mRNA Expression Levels Of Osteocalcin Research Articles

Inhibition of Poly (ADP-Ribose) Glycohydrolase Accelerates Osteoblast Differentiation in Preosteoblastic MC3T3-E1 Cells.

Poly ADP-ribosylation (PARylation) is a post-translational modification catalyzed by poly (ADP-ribose) polymerase (PARP) family proteins such as PARP1. Although PARylation regulates important biological phenomena such as DNA repair, chromatin regulation, and cell death, little is known about the relationship between osteoblast differentiation and the PARylation cycle involving PARP1 and the poly (ADP-ribose)-degrading enzyme poly (ADP-ribose) glycohydrolase (PARG). Here, we examined the effects of PARP inhibitor olaparib, an approved anti-cancer agent, and PARG inhibitor PDD00017273 on osteoblast differentiation. Olaparib decreased alkaline phosphatase (ALP) activity and suppressed mineralized nodule formation evaluated by Alizarin Red S staining in preosteoblastic MC3T3-E1 cells, while PDD00017273 promoted ALP activity and mineralization. Furthermore, PDD00017273 up-regulated the mRNA expression levels of osteocalcin and bone sialoprotein, as osteoblast differentiation markers, and osterix as transcription inducers for osteoblast differentiation, whereas olaparib down-regulated the expression of these genes. These findings suggest that PARG inhibition by PDD00017273 accelerates osteoblast differentiation in MC3T3-E1 cells. Thus, PARG inhibitor administration could provide therapeutic benefits for metabolic bone diseases such as osteoporosis.

Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.

Abnormal expression of miR-135b-5p in bone tissue of patients with osteoporosis and its role and mechanism in osteoporosis progression.

Osteoporosis (OP) is an age-related bone disease occurring worldwide. Osteoporotic fracture is one of the leading causes of disability and death in elderly patients. MicroRNAs (miRNAs/miRs) are key molecular regulatory factors in bone remodeling processes. The present study investigated the expression and mechanism of miR-135b-5p in patients with osteoporosis. The present results suggested that miR-135b-5p was upregulated in bone tissue fragments of patients with osteoporosis compared with the control patients. MC3T3-E1 cells were used to perform osteogenic differentiation induction. Reverse transcription-quantitative PCR and western blot assay were used to detect the mRNA and protein expression levels of the osteogenic markers osteocalcin (OC), Osterix and alkaline phosphatase (ALP). A specific kit was used for detecting ALP activity. The present results indicated that the mRNA expression levels of OC, Osterix and ALP significantly increased on the 7 and 14th day after osteogenic differentiation induction compared with the control group. Protein expression levels of OC, Osterix and ALP also increased on the 7 and 14th day after induction. ALP assay showed that ALP activity was significantly increased on the 7 and 14th day after induction. In addition, the present study found that miR-135b-5p was downregulated in MC3T3-E1 cells 7 and 14 days after osteogenic differentiation induction. The results of TargetScan analysis and dual luciferase reporter gene assay indicated that runt-related transcription factor 2 (RUNX2) was a direct target gene of miR-135b-5p. RUNX2 was upregulated in MC3T3-E1 cells on the 7 and 14th day after induction. Moreover, the present study found that compared with the osteogenic differentiation induction group, miR-135b-5p mimic significantly decreased OC, Osterix and ALP expression, and reduced ALP activity in MC3T3-E1 cells. However, these reductions were reversed following overexpression of RUNX2. The present results showed that miR-135b-5p mimic significantly reduced cell viability in MC3T3-E1 cells and induced cell apoptosis, and these effects were significantly reversed following RUNX2 overexpression. In summary, the present results suggested that miR-135-5p participated in the occurrence and development of osteoporosis via inhibition of osteogenic differentiation and osteoblast growth by targeting RUNX2. The present study suggested a novel potential target that may faciliate the treatment of osteoporosis, and further study is required to examine this possibility.

Effect of porous tantalum on promoting the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the MAPK/ERK signal pathway

BackgroundAs an ideal new graft material, porous tantalum (pTa) has excellent mechanical properties and corrosion resistance and has received increased attention in the biomedical field because of its excellent cytocompatibility and ability to induce bone formation. However, the molecular mechanism of its potential to promote osteogenesis remains unclear, and very few reports have been published on this topic. MethodsIn this study, we first produced porous Ti6Al4V (pTi6Al4V) and pTa with the same pore size by three-dimensional printing combined with chemical vapour deposition. The number of adhesions between pTa and pTi6Al4V and bone marrow mesenchymal stem cells (BMSCs) after 1 day of culture was detected by the live/dead cell staining method. The proliferation activity of the two groups was determined after culture for 1, 3, 5 and 7 days by the cell counting kit-8 method. In addition, the osteogenic activity, mRNA expression levels of osteogenic genes alkaline phosphatase (ALP), osterix (OSX), collagen-I (Col-I), osteonectin (OSN) and osteocalcin (OCN) and protein expression levels of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling pathway marker p-ERK of the two groups cultured for 7, 14 and 21 days were determined by the ALP activity assay, real-time quantitative polymerase chain reaction (Q-PCR) and Western blotting, respectively. Subsequently, the two groups were treated with the MAPK/ERK–specific inhibitor U0126, and then, the mRNA expression levels of osteogenic genes and protein expression levels of p-ERK in the cultures were determined by Q-PCR and Western blotting, respectively. ResultsThe live/dead cell staining and cell counting kit-8 assays showed that the adhesion and proliferation activities of BMSCs on pTa were significantly better than those on pTi6Al4V. In addition, the ALP activity assay and Q-PCR showed that pTa harboured osteogenic activity and that the osteogenic genes ALP, OSX, Col-I, OSN and OCN were highly expressed, and by Western blotting, the expression of p-ERK protein in the pTa group was also significantly higher than that in the pTi6Al4V group. Subsequently, using the MAPK/ERK–specific inhibitor U0126, Western blotting showed that the expression of p-ERK protein was significantly inhibited and that there was no difference between the two groups. Furthermore, Q-PCR showed that osteogenic gene expression and ALP expression levels were significantly increased in the pTa group, and there were no differences in the OSX, Col-I, OSN and OCN mRNA expression levels between the two groups. ConclusionOverall, our research found that compared with the widely used titanium alloy materials, our pTa can promote the adhesion and proliferation of BMSCs, and the molecular mechanism of pTa may occur via activation of the MAPK/ERK signalling pathway to regulate the high expression of OSX, Col I, OSN and OCN osteogenic genes and promote the osteogenic differentiation of BMSCs in vitro.The translational potential of this article: Our self-developed pTa material produced by three-dimensional printing combined with the chemical vapour deposition method not only retains excellent biological activity and osteoinductive ability of the original tantalum metal but also saves considerably on material costs to achieve mass production of personalised orthopaedic implants with pTa as a stent and to accelerate the wide application of pTa implants in clinical practice, which have certain profound significance.

Isolation and characterization of minipig perivascular stem cells for bone tissue engineering.

Human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC) in recent years. The current study was designed to sort the minipig (mp) perivascular stem cells (PSCs) and investigate the osteogenic potential. Purification of human PSCs was achieved via fluorescence-activated cell sorting (FACS) from human liposuction samples [cluster of differentiation (CD)45−CD34−CD146+ perithelial cells and CD45−CD34+CD146− adventitial cells]. Subsequently, PSCs were isolated from mp adipose tissue samples (n=9), characterized and, using purified mpPSCs (obtained by FACS, which is used in human PSC purification), the mpPSC osteogenic and adipogenic potential was evaluated by Alizarin Red S and Oil Red O staining in vitro, respectively. The cell morphometry was observed following cell isolation and culture, and hematoxylin and eosin staining was performed to identify the fat tissue structure and vascular distribution. Osteogenic and adipogenic differentiation-associated gene expression levels were analyzed by reverse transcription-quantitative polymerase chain reaction. The results demonstrated that the same antigens used for human PSC identification and isolation were working in mp tissue (CD45, CD146 and CD34). The two cell groups: CD45−CD34−CD146+ pericytes and CD45−CD34+CD146− adventitial cells were successfully isolated from the subcutaneous fat in the posterior neck of mps, mpPSCs accounted for 8.6% of the stromal vascular fraction (SVF) with 1.4% pericytes and 7.2% adventitial cells. mpPSCs demonstrated characteristics of MSCs, including cell surface marker expression, colony forming unit-fibroblast inclusion, and the stronger osteogenic and adipogenic differentiation potential than that of the non-selected vascular stromal cells. The mRNA expression levels of osteocalcin, collagen, type I, α1 and peroxisome proliferator-activated receptor-γ in the mpPSCs group were significantly higher than those of the unsorted pSVF group (P<0.05). Thus, the current study successfully isolated and cultured CD146+ and CD34+ cell populations from mp tissues, characterized the cells' PSC-like phenotype and identified their distinctly osteogenic and adipogenic potential.

Supplying osteogenesis to dead bone using an osteogenic matrix cell sheet

To evaluate whether osteogenic matrix cell sheets can supply osteogenesis to dead bone. Femur bone fragments (5mm in length) were obtained from Fisher 344 rats and irradiated by a single exposure of 60Gy to produce bones that were no longer viable. Osteogenic matrix cell sheets were created from rat bone marrow-derived stromal cells (BMSCs). After wrapping the dead bone with an osteogenic matrix cell sheet, it was subcutaneously transplanted into the back of a rat and harvested after 4 weeks. Bone formation around the dead bone was evaluated by X-ray imaging and histology. Alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression levels were measured to confirm osteogenesis of the transplanted bone. The contribution of donor cells to bone formation was assessed using the Sry gene and PKH26. After the cell sheet was transplanted together with dead bone, X-ray images showed abundant calcification around the dead bone. In contrast, no newly formed bone was seen in samples that were transplanted without the cell sheet. Histological sections also showed newly formed bone around dead bone in samples transplanted with the cell sheet, whereas many empty lacunae and no newly formed bone were observed in samples transplanted without the cell sheet. ALP and OC mRNA expression levels were significantly higher in dead bones transplanted with cell sheets than in those without a cell sheet (P<0.01). Sry gene expression and cells derived from cell sheets labeled with PKH26 were detected in samples transplanted with a cell sheet, indicating survival of donor cells after transplantation. Our study indicates that osteogenic matrix cell sheet transplantation can supply osteogenesis to dead bone.

99Tc-MDP-induced human osteoblast proliferation, differentiation and expression of osteoprotegerin.

The aim of the present study was to examine the influence of technetium methylenediphosphonate (99Tc-MDP) on the proliferation and differentiation of human osteoblasts. Human iliac cancellous bone was isolated and cultured with either 99Tc-MDP, β fibroblast growth factor (as a positive control) or medium only (as a negative control). Proliferation was assessed by direct cell counting, CCK-8 assay and bromodeoxyuridine staining. The cell cycle and rate of apoptosis was assessed by propidium iodide staining and flow cytometry. Alkaline phosphatase (ALP) activity was assessed by the p-nitrophenyl phosphate method and mineralized nodules were stained with Alizarin Red. Expression of osteocalcin (OCN) and bone morphogenetic protein-2 (BMP-2) was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and expression levels of osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) were assessed by RT-qPCR and ELISA. Isolated human osteoblasts stained positively for ALP and developed mineralized nodules. Treatment with 10−5-10−10 M 99Tc-MDP enhanced proliferation and 48 h incubation with 10−8 M 99Tc-MDP increased the proportion of cells in S-phase, decreased the proportion in G0/G1 phase, and increased the cell proliferation index. The rate of apoptosis also increased, but the increase was not significant. Cells incubated with 10−6-10−9 M 99Tc-MDP for 3–9 days exhibited increased ALP activity and mineralized nodule development. 10−8 M 99Tc-MDP increased BMP-2 and OPG expression levels and OPG secretion, but OCN mRNA expression levels and RANKL secretion were not significantly altered at 72 h. 99Tc-MDP treatment induced osteoblast proliferation and differentiation without affecting apoptosis. These findings provide proof of concept for the future use of 99Tc-MDP in the treatment of bone-destructive diseases.

Attenuation by incretins of thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

Incretins, the polypeptide hormone glucose- dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) secreted from the small intestine after nutrient ingestion, are generally known to stimulate insulin secretion from pancreatic β-cells. We previously demonstrated that triiodothyronine (T3) stimulates osteocalcin synthesis at least in part through p38 mitogen-activated protein kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of GIP and GLP-1 on T3-stimulated osteocalcin synthesis and the mechanism of action involved in MC3T3-E1 cells. GIP and GLP-1 markedly suppressed the T3-stimulated osteocalcin release. GIP and GLP-1 significantly attenuated the expression levels of osteocalcin mRNA induced by T3. The T3-stimulated transactivation activity of the thyroid hormone-responsive element was reduced by GIP and GLP-1. These results suggest that incretins repressed the T3-stimulated osteocalcin synthesis in osteoblast-like MC3T3-E1 cells, and the suppressive effect of incretins was mediated through transcriptional levels.

Naringin promotes differentiation of bone marrow stem cells into osteoblasts by upregulating the expression levels of microRNA-20a and downregulating the expression levels of PPARγ.

Naringin is a dihydrotestosterone flavonoid compound that significantly inhibits bone loss, improves bone density, and enhances biomechanical anti‑compression performance. Previous studies have demonstrated that naringin improves the activity levels of osteocalcin (OC) and alkaline phosphatase (ALP) in MC3T3‑E1 osteoblast precursor cells. The present study investigated the effects of naringin on osteoblastic differentiation and inhibition of adipocyte formation in bone marrow stem cells (BMSCs). The levels of osteogenesis were modulated via upregulation of the expression levels of microRNA (miR)‑20a, and downregulation of the expression levels of peroxisome proliferator‑activated receptor γ (PPARγ). The results indicated that naringin significantly enhanced BMSC proliferation in a dose‑dependent manner. In addition, naringin significantly increased the mRNA expression levels of OC, ALP, and collagen type I. Furthermore, naringin decreased the protein expression levels of PPARγ, and increased the expression levels of miR‑20a in the BMSCs. These results suggested that miR‑20a may regulate the expression of PPARγ in BMSCs. To our knowledge, this is the first study to report naringin‑induced osteogenesis via upregulation of the expression levels of miR‑20a, and downregulation of the expression levels of PPARγ. These results indicated the important role of naringin in BMSC differentiation.

In vivo ossification of a scaffold combining β-tricalcium phosphate and platelet-rich plasma

Tricalcium phosphate (TCP) and platelet-rich plasma (PRP) are commonly used in bone tissue engineering. The aim of the present study was to investigate a composite that combined TCP with PRP and assess its effectiveness in the treatment of bone defects. Cavity-shaped bone defects were established on the tibiae of 27 beagle dogs, and were repaired by pure β-TCP with bone marrow stromal cells (BMSCs), β-TCP/PRP with BMSCs and autogenic ilium. The samples were harvested at 4, 8 and 12 weeks, and bone regeneration was evaluated using X-ray radiography, immunocytochemical staining of osteocalcin (OCN), hematoxylin and eosin staining and reverse transcription-polymerase chain reaction analyses. Biomechanical tests of the scaffolds were performed at the 12th week after scaffold implantation. When using pure β-TCP as a scaffold, the scaffold-bone interface was clear and no material adsorption and bone healing was observed. Substantial bone regeneration was observed when the tibial defects were restored using β-TCP/PRP and autogenic ilium. Furthermore, the mRNA expression levels of OCN, alkaline phosphatase and collagen type I α1 were significantly higher in the animals with β-TCP/PRP scaffolds at 8 and 12 weeks following implantation compared with those in the animals with the pure β-TCP scaffolds. The maximum load and compressive strength of the β-TCP/PRP scaffolds were similar to those of the autogenic ilium; however, they were significantly higher than those of the pure β-TCP scaffold. Thus, the β-TCP/PRP composite may be used as a potential scaffold to carry in vitro cultured BMSCs to treat bone defects.

Down-regulated expression of vimentin induced by mechanical stress in fibroblasts derived from patients with ossification of the posterior longitudinal ligament.

The aim of this study was to investigate the potential role of vimentin in the signal transduction pathways initiated by mechanical stimulation that contribute to ossification of the posterior longitudinal ligament of the spine (OPLL). We investigated the effects of in vitro cyclic stretch on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The expression levels of the osteoblast-specific genes encoding osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) were assessed by semi-quantitative reverse transcription-polymerase chain reaction. Vimentin protein expression in OPLL cells was detected by Western blotting. Small interfering RNA (siRNA) interference targeting vimentin was performed in OPLL cells induced by mechanical stress, and the expression levels of OCN, ALP and COL I were assessed. In response to mechanical stretch, the expression levels of OCN, ALP, and COL I were increased in OPLL cells, whereas no change was observed in non-OPLL cells. Furthermore, knockdown of vimentin protein expression by siRNA resulted in an increase in the mRNA expression levels of OCN, ALP, and COL I. The down-regulation of vimentin induced by mechanical stress plays an important role in the progression of OPLL through the induction of osteogenic differentiation in OPLL cells.

Effects of simvastatin on the osteogenic differentiation and immunomodulation of bone marrow mesenchymal stem cells.

The present study aimed to investigate the effects of simvastatin on the bone differentiation capacity and immunological characteristics of bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and cultured in medium containing 1.0µmol/mlsimvastatin. The alkaline phosphatase activity, mRNA expression levels of osteocalcin and bone sialoprotein, and calcium nodule formation were assessed to determine the osteogenic differentiation capability of BMSCs. To investigate alterations in the immunological properties of simvastation‑treated BMSCs, the immunogenicity of these cells and the effect of BMSCs on phytohemagglutinin‑stimulated lymphocyte proliferation were also assessed. Following treatment with simvastatin, the alkaline phosphatase activity, and mRNA expression levels of osteocalcin and bone sialoprotein were increased significantly in the BMSCs. In addition, von Kossa staining revealed a brown calcium‑positive reaction zone in simvastatin‑treated cells. Simvastatin‑induced BMSCs revealed no affect on the proliferation of allogeneic lymphocytes, however, inhibited phytohemagglutinin‑induced lymphocyte proliferation. Collectively, simvastatin promoted the osteogenic differentiation of BMSCs significantly without affecting their immunosuppressive properties.

Effect of simvastatin on the osteogenetic behavior of alveolar osteoblasts and periodontal ligament cells

Statins are routinely used in the clinic as cholesterol lowering drugs, but recently they were reported to also have anabolic effects on bone tissue. Since regeneration of alveolar bone is one of the primary aims of periodontal treatment, in the present study we investigated the effects of simvastatin, a lipophilic statin, on primary alveolar osteoblasts (AOBs) and periodontal ligament cells (PDLs) in vitro. The effect of simvastatin (1-100 nM) on the cells proliferation/viability after 24, 48, and 72 h stimulation was measured using 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT)-assay. The alkaline phosphatase (ALP) activity was measured after stimulation with simvastatin using specific colorimetric assay. Finally, the mRNA expression levels of osteocalcin (OC), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) were measured by real-time PCR. The proliferation/viability of AOBs was significantly decreased by all simvastatin concentrations after 72 h stimulation. The proliferation/viability of PDLs was not influenced by simvastatin. ALP activity of AOBs and PDLs was increased by 1 and 100 nM simvastatin, respectively. Simvastatin induced a dose-dependent increase in OC mRNA expression of AOBs and did not influence that in PDLs. RANKL expression of AOBs was increased at all tested simvastatin concentrations and that in PDLs was increased by higher simvastatin concentrations (10-100 nM). Finally, the expression of OPG in AOBs and PDLs was stimulated by 1-10 and 100 nM simvastatin, respectively. Simvastatin seems to slightly increase the expression of osteogenic markers in AOBs and PDLs, indicating its ability to influence alveolar bone formation and periodontal regeneration.

Involvement of sensory neurons in bone defect repair in rats

We investigated bone repair in sensory-denervated rats, compared with controls, to elucidate the involvement of sensory neurons. Nine-week-old male Wistar rats received subcutaneous injections of capsaicin to denervate sensory neurons. Rats treated with the same amount of vehicle served as controls. A standardized bone defect was created on the parietal bone. We measured the amount of repaired bone with quantitative radiographic analysis and the mRNA expressions of osteocalcin and cathepsin K with real-time polymerase chain reaction (PCR). Quantitative radiographic analysis showed that the standard deviations and coefficients of variation for the amount of repaired bone were much higher in the capsaicin-treated group than in the control group at any time point, which means that larger individual differences in the amount of repaired bone were found in capsaicin-treated rats than controls. Furthermore, radiographs showed radiolucency in pre-existing bone surrounding the standardized defect only in the capsaicin-treated group, and histological observation demonstrated some multinuclear cells corresponding to the radiolucent area. Real-time PCR indicated that there was no significant difference in the mRNA expression levels of osteocalcin and cathepsin K between the control group and the capsaicin-treated group. These results suggest that capsaicin-induced sensory denervation affects the bone defect repair.

Effects of exposure to space relevant radiation on the differentiation and mineralization process of murine osteoblastic cell lines

During spaceflight, astronauts experience both solar and galactic cosmic radiation as well as the absence of gravity, especially during planned long term missions to Moon and Mars. Potential deleterious effects under these conditions include specific impact on the skeletal, central nervous and immune systems as well as the induction of cancer. However, until now little is known about the effects of space radiation on bone cells, especially on the bone forming osteoblasts. Heavy ion accelerators are ground based facilities, used to simulate single components of the radiation environment in space. Within our experimental setup we examined the cellular survival as well as the differentiation and mineralization capacities of osteoblastic cell lines after exposure to carbon ( C-13 75MeV/A), neon ( Ne-22 80 MeV/A), and nickel ions ( Ni-58 1 GeV/A) in comparison to low LET X-rays. Modifications in the differentiation process were monitored by measuring mRNA expression levels of osteocalcin, a key marker of osteoblastic differentiation and transforming growth factor-β1 (TGF-β1) which offers an important role during bone remodeling. Additionally, the mineralization process after radiation exposure was quantified after staining of hydroxyapatite containing bone-like nodules deposited by osteoblastic cells. The results suggest that ionizing radiation significantly modulates the differentiation process and effectively modulates the gene expression levels of marker genes involved in the differentiation of osteoblasts. In conclusion, the presented data allow the suggestion that exposure to heavy ions interferes with bone formation at the level of cell differentiation. Further experiments are needed to investigate gene expression patterns in mammalian cells that respond to differentiation after exposure to high LET radiation.

Prevention of alcohol-induced osteonecrosis of femoral head in rabbits by small interfering RNA targeting specific gene by gavage

Objective To observe the effect of small interfering RNA (siRNA) adenovirus vectors targeting PPARγto prevent the alcohol-induced osteonecrosis of the femoral head (ONFH) in rabbits.Methods Ninety-six rabbits were randomly divided into four groups. In group N, normal saline ( 10 ml/kg every day) was poured into stomach. In group M, the strong wine (the volume fraction was 46% alcohol,10 ml/kg every day) was poured into stomach. In group S, under the randomly selected side and anesthesia, the animals were injected with 25 μl siRNA adenovirus drip into the femoral head and then the puncture hole was closed on the 1st day at the 1st, 3rd and 5th month of the experiment, and at the same time the strong wine (the volume fraction was 46% alcohol, 10 ml/kg every day) was poured into stomach. In group CO, animals were treated with the same method as the group S, but not injected with siRNA adenovirus drip. The animals were sacrificed in batches at 2nd, 4th and 6th month after the experiment. The serology and pathological changes of the femoral head were studied. Results At the 6th month, the triglyceride (TG, mmol/L) contents in groups N, M, CO and S were (1.21 ±0. 12), (4.59 ± 1.58), (4.63 ±1.17) and (4. 32 ± 1.20), the cholesterol levels ( CHO, mmol/L) were (2. 35 ± 0. 33), ( 19. 59 ±1.58), (20. 13 ± 1. 17) and ( 18.32 ± 1.20), the average diameter of the max adipocyte (μm) was (40. 89 ± 2. 41 ), (48.65 ± 2.93 ), (49. 45 ± 2. 63 ) and ( 42. 52 ± 2. 57 ), the trabeculace area fraction was (41.80 ±2. 47)%, (30. 70 ±2. 86)%, (29. 80 ±2. 69)% and (41.60 ±2. 87)%, the percentage of empty osteocyte lacunae was (12.30 ± 1.73)%, (22.40 ± 1.52)%, (23. 10 ± 1.62)% and (11.70 ±1.46)%, the expreasion levels of PPARγ mRNA were (0.29 ±0.05), (0.66±0. 12), (0.61±0.15) and (0. 33 ± 0. 05 ), the expression levels of osteocalcin mRNA were (0. 92 ± 0. 07 ), (0. 19 ± 0. 11 ), (0. 23 ±0. 08) and (0. 88 ± 0. 11 ), the expression levels of PPARγprotein were ( 0. 75 ± 0. 08 ), ( 1.60 ± 0. 11 ),(1.55 ±0. 12) and (0.65 ±0.05), respectively. In groups M and CO, the pathological changes were obvious; there was decreased hematopoietic tissue, proliferation and hypertrophy of the adipocytes, increased fatty tissue, and thinned, sparse or breoken bone trabeculae in the femoral head; the area fraction of the trabeculae was reduced. In group S, the average diameter of the max adipocyte, percentage of empty osteocyte lacunae, incidence of ONFH, the expressions of PPARγmRNA and protein were obviously reduced as compared with groups M and CO (P ＜0. 01,P ＜0. 05). There was no statistically significant difference between group S and group N (P ＞ 0. 05). Conclusion siRNA adenovirus vectors targeting PPARγcan efficaciously suppress the expression of PPARγ gene and adipogenic differentiation of the MSCs in the femoral head induced by alcohol, which may prevent the development of the alcohol-induced ONFH in rabbits. Key words: siRNA; PPARγ; Alcohol; Osteonecrosis of femoral head; Rabbit; Prevent

Static Electromagnetic Fields Generated by Corrosion Currents Inhibit Human Osteoblast Differentiation

Human osteoblast cultures were exposed to a very low intensity static magnetic fields (SMF) to investigate its effects on osteoblast growth and differentiation. Analysis of the effects of periprosthetic SMF on the growth and differentiation of human osteoblast cell cultures in vitro. The effects of pulsed electromagnetic fields (PEMF) on cell proliferation, especially in human osteoblast-like cells is well described, whereas few data are available on the effects of SMF on osteoblast cell culture. We previously demonstrated that the proliferation of human osteoblast cultures is reduced when cells are exposed to a continuous low intensity SMF comparable to the one that occurs around metal devices (Ti spinal implant) because of the generation of electric currents between the screw (Ti6Al4V) and the rod (Ti). Primary osteoblastic cells were isolated from a human femoral head. Osteoblast cultures were exposed to SMF and alkaline phosphatase activity was evaluated in the osteoblast cell cultures at different time points. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression levels of osteocalcin, Runx2, and collagen I genes. The SMF-treated cells showed a progressive increase in the alkaline phosphatase activity which, however, remained always lower than the one observed in the control group at each observation time (72 hours, 7 and 14 days). RT-PCR demonstrated that Runx2 and collagen I mRNA were downregulated following SMF stimulation, whereas no change in osteocalcin mRNA was observed. Continuous low-intensity electromagnetic field comparable to the one that generates around metal devices because of the generation of corrosion currents inhibits osteoblasts differentiation pattern and might contribute at least in part to a decrease in periprosthetic bone formation occurring in vivo.

Preventive effects of puerarin on alcohol-induced osteonecrosis.

Alcohol can induce adipogenesis by bone marrow stromal cells and may cause osteonecrosis of the femoral head. Currently, there are no medications available to prevent alcohol-induced osteonecrosis. We hypothesized puerarin, a Chinese herbal medicine with antioxidative and antithrombotic effects, can prevent alcohol-induced adipogenesis and osteonecrosis. Both bone marrow stromal cells (in vitro) and mice (in vivo) were treated either with ethanol or with ethanol and puerarin, with an untreated group serving as a control. In the in vitro study, the number of adipocytes, contents of triglycerides, and levels of PPAR gamma mRNA expression were decreased and alkaline phosphatase activity, contents of osteocalcin, and levels of osteocalcin mRNA expression were increased in cells treated with both alcohol and puerarin, compared with cells treated with alcohol only. In the in vivo study, marrow necrosis, fat cell hypertrophy and proliferation, thinner and sparse trabeculae, diminished hematopoiesis, and increased empty osteocyte lacunae in the subchondral region of the femoral head were observed in mice treated with alcohol. However, no such changes were seen in femoral heads of mice treated with alcohol and puerarin. The data suggest puerarin can inhibit adipogenic differentiation by bone marrow stromal cells both in vitro and in vivo and prevents alcohol-induced osteonecrosis in this model.

Effect of polyphosphoric acid pre‐treatment of titanium on attachment, proliferation, and differentiation of osteoblast‐like cells (MC3T3‐E1)

To evaluate the effect of polyphosphoric acid (PPA) pre-treatment of titanium (Ti) on the initial attachment, proliferation, and differentiation of mouse osteoblast-like cells (MC3T3-E1). Adsorption of PPA to Ti was achieved by immersing Ti disks (15 mm in diameter) into 0, 1, and 10 wt% PPA and 10 wt% orthophosphoric acid (OPA) for 24 h. On each pre-treated Ti disk, 5.0 x 10(4) MC3T3-E1 cells were seeded, and 1, 3, and 5 h later cell attachment was evaluated. Cell proliferation was also determined 1, 3, and 5 days after cell seed. Cell differentiation was evaluated 5, 10, and 15 days after cell seed using osteoblast marker gene expression. Total RNA was purified from each culture and Type-I collagen, alkaline phosphatase, and osteocalcin mRNA expression levels were measured by real-time reverse transcription polymerase chain reaction. Adsorption of PPA or OPA to Ti significantly accelerated initial cell attachment at every time point (P<0.0001). As with cell attachment, cell proliferation was also accelerated on the PPA-treated Ti disks in a dose-dependent manner at every time point (P<0.0001). However, OPA-treated Ti disks did not enhance the cell proliferation. Regarding osteoblastic differentiation, PPA-treated Ti significantly accelerated the Type-I collagen gene expression at 5 and 10 days after cell seed. Regarding alkaline phosphatase and osteocalcin gene expression, no significant difference was found among the different Ti surface conditions. The accelerated cell behavior following Ti pre-treatment with PPA is a promising new strategy to fabricate bioactive Ti implants.

FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-β signaling

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days' culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days' culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-beta signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.