There is an increasing interest in phytoestrogens due to their potential medical usage in hormone replacement therapy (HRT). The present study was designed to investigate the in vitro effects of estrogen-like activities of two widespread coumarins, osthole and imperatorin, using the MCF-7 cell proliferation assay and their alkaline phosphatase (ALP) activities in osteoblasts Saos-2 cells. The two compounds were found to strongly stimulate the proliferation of MCF-7 cells. The estrogen receptor-regulated ERα, progesterone receptor (PR) and PS2 mRNA levels were increased by treatment with osthole and imperatorin. All these effects were significantly inhibited by the specific estrogen receptor antagonist ICI182, 780. Cell cycle analysis revealed that their proliferation stimulatory effect was associated with a marked increase in the number of MCF-7 cells in S phase, which was similar to that observed with estradiol. It was also observed that they significantly increased ALP activity, which was reversed by ICI182,780. These results suggested that osthole and imperatorin could stimulate osteoblastic activity by displaying estrogenic properties or through the ER pathway. In conclusion, osthole and imperatorin may represent new pharmacological tools for the treatment of osteoporosis.
Alkaline Phosphatase ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cnidium ; chemistry ; Coumarins ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Furocoumarins ; pharmacology ; Humans ; MCF-7 Cells ; Osteoblasts ; cytology ; drug effects ; enzymology ; Phytoestrogens ; pharmacology ; Receptors, Estrogen ; genetics ; 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

OBJECTIVETo investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture.

METHODSROB 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.

RESULTSROB 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.

CONCLUSIONIcariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Collagen ; chemistry ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Drugs, Chinese Herbal ; Flavonoids ; pharmacology ; Hydrogels ; chemistry ; Osteoblasts ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; Transcription Factors ; metabolism

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

Natural products especially flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. The present study is to investigate the effects of isoquercitrin from Craibiodendron yunnanense with different concentrations at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) on proliferation, differentiation and mineralization of MC3T3-E1. Cell proliferation was assessed by CCK-8 kit at 1, 3, 5 and 7 days of culture. Alkaline phosphatase (ALP) activity were performed qualitatively and quantitatively on day 7, and alizarin red S staining was employed to access the mineralization of cells on day 21. The osteogenic markers ALP, collagen type I (COL 1A1), runt-related transcription factor 2 (Runx-2) and Osterix were detected to analysis early osteogenic differentiation of cells on day 3 by RT-PCR. The results showed that isoquercitrin had a dose-dependent effect on the proliferation, osteogenic differentiation, mineralization and gene expression of MC3T3-E1 in the range from 1 x 10(-7) to 1 x 10(-5) mol x L(-1). At concentrations above 1 x 10(-4) mol x L(-1) isoquercitrin showed cytotoxicity, while 1 x 10(-6) mol x L(-1) is the optimal concentration of isoquercitrin to improve the osteoblastic activity. All these results implied that isoquercitrin might be the major composition of traditional Chinese medicine C. yunnanense to treat bone fractures.
Alkaline Phosphatase ; metabolism ; Animals ; Cell Line ; Collagen Type I ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Ericaceae ; chemistry ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteogenesis ; drug effects ; Quercetin ; analogs & derivatives ; pharmacology

BACKGROUNDThe initial osteoblastic adhesion to materials characterizes the first phase of cell-material interactions and influences all the events leading to the formation of new bone. In a previous work, we developed a novel amorphous calcium phosphate (ACP)/poly(L-lactic acid) (PLLA) material that demonstrated morphologic variations in its microstructure. The aim of this study was to investigate the initial interaction between this material and osteoblastic cells. Cellular attachment and the corresponding signal transduction pathways were investigated.

METHODSA porous ACP/PLLA composite and PLLA scaffold (as a control) were incubated in fetal bovine serum (FBS) containing phosphate-buffered saline (PBS), and the protein adsorption was determined. Osteoblastic MG63 cells were seeded on the materials and cultured for 1, 4, 8, or 24 hours. Cell attachment was evaluated using the MTS method. Cell morphology was examined using scanning electron microscopy (SEM). The expression levels of the genes encoding integrin subunits α1, α5, αv, β1, focal adhesion kinase (FAK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using real-time reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe ACP/PLLA material significantly increased the protein adsorption by 6.4-fold at 1 hour and 2.4-fold at 24 hours, compared with the pure PLLA scaffold. The attachment of osteoblastic cells to the ACP/PLLA was significantly higher than that on the PLLA scaffold. The SEM observation revealed a polygonal spread shape of cells on the ACP/ PLLA, with the filopodia adhered to the scaffold surface. In contrast, the cells on the PLLA scaffold exhibited a spherical or polygonal morphology. Additionally, real-time RT-PCR showed that the genes encoding the integrin subunits α1, αv, β1, and FAK were expressed at higher levels on the ACP/PLLA composite.

CONCLUSIONSThe ACP/PLLA composite promoted protein adsorption and osteoblastic adhesion. The enhanced cell adhesion may be mediated by the binding of integrin subunits α1, αv, and β1, and subsequently may be regulated through the FAK signal transduction pathways.

Biocompatible Materials ; chemistry ; Calcium Phosphates ; chemistry ; Cell Adhesion ; physiology ; Cells, Cultured ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Integrin alpha1 ; metabolism ; Integrin alpha5 ; metabolism ; Integrin alphaV ; metabolism ; Integrin beta1 ; metabolism ; Integrins ; genetics ; metabolism ; Lactic Acid ; chemistry ; Osteoblasts ; cytology ; Porosity ; Tissue Engineering ; methods

BACKGROUNDMechanical stress plays an important role in the maintenance of bone homeostasis. Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone. This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress (FSS) is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells.

METHODSMC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm(2) using a parallel plate flow system. After FSS treatment, cytoskeleton immunohistochemical staining and microRNAs (miRNAs) were detected immediately. Osteogenic gene expression and immunohistochemical staining for collagen type I were tested at the 24th hour after treatment, alkaline phosphatase (ALP) activity assay was performed at 24th, 48th, and 72 th hours after FSS treatment, and Alizarin Red Staining was checked at day 12.

RESULTSOne hour of FSS at 12 dyn/cm(2) induced actin stress fiber formation and rearrangement, up-regulated osteogenic gene expression, increased ALP activity, promoted synthesis and secretion of type I collagen, enhanced nodule formation, and promoted terminal differentiation in MC3T3-E1 cells. During osteogenic differentiation, expression levels of miR-20a, -21, -19b, -34a, -34c, -140, and -200b in FSS-induced cells were significantly down-regulated.

CONCLUSIONThe short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be involved in FSS-induced pre-osteoblast differentiation.

Actins ; chemistry ; Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; genetics ; Cyclooxygenase 2 ; genetics ; Gene Expression Profiling ; Mice ; MicroRNAs ; physiology ; Osteoblasts ; cytology ; Osteogenesis ; Stress, Mechanical ; Stress, Physiological

OBJECTIVETo evaluate the adhesion, proliferation and differentiation of osteoblast-like cells on the ultraviolet (UV)-treated titanium in different storing environment, and to find a way to enhance the bioactivity of titanium and to prevent its age-related degradation.

METHODSAcid-etched titanium disks stored under ambient conditions for 4 weeks and treated with UV light for 48 h.Then disks were divided into three groups and placed in a sealed container for 0 h (no-stored,NO group) , 4 weeks (air-stored, AS group) or in a sealed container filled with nitrogen for 4 weeks (nitrogen-stored,NS group) respectively. A group of UV-untreated titanium served as negative control (NC group).The surface morphology was evaluated using scanning electron microscopy (SEM), and hydrophilicity of disks were measured using contact angle measuring device. Cell counting kit-8 was used to measure the cell adhesion and proliferation. Cell differentiation was evaluated by testing alkaline phosphatase (ALP) activity using ALP reagent kit.

RESULTSThere was no difference in surface topography among groups.Contact angels in NS group [(67.70 ± 3.59)°] and NO group [(0.70 ± 0.28)°] were smaller than the others (P < 0.05). Cell adhesion in NS group at 2 h and 4 h point was (0.237 ± 0.006) and (0.578 ± 0.039), respectively, and proliferation at 3 d and 5 d point was (0.743 ± 0.026) and (1.548 ± 0.046) respectively, which were significantly higher than those in AS group [(0.158 ± 0.036), (0.400 ± 0.010), (0.499 ± 0.019) and (1.174 ± 0.062)] and in NC group [(0.161 ± 0.024), (0.390 ± 0.011), (0.508 ± 0.015) and (1.209 ± 0.025)] at the same time point (P < 0.05). How ever the results mention above in the NS group were lower than those in the NO group (P < 0.05). No difference were found between data from the AS group and NS group (P > 0.05). Osteoblast-like cells had an abundant spread on NS and NO group during 2 h incubation, but did not exactly spread on AS and NC group after incubation for 4 h. No difference were found in ALP among groups.

CONCLUSIONSUV treatment can enhance bioactivity of titanium, and nitrogen storage can slow down its biological aging.

Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; radiation effects ; Cell Differentiation ; radiation effects ; Cell Proliferation ; radiation effects ; Cells, Cultured ; Mice ; Microscopy, Electron, Scanning ; Nitrogen ; chemistry ; Osteoblasts ; cytology ; metabolism ; Surface Properties ; Titanium ; chemistry ; radiation effects ; Ultraviolet Rays

OBJECTIVETo evaluate the effect of platelet-rich fibrin extract (PRFe) on proliferation and differentiation and F-actin cytoskeleton of osteoblasts.

METHODSThe experimental group used the α-minimum essential medium (α-MEM) containing PRFe (10% fetal bovine serum), and the control group used the α-MEM (10% fetal bovine serum). The number of the osteoblasts at 1st, 3rd, 5th d was detected by methyl thiazolyl tetrazolium (MTT) assay, and the differentiation of osteoblast at 1st, 3rd, 5th,7 th d detected by the activity of alkaline phosphatase (ALP).The alizarin red dye was used to observe the number of calcium nodus at 14th, 21st d. The F-actin cytoskeleton was evaluated by confocal laser scanning microscope(CLSM) at 3rd,6th,9th,12th h. The level of osteogenetic biomarkers osteocalcin (OCN) and core-binding factor α1(Cbfα1) at 3rd,7th d were quantified by real-time PCR.

RESULTSA significant increase of absorbance at 1st, 3rd, 5th d was showed in experimental group (0.336 ± 0.011, 0.571 ± 0.039, 0.787 ± 0.050) compared to control group (0.300 ± 0.021, 0.387 ± 0.040, 0.527 ± 0.034) (P < 0.05). The absorbance of experimental group at 1st, 3rd, 5th, 7th d (0.146 ± 0.014, 0.199 ± 0.017, 0.390 ± 0.020, 0.492 ± 0.019) was significantly higher than that of control group (0.115 ± 0.014, 0.145 ± 0.015, 0.190 ± 0.015, 0.230 ± 0.026) (P < 0.05). The integrated absorbance of the calcium nodus in experimental group at 14th, 21st d (22.119 ± 3.694, 31.528 ± 3.162) was significantly higher than in control group (8.498 ± 2.041, 15.162 ± 2.526) (P < 0.05). The Cbfα1 and OCN gene expression in experimental group was higher than in control group (P < 0.05).

CONCLUSIONSPRFe could enhance the proliferation and differentiation of osteoblasts and promote the spread of F-actin cytoskeleton.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Cytoskeleton ; drug effects ; Fibrin ; isolation & purification ; pharmacology ; Male ; Mice ; Osteoblasts ; cytology ; Osteocalcin ; metabolism ; Platelet-Rich Plasma ; chemistry ; Rats ; Rats, Sprague-Dawley

The temporal expression of estrogen receptor (ER)-alpha and ER-beta mRNA was examined in male Japanese quails. Femurs of quails receiving 17beta-estradiol underwent RTPCR and histochemical analysis 1 to 15 days after treatment. Untreated quails were used as controls (day 0). Between days 0 and 5, cells lining the bone endosteal surface differentiated into osteoblasts, which in turn formed medullary bone. Expression of ER-alpha was already observed on day 0 and increased slightly during bone formation whereas ER-beta was hardly detected throughout this process. After osteoclasts appeared on the medullary bone surface, this type of bone disappeared from the bone marrow cavity (days 7~15). ER-alpha expression simultaneously decreased slightly and ER-beta levels remained very low. These results suggest that estrogen activity mediated by ER-alpha not only affects medullary bone formation but also bone resorption.
Animals ; Bone Resorption/genetics ; Bone and Bones/chemistry/cytology/*metabolism ; Cells, Cultured ; Coturnix/*metabolism ; Estradiol/*pharmacology ; Estrogen Receptor alpha/genetics/*metabolism ; Estrogen Receptor beta/genetics/*metabolism ; Gene Expression Regulation ; Male ; Osteoblasts/chemistry/cytology/*metabolism ; Osteogenesis/genetics ; RNA, Messenger/metabolism ; Reverse Transcriptase Polymerase Chain Reaction