RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation

RANKL signaling is essential for osteoclastogenesis. Its role in osteoblastic differentiation and bone formation is unknown. Here we demonstrate that RANK is expressed at an early stage of bone marrow mesenchymal stem cells(BMSCs) during osteogenic differentiation in both mice and human and decreased rapidly. RANKL signaling inhibits osteogenesis by promoting β-catenin degradation and inhibiting its synthesis. In contrast, RANKL signaling has no significant effects on adipogenesis of BMSCs.Interestingly, conditional knockout of rank in BMSCs with Prx1-Cre mice leads to a higher bone mass and increased trabecular bone formation independent of osteoclasts. In addition, rank: Prx1-Cre mice show resistance to ovariectomy-(OVX) induced bone loss. Thus, our results reveal that RANKL signaling regulates both osteoclasts and osteoblasts by inhibition of osteogenic differentiation of BMSCs and promotion of osteoclastogenesis.

miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells

Age-related osteoporosis is associated with the reduced capacity of bone marrow mesenchymal stem cells （BMSCs） to differentiate into osteoblasts instead of adipocytes. However, the molecular mechanisms that decide the fate of BMSCs remain unclear. In our study, microRNA-23a, and microRNA-23b （miR-23a/b） were found to be markedly downregulated in BMSCs of aged mice and humans. The overexpression of miR-23a/b in BMSCs promoted osteogenic differentiation, whereas the inhibition of miR-23a/b increased adipogenic differentiation. Transmembrane protein 64 （Tmem64）, which has expression levels inversely related to those of miR-23a/b in aged and young mice, was identified as a major target of miR-23a/b during BMSC differentiation. In conclusion, our study suggests that miR-23a/b has a critical role in the regulation of mesenchymal lineage differentiation through the suppression of Tmem64.

Enzymatic Cell Isolation and Explant Cultures of Rat Calvarial Osteoblast Cells

Osteoblast cells were isolated from the calvarial bones of newborn Wistar rats and cultured in vitro via both collagenase digestion method and explant technique, and a comparative study was carried out on the two culture methods. The biologic charwteristics of the osteoblast cells were studied via cell number counting, morphology observation, alkaline phosphatase staining of the cells and alizarine- red staining of the calcified nodules. The results show that osteoblast cells can be cultured in vitro via collagenase digestion method and explant technique, and the obtained cells ure of good biologic characteristics. In comparison with the explant technique, the operative procedure of the enzymatic digestion method is more complicated. The digestion time must be carefully controlled. However, with this method, one can obtain a lager number of cells in a short time. The operative procedure of the explant technique is simpler, but it usually takes longer time to obtain cells of desirable number.

Effect of Phytoestrogen Activity on hFOB 1.19 Osteoblast Cells of Vanilla Siamensis

The unsaponifiable compounds derived from the fresh green beans of Vanilla siamens＆ Rol. ex. Dow were assayed for the first time to detect their estrogenic activity. We used a simple screening method using the yeast two hybrid system based on the binding of a ligand to estrogen receptors. Yeast cells carrying the hER （human estrogen receptor） gene, ERE （estrogen response elements） and lacZ （β-galactosidase gene） are very suitable for screening and sensitive analysis of estrogenic compound. Our results showed that V. siamensis plant extracts bind with relatively affinity to YES- hERa was 2.27-fold the relative potency ofestradiol （E2） in YES-hERa. The effects of phytoestrogen activity on the osteoblast cells were examined on the proliferation of hFOB 1.19 cells and the bone mineralization process. V. siamens＆ was a positive screening result and induced mineralization ofosteoblasts. This study indicated that V. siamensis plant extract exhibited the characteristic effects of a nature bone promoter compound as phytoestrogen.

A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

We report here a method for the use of poly-L-lysine （PLL） to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells （BMSCs） in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein （GFP）-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research.

In Vitro Comparative Effect of Three Novel Borate Bioglasses on the Behaviors of Osteoblastic MC3T3-E1 Cells

Most related investigations focused on the effects of borate glass on cell proliferation/biocompatibility in vitro or bone repair in vivo; however, very few researches were carried out on other cell behaviors. Three novel borate bioglasses were designed as scaffolds for bone regeneration in this wok. Comparative effects of three bioglasses on the behaviors of osteoblastic MC3T3-E1 cells were evaluated. Excellent cytocompatibility of these novel borate bioglasses were approved in this work. Meanwhile, the promotion on cell proliferation, protein secretion and migration with minor cell apoptosis were also discussed in details, which contributed to the potential clinical application as a new biomaterial for orthopedics.

Differentiation of Osteoblast in vitro Is Regulated by Progesterone

The regulation of cellular differentiation by progesterone in fetal rat calvarial osteoblasts was investigated. Our results showed that cells cultured in the presence of progesterone had a 7% increase in the alkaline phosphatase activity when compared to untreated cells. The concentration of osteocalcin in the conditioned medium from progesterone treated osteoblasts was 28% higher than that of untreated controls. In addition,administration of progesterone significantly enhanced the number and area of bone nodules. In conclusion, progesterone stimulates the differentiation of fetal rat calvarial osteoblastic cells in vitro

Modification of Titanium Surfaces via Surface-initiated Atom Transfer Radical Polymerization to Graft PEG-RGD Polymer Brushes to Inhibit Bacterial Adhesion and Promote Osteoblast Cell Attachment

Implant-related infection is one of the key concerns in clinical medicine, so the modification of titanium to inhibit bacterial adhesion and support osteoblast cell attachment is important. In this article, two strategies were used to examine the above effects. First, modification of titanium via surface-initiated atom transfer radical polymerization（ATRP） was performed. The surface of the titanium was activated initially by a silane coupling agent. Well-defined polymer brushes of poly（ethylene glycol） methacrylate were successfully tethered on the silane-coupled titanium surface to form hydration shell to examine the anti-fouling effect. Second, functionalization of the Ti-PEG surface with RGD was performed to examine the anti-bacterial adhesion and osteoblast cell attachment ability. The chemical composition of modified titanium surfaces was characterized by X-ray photoelectron spectroscopy（XPS）. Changes in surface hydrophilicity and hydrophobicity were characterized by static water contact angle measurements. Results indicated that PEG-RGD brushes were successfully tethered on the titanium surface. And anti-bacterial adhesion ability and osteoblast cell attachment ability were confirmed by fluorescence microscopy and scanning electron microscopy. Results indicated that PEG can inhibit both bacterial adhesion and osteoblast cell attachment, while PEG-RGD brushes can not only inhibit bacterial adhesion but also promote osteoblast cell attachment.

Comparative Evaluation of Ozone Treatment in Critical Size Bone Defects Reconstructed with Alloplastic Bone Grafts

The purpose of this study was to investigate osteogenesis promoted by osteoconductive properties of bone grafting materials and the histopathological effects of ozone on osteogenesis. In total, 56 Winstar rats were equally divided into 4 groups. In control group, calvarial bone defect was created in 14 rats. For second group, 8 mm calvarial bone defect with ozone treatment was applied in 14 rats. For third group, an alloplastic bone graft was implanted on 8 mm calvarial bone defect. In fourth group, alloplastic bone graft was inserted in calvarial defect and ozone was treated additionally. Seven of the rats were sacrificed at the end of 4th week and the remaining 7 were sacrificed at the end of 8th week of experiment. In the study, the periosteal flaps were removed with a thin periosteal elevator and averagely 0.8 cm diameter-circular full bone defect was created with a specially designed trephine drill. The bone from the calvarial region was fixed in 10% formalin solution. After decalcification, bones were taken for routine paraffin blocking. Sections were stained with Hematoxylin-Eosin and Masson Trichrome. Histopathological findings of 4th and 8th weeks rats showed that best result for new bone formation was observed in graft + ozone treatment. It is concluded that ozone treatment increases the hemostasis in graft region, induces angiogenesis, promotes cell proliferation by preventing infiltration, induces matrix formation by influencing osteoblastic activity and has a positive effect in osteogenesis.

Use of Laser Microbeam and Ultrasound Methods to Enhance the Efficiency of Gene Transfer into Osteoblast Cells

Gene transfer introduced by physical methods is widely used for its convenient operation, high transfer efficiency and no host distinctive limitation. In this article pSV β Gal plasmid was induced into osteoblast cells successfully by laser microbeam method and ultrasound method, and β Gal gene was highly expressed. The optimal conditions for the ultrasound and laser microbeam methods of gene transfer were obtained.

Algal oligosaccharides ameliorate osteoporosis via up-regulation of parathyroid hormone 1-84 and vascular endothelial growth factor

OBJECTIVE: To determine whether algal oligosaccharide affects the levels of parathyroid hormone 1-84(PTH1-84) and vascular endothelial growth factor(VEGF).METHODS: An osteoporosis rat model was established via bilateral ovariectomy. The model rats were fed algal oligosaccharides(molecular weights:600-1, 200 Da) for 4 months. Bone mineral density(BMD) was then measured. MG-63 human osteoblastic cells were treated with algal oligosaccharides. The expression of PTH1-84 and VEGF was then examined. Oligosaccharide-treated cells were transfected with PTH1-84 short hairpin RNA(sh RNA), VEGF sh RNA, and PTH1-84-VEGF small interfering RNA(si RNA). The growth rates were then compared between transfected and non-transfected cells.RESULTS: Algal oligosaccharides increased the BMD of the osteoporosis rat model compared with untreated controls(P < 0.05). When MG-63 cells were treated with algal oligosaccharides, the growth rate increased by 25% compared with the control group at day 3(P < 0.05). In addition, the expression of PTH84 and VEGF was enhanced. Con-versely, when the cells were transfected with PTH84 sh RNA, VEGF sh RNA, or PTH1-84-VEGF si RNA, the growth rate was decreased by 17%, 35% and 70%, respectively, compared with controls at day 3(P < 0.05).CONCLUSION: Algal oligosaccharides ameliorate osteoporosis via up-regulation of PTH1-84 and VEGF. Algal oligosaccharides should be developed as a potential drug for osteoporosis treatment.

Effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro

Objective To observe the effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro.Methods Iliac trabecular bone specimens were obtained from adult patients undergoing necessary surgery. After the bone pieces were digested with collagenase-trypsin, osteoblasts were released and incubated at 37℃in a relative humidity of 95% and 5% CO2. Then, the cells were purified, and their passages were given DMEM-F12 and fetal bovine serum medium. Subsequently, 10^(-8) mol/L dexamethasone was added into the culture medium to incubate the osteoblasts for three days, and the cells from control groups were incubated without any drugs. All cells were observed continually with phase contrast microscope and transmission electron microscope. Finally, apoptosis was detected by the use of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and biochemical indices, alkaline phosphatase (ALP) and osteocalcin (OCN) were used to determine the effects of dexamethasone on proliferation, differentiation and apoptosis of adult osteoblasts in vitro.Results In the adult osteoblasts obtained by collagenase-trypsin digestion, it achieved high survial, stable biochemical indices and excellent purification. Under the condition of dexamethasone 10^(-8) mol/L and osteoblasts 10 000/ml, there was significant promotion of ALP and OCN secretion without cell apoptosis.Conclusions Dexamethasone has a significant effect on the proliferation and differentiation of adult osteoblasts in vitro without apoptosis, and dexamethasone at the suggested concentration can be used as positive control in drug studies for osteoporosis treatment.

Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro

Purpose： To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods： Primary osteoblasts, bone marrow-derived mesenchymal stem cells （BMSCs, cultured in osteoinductive medium） and RAW264.7 cells cultured in osteoclast inductive medium were all subjected to a 1000μstrain （μs） at 1 Hz cyclic mechanical stretch for 30 min （twice a day）. Results： After mechanical stimulation, the alkaline phosphatase （ALP） activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the mRNA levels of ALP and collagen type I increased. Additionally, extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant acid phosphatase-positive multinucleated cells, matrix metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium solvency all decreased. Conclusion： The results demonstrated that 1000 μs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.