Objective: To compare the effects of 50 Hz 1.8 mT sinusoidal magnetic field (SEMF) and 50 Hz 0.6 mT pulsed electromagnetic field(PEMF) on the maturation and mineralization of rat calvaria osteoblasts. Methods: Primary cultured rat calvarial osteoblasts were divided into 3 groups:blank control group, SEMF group and PEMF group. The rats in SEMT and PEMT groups were treated with 50 Hz 1.8 mT SEMF or 50 Hz 0.6 mT PEMF for 90 min/d, respectively. Western blotting and Real-time RT-PCR were used to detect the protein and mRNA expressions of Collagen-1, bone morphogenetic protein 2 (BMP-2), osterix (OSX) and Runt-associated transcription factor 2(Runx-2). The alkaline phosphatase(ALP) activity was detected by ALP test kits at d6 and d9 after treatment, and by ALP staining using azo coupling at d10 after treatment. The formation of calcium nodules was observed by alizarin red staining. Results: Compared with blank control group, the protein and mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in SEMT and PEMT groups were significantly increased (P <0.01 or P <0.05); while the mRNA expressions of Collagen-1 and BMP-2 in PEMF group were significantly higher than those in SEMF group. After 6 days treatment, the activity of ALP in PEMF group was significantly higher than that in blank control group (P<0.05), while such difference was not observed in SEMF group (P0.05); after 9 days treatment, the activities of ALP in both PEMF and SEMP groups were significantly higher than that in blank control group (all P<0.05), but the difference between PEMF and SEMF groups was not significant (P0.05). After 10 days treatment, ALP staining was increased in both PEMF and SEMF groups compared with that in blank control group (all P<0.01), and the stained area was bigger in PEMF group than that in SEMF group (P<0.05). After 12 days treatment, calcium nodules were increased in PEMF and SEMF groups compared with that in blank control group (all P<0.01), and more calcium nodules were observed in PEMF group than SEMF group (P<0.05). Conclusion: Both 50 Hz 1.8 mT that in SEMF and 50 Hz 0.6 mT PEMF can promote the maturation and mineralization of osteoblasts, and the effect of PEMF is more marked.
Animals ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; Cells, Cultured ; Electromagnetic Fields ; Gene Expression Regulation, Developmental ; radiation effects ; Magnetic Fields ; Osteoblasts ; cytology ; radiation effects ; Rats ; Skull ; drug effects

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

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

To establish an experimental model of osteoblasts to easily cause calcification of bone matrix in vitro, we took cranium of a newborn rabbit out under an aseptic condition, removed the connective tissue of the bony suture, and cut the cranium freely into the fragments of not more than 1 mm2. The we isolated and cultured the osteoblasts using tissue explant method. We observed growth status of primary osteoblasts and subcultured osteoblasts using inverted microscope. Then we conducted enzyme staining and alizarin red staining for the third generation of osteoblasts to detect the alkaline phosphatase (ALP) expression and calcified nodules. The result showed that there were calcified nodules or calcification formed after the primary osteoblasts climbing out from the bone for 1 week, and each generation of osteoblasts had the similar calcification with the primary osteoblasts, and there was an increase in calcified nodules after the continuous culture. There was a strong expression of ALP in the plasma membrane of osteoblasts. The calcified nodules were red with alizarin red staining. It is well concluded that osteoblasts isolated with this method easily cause calcification, and can be used as a new experimental model.
Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Cell Separation ; methods ; Osteoblasts ; cytology ; Primary Cell Culture ; methods ; Rabbits ; Skull ; cytology

OBJECTIVETo investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial osteoblasts (ROB).

METHODSegregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differentiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERbeta was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERbeta and collagen I was examined by Western blot. After the addition of ICI 182.780, an inhibitor of the estrogen signal pathway, these index also was examined and the changes were compared.

RESULTThe ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 x 10(-5) mol x L(-1) is the best concentration. Naringin improved the secretion of osteocalcin, osteopontin, bone morphogenetic protein-2 and collagen I significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERalpha and ERbeta. While all these effects can be restrained by ICI 182.780.

CONCLUSIONThe naringin with final concentration of 1 x 10(-5) mol x L(-1) enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182.780. These results suggesting that naringin is one of the phytoestrogens and have the activity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.

Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Flavanones ; pharmacology ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteocalcin ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; drug effects ; metabolism

To study the biomechanical behaviors of the cells, reliable fluid shear stress loading system is needed. Compared to the traditional parallel plate flow chamber (PPFC) system, a rocking system presented by Zhou offers some advantages such as easier operation, lower cost and higher quantity of pocessing. But the feasibility of it has not been practically studied. To investigate the feasibility whether the rocking system can be used to apply quantified fluid shear stress loading, primary osteoblasts of mouse were loaded with fluid shear stress based on rocking system and traditional PPFC system, respectively. Another group of cells was unloaded as control. The cytoskeleton was observed with laser scanning confocal microscope (LSM) and average fluorescence of F-actin was recorded. Cell cycle was also measured by flow cytometry and percentage of S-phase cells was recorded. The result showed that average fluorescence of F-actin was enhanced after rocking system loading (46.8 +/- 4.5) compared to the control (20.4 +/- 1 8) and the percentage of S-phase cells was increased (10.6 +/- 1.04) after rocking system loading as well (which was 4.1 +/- 0.54 in control group). Furthermore, the fluid shear stress generated by rocking system could induce more significant biological effects compared to PPFC system. This study demonstrated that fluid shear stress generated by rocking system could induce biological effects of osteoblasts, and it could simulate the micro environment of cells in vioe better than PPFC. Rocking system is a convenient and feasible method for fluid shear stress loading.
Animals ; Animals, Newborn ; Cell Proliferation ; Cells, Cultured ; Cytoskeleton ; ultrastructure ; Feasibility Studies ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; cytology ; Shear Strength ; Skull ; chemistry ; Stress, Mechanical

OBJECTIVETo evaluate the effect of endothelial progenitor cells (EPCs) mobilization on the healing of calvarial defect in diabetic mice.

METHODS55 type II adult male diabetic mice were included in this study. They were randomly divided into three groups: the non-operative group (n = 5), the experimental group (n = 25) and the control group (n = 25). Two circular bony defects, 3 mm in diameter, were created on the parietal bones of the diabetic mice. Intraperitoneal AMD3100 (10 mg/kg; n = 25) or sterile saline (control group) was administered daily beginning at post-operative day 3 and continuing for 15 days. 5 mice were sacrificed in each group at non-operation, post-operative week 1,2,4, 8,12. Circulation EPC level was measured at pre-operation, post-operative day 7 and day 14. Bony regeneration was assessed with micro-CT at post-operative week 4, 8 and 12. HE staining was performed on all the decalcified bone samples. Immunofluorescent CD31 and osteocalcin staining was performed on calvarial defects at weeks 1, 2, and 4 to assess the vascularity and osteoblast density, respectively.

RESULTSThe mobilization of EPC in diabetic mice almost disappeared one week after trauma, while AMD3100 could dramatically increase the circulation EPC level for a long time after trauma. Compared to control group, the healing percentage of bony defect in the diabetic mice treated with AMD3100 was obviously increased at post-operative week 8 (50.5% vs. 34.8%) and week 12 (50.9% vs. 40.2%). Calvarial defects of AMD3100-treated mice harvested at 1, 2, and 4 weeks demonstrated increased vascularity and osteoblast density, compared to the controls. The difference was most marked in postoperative week 2 (vascularity: 6.11% vs. 2.47%; osteoblast density 2.81% vs. 1.22%, P < 0.01).

CONCLUSIONAMD3100 can improve the healing of calvarial defect in diabetic mice by increasing the vascularity and osteoblast density at the regeneration area.

Animals ; Bone Regeneration ; Diabetes Mellitus, Experimental ; Endothelial Cells ; cytology ; drug effects ; Endothelium, Vascular ; cytology ; Heterocyclic Compounds ; pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; Osteoblasts ; cytology ; Parietal Bone ; Skull ; pathology ; Stem Cells ; cytology ; drug effects ; Wound Healing

OBJECTIVETo investigate the feasibility of using human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) and demineralized bone matrix (DBM) scaffolds to repair critical-sized calvarial defects in athymic rats.

METHODSHuman UCB-MSCs were isolated, expanded and osteogenically induced in vitro. Osteogenic differentiation of UCB-MSCs was evaluated by Alizarin Red staining and measurement of calcium content respectively, and then the cells were seeded onto DBM scaffolds. Bilateral full-thickness defects (5 mm in diameter) of parietal bone were created in an athymic rat model. The defects were either repaired with UCB-MSC/DBM constructs (experimental group) or with DBM scaffolds alone (control group). Animals were harvested at 6 and 12 weeks post-implantation respectively, and defect repair was evaluated with gross observation, micro-CT measurement and histological analysis.

RESULTSMicro-CT showed that new bone was formed in the experimental group at 6 weeks post-implantation, while no sign of new bone formation was observed in the control group. At 12 weeks post-transplantation, scaffolds had been degraded almost completely in both sides. It was shown that an average of (78.19 +/- 6.45)% of each defect volume had been repaired in experimental side; while in the control side, only limited bone formed at the periphery of the defect. Histological examination revealed that the defect was repaired by trabecular bone tissue in experimental side at 12 weeks, while only fibrous connection was observed in the control group.

CONCLUSIONSTissue-engineered bone composed of osteogenically-induced human UCB-MSCs on DBM scaffolds could successfully repair the critical-sized calvarial defects in athymic rat models.

Animals ; Bone Regeneration ; Bone Substitutes ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Nude ; Rats, Sprague-Dawley ; Skull ; injuries ; surgery ; Tissue Engineering ; Tissue Scaffolds ; Transplantation, Autologous

AIMThe piezoelectric properties and cytotoxicity of a porous lead-free piezoelectric ceramic for use as a direct bone substitute were investigated.

METHODOLOGYCold isostatic pressing (CIP) was applied to fabricate porous lithium sodium potassium niobate (Li0.06Na0.5K0.44) NbO3 specimens using a pore-forming method. The morphologies of the CIP-processed specimens were characterized and compared to those of specimens made by from conventional pressing procedures. The effects of the ceramic on the attachment and proliferation of osteoblasts isolated from the cranium of 1-day-old Sprague-Dawley rats were examined by a scanning electron microscopy (SEM) and methylthiazol tetrazolium (MTT) assay.

RESULTSThe results showed that CIP enhanced piezoelectricity and biological performance of the niobate specimen, and also promoted an extracellular matrix-like topography of it. In vitro studies showed that the CIP-enhanced material had positive effects on the attachment and proliferation of osteoblasts.

CONCLUSIONNiobate ceramic generated by CIP shows a promise for being a piezoelectric composite bone substitute.

Animals ; Biocompatible Materials ; chemistry ; toxicity ; Bone Substitutes ; chemical synthesis ; toxicity ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Ceramics ; chemical synthesis ; toxicity ; Coloring Agents ; Electrochemistry ; Materials Testing ; Microscopy, Electron, Scanning ; Niobium ; toxicity ; Osteoblasts ; drug effects ; Oxides ; chemical synthesis ; toxicity ; Porosity ; Potassium ; toxicity ; Pressure ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; Stress, Mechanical ; Surface Properties ; Tetrazolium Salts ; Thiazoles

OBJECTIVETo investigate the application of tissue-engineering bone with ADSCs (adipose-derived stem cells) and coral scaffold for repairing of cranial bone defect in canine.

METHODSAutologous ADSCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm x 20 mm) of parietal bone were created (n = 7). The defects were either repaired with ADSC-coral constructs (experimental group) or with coral alone (control group). Radiological, gross, biomechanical and histological observations were done to evaluate the bone regeneration.

RESULTSThree-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks after implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it showed that an average repair percentage of each defect was (84.19 +/- 6.45)% in experimental group, and (25.04 +/- 18.82)% in control group (P < 0.01). The maximum compression loading was (73.45 +/- 17.26) N in experimental group, and (104.27 +/- 22.71) N in control group (P <0.01). Histological examination revealed that the defect was repaired by typical bone tissue in experimental group, while only minimal bone formation with fibrous connection in the control group.

CONCLUSIONSThe tissue-engineering bone with autologous osteogenic ADSCs and scaffold could successfully repair the cranial defects in canine models.

Adipocytes ; cytology ; transplantation ; Animals ; Anthozoa ; Bone Regeneration ; Bone Substitutes ; Bone and Bones ; Cell Culture Techniques ; Cells, Cultured ; Dogs ; Female ; Male ; Skull ; surgery ; Stem Cell Transplantation ; Tissue Engineering ; methods ; Tissue Scaffolds ; Transplantation, Autologous