolism ; Strontium ; chemistry

Heme oxygenase-1 (HO-1) plays important roles in anti-oxidant, anti-inflammatory and immunoregulative activities. The aim of this study was to observe if HO-1 transfection could inhibit the damage of osteoblasts induced by ethanol. HO-1 was transfected into osteoblasts via constructed plasmid. After exposure to ethanol for 24 h, cytoactivity and apoptosis of osteoblasts were measured by MTT assay and flow cytometry, respectively. Furthermore, the oxidative stress and inflammatory factors in osteoblasts were measured. Compared to positive control group, the cytoactivity of transfected osteoblasts was significantly increased, and the apoptosis rate was significantly decreased (P<0.05). At the same time, the levels of reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA), tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) were significantly decreased (P<0.05), and superoxide dismutase (SOD) level was increased (P<0.05) in the transfected osteoblasts as compared with positive controls. These results suggest that HO-1 plays a protective role in osteoblasts, and HO-1 transfection can effectively inhibit bone damage induced by ethanol.
Cells, Cultured ; Ethanol ; toxicity ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Osteoblasts ; cytology ; drug effects ; Oxidative Stress ; drug effects ; Transfection

With the development of cell culture technology in vitro, people have successfully cultivated osteoblast cells of typical characteristics from a number of animal skull, bone marrow, periosteum and bone tissues; studies have shown that these osteoblasts have good biological characteristics, can create bone tissue in different environments, can apply joint stand for the construction of tissue engineered bone, be implanted in the body to repair bone defects. In this article, the source of osteoblast, regulatory factor, composite graft and Chinese medicine research progress were reviewed.
Animals ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Humans ; Osteoblasts ; cytology ; drug effects ; metabolism ; Tissue Engineering

OBJECTIVETo investigate the effects of Osthol on the proliferation and differentiation of osteoblasts of rats (rat calvarial osteoblasts, ROB) cultured in vitro.

METHODSThe neonatal SD rat skull was segregated, and enzyme digestion was used to obtain bone cells which were cultured in MEM containing 10% FBS. The medium was changed every three days, and serial subcultivation was performed when cells covered with 90% of the culture dish. The Osthol was added to 96-well plates with final concentration of 1 x 10(-4) mol/L, 1 x 10(-5) mol/L, 1 x l0(-6) mol/L and 1 x10(-7) mol/L, and MTT method was used to evaluate the proliferation. Differentiation analysis: the alkaline phosphatase (ALP) activity was determined at the 3rd, 6th, 9th, 12th and 15th days separately after osteogenic induction culture. The synthesis of type I collagen was observed using immunohistochemical method at the 8th day. The ALP stain was performed at the 12th day. The alizarin red staining was done and calcified nodules was counted at the 14th day.

RESULTSThe Osthol with final concentration of 1 x 10(-4) mo/L inhibit the proliferation of ROB. The Osthol with final concentration of 1 x 10(-5) mol/L had no obvious influence on the proliferation of ROB, but it significantly promoted the activity of ALP, enhanced the synthesis of collagen type I and increased the number of calcified nodules.

CONCLUSIONThe Osthol with final concentration of 1 x 10(-5) mol/L can promote differentiation and maturation of ROB, which may be active ingredients of Chinese drugs for the osteoporosis prophylaxis.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coumarins ; pharmacology ; Female ; Male ; Osteoblasts ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo determine the effects of KH2PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae (SCAP) in vitro.

METHODSSCAP were isolated and cultured respectively in alpha minimum essential medium (α-MEM) or α-MEM containing 1.8 mmol/L KH2PO4. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine the odonto and osteogenic potential of SCAP in the two media.

RESULTSSCAP cultured in α-MEM containing 1.8 mmol/L KH2PO4 exhibited a higher ALP activity [(0.370 ± 0.013) Sigma unit×min(-1)×mg(-1)] at day 3 than control group [(0.285 ± 0.008) Sigma unit×min(-1)×mg(-1)] and KH2PO4-treated SCAP formed more calcified nodules at day 5 [(0.539 ± 0.007) µg/g] and day 7 [(1.617 ± 0.042) µg/g] than those in normal medium [(0.138 ± 0.037) µg/g, P < 0.01]. The expression of odonto- and osteogenic markers were significantly up-regulated after the stimulation of KH2PO4 at day 3 and 7 respectively, as compared with control group.

CONCLUSIONS1.8 mmol/L KH2PO4 can promote the odonto and osteogenic differentiation potential of human SCAP.

Cell Differentiation ; drug effects ; Cells, Cultured ; Dental Pulp ; cytology ; Extracellular Matrix Proteins ; metabolism ; Humans ; Osteoblasts ; cytology ; Osteocalcin ; metabolism ; Phosphates ; pharmacology ; Phosphoproteins ; metabolism ; Potassium Compounds ; pharmacology ; Sialoglycoproteins ; metabolism ; Stem Cells ; cytology ; drug effects ; 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