Rifampin suppresses osteoclastogenesis and titanium particle-induced osteolysis via modulating RANKL signaling pathways.

Wear particles liberated from the surface of prostheses are considered to be main reason for osteoclast bone resorption and that extensive osteoclastogenesis leads to peri-implant osteolysis and subsequent prosthetic loosening. The aim of this study was to assess the effect of rifampin on osteoclastogenesis and titanium (Ti) particle-induced osteolysis. The Ti particle-induced osteolysis mouse calvarial model and bone marrow-derived macrophages (BMMs) were used. Rifampin, at dose of 10 or 50 mg/kg/day, was respectively given intraperitoneally for 14 days in vivo. The calvariae were removed and processed for Further histological analysis. In vitro, osteoclasts were generated from mouse BMMs with receptor activator of nuclear factor-κB ligand (RANKL) and the macrophage colony stimulating factor. Rifampin at different concentrations was added to the medium. The cell viability, tartrate-resistant acid phosphatase (TRAP) staining, TRAP activity and resorption on bone slices were analysis. Osteoclast-specific genes and RANKL-induced MAPKs signaling were tested for further study of the mechanism. Rifampin inhibited Ti-induced osteolysis and osteoclastogenesis in vivo. In vitro data indicated that rifampin suppressed osteoclast differentiation and bone resorption in a dose-dependent manner. Moreover, rifampin significantly reduced the expression of osteoclast-specific markers, including TRAP, cathepsin K, V-ATPase d2, V-ATPase a3, c-Fos, and nuclear factor of activated T cells (NFAT) c1. Further investigation revealed that rifampin inhibited osteoclast formation by specifically abrogating RANKL-induced p38 and NF-κB signaling. Rifampin had significant potential for the treatment of particle-induced peri-implant osteolysis and other diseases caused by excessive osteoclast formation and function.

Deferoxamine released from poly(lactic-co-glycolic acid) promotes healing of osteoporotic bone defect via enhanced angiogenesis and osteogenesis.

The regeneration capacity of osteoporotic bones is generally lower than that of normal bones. Current methods of osteoporotic bone defect treatment are not always satisfactory. Recent studies demonstrate that activation of the hypoxia inducible factor-1α (HIF-1α) pathway, by genetic methods or hypoxia-mimicking agents, could accelerate bone regeneration. However, little is known as to whether modulating the HIF-1α pathway promotes osteoporotic defect healing. To address this problem in the present study, we first demonstrated that HIF-1α and vascular endothelial growth factor expression levels are lower in osteoporotic bones than in normal bones. Second, we loaded poly(Lactic-co-glycolic acid) (PLGA) with the hypoxia-mimetic agent deferoxamine (DFO). DFO released from PLGA had no significant effect on the proliferation of mesenchymal stem cells (MSCs); however, DFO did enhance the osteogenic differentiation of MSCs. In addition, DFO upregulated the mRNA expression levels of angiogenic factors in MSCs. Endothelial tubule formation assays demonstrate that DFO promoted angiogenesis in human umbilical vein endothelial cells. Third, untreated PLGA scaffolds (PLGA group) or DFO-containing PLGA (PLGA + DFO group) were implanted into critically sized osteoporotic femur defects in ovariectomized rats. After treatment periods of 14 or 28 days, micro-CT, histological, CD31 immunohistochemical, and dynamic bone histomorphometric analyses showed that DFO dramatically stimulated bone formation and angiogenesis in a critically sized osteoporotic femur defect model. Our in vitro and in vivo results demonstrate that DFO may promote the healing of osteoporotic bone defects due to enhanced angiogenesis and osteogenesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2515-2527, 2016.

mTORC1 maintains the tumorigenicity of SSEA-4(+) high-grade osteosarcoma.

Inactivation of p53 and/or Rb pathways restrains osteoblasts from cell-cycle exit and terminal differentiation, which underpins osteosarcoma formation coupled with dedifferentiation. Recently, the level of p-S6K was shown to independently predict the prognosis for osteosarcomas, while the reason behind this is not understood. Here we show that in certain high-grade osteosarcomas, immature SSEA-4(+) tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4(+) cell self-renewal through S6K but also the regeneration of SSEA-4(+) TICs by SSEA-4(-) osteosarcoma cell dedifferentiation. Mechanistically, active mTORC1 is required to prevent a likely upregulation of the cell-cycle inhibitor p27 independently of p53 or Rb activation, which otherwise effectively drives the terminal differentiation of SSEA-4(-) osteosarcoma cells at the expense of dedifferentiation. Thus, mTORC1 is shown to critically regulate the retention of tumorigenicity versus differentiation in discrete differentiation phases in SSEA-4(+) TICs and their progeny.

Activation of HIFa pathway in mature osteoblasts disrupts the integrity of the osteocyte/canalicular network.

The hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, are the central mediators of the homeostatic response that enables cells to survive and differentiate in low-oxygen conditions. Previous studies indicated that disruption of the von Hippel-Lindau gene (Vhl) coincides with the activation of HIFα signaling. Here we show that inactivation of Vhl in mature osteoblasts/osteocytes induces their apoptosis and disrupts the cell/canalicular network. VHL-deficient (ΔVHL) mice exhibited a significantly increased cortical bone area resulting from enhanced proliferation and osteogenic differentiation of the bone marrow stromal cells (BMSCs) by inducing the expression of ß-catenin in the BMSC. Our data suggest that the VHL/HIFα pathway in mature osteoblasts/osteocytes plays a critical role in the bone cell/canalicular network and that the changes of osteocyte morphology/function and cell/canalicular network may unleash the bone formation, The underlying mechanism of which was the accumulation of ß-catenin in the osteoblasts/osteoprogenitors of the bone marrow.

Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro.

AIM: Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro. METHODS: Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression. The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alkaloid phosphotase (ALP) was assessed using a commercial ALP staining kit. RESULTS: Microarray analyses revealed that 10 genes were up-regulated by NMDA (0.5 mmol/L) and down-regulated by MK801 (100 µmol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 µmol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 µmol/L), the PKA inhibitor H-89 (20 nmol/L), or the PI3K inhibitor wortmannin (100 nmol/L) blocked the ALP activity increase caused by NMDA (0.5 mmol/L). Furthermore, NMDA (0.5 mmol/L) rapidly increased PI3K phosphorylation, which could be blocked by pretreatment of wortmannin (100 nmol/L). CONCLUSION: The results suggest that activation of NMDA receptors stimulates osteoblasts differentiation through PKA, PKC, and PI3K signaling pathways, which is a new role for glutamate in regulating bone remodeling.

Prolyl hydroxylase inhibitors increase neoangiogenesis and callus formation following femur fracture in mice.

Skeletal trauma and impaired skeletal healing is commonly associated with diminished vascularity. Hypoxia inducible factor alpha (HIF-1) is a key transcription factor responsible for activating angiogenic factors during development and tissue repair. Small molecule inhibitors of the prolyl hydroxylase enzyme (PHD), the key enzyme responsible for degrading HIF-1, have been shown to activate HIF-1, and are effective in inducing angiogenesis. Here we examined the effects of several commercially available PHD inhibitors on bone marrow mesenchymal stromal cells (MSCs) in vitro and in a stabilized fracture model in vivo. Three PHD inhibitors [Desferrioxamine (DFO), L-mimosine (L-mim), and Dimethyloxalylglycine (DMOG)] effectively activated a HIF-1 target reporter, induced expression of vascular endothelial growth factor (VEGF) mRNA in vitro, and increased capillary sprouting in a functional angiogenesis assay. DFO and DMOG were applied by direct injection at the fracture site in a stabilized murine femur fracture model. PHD inhibition increased the vascularity at 14 days and increased callus size as assessed by microCT at 28 days. These results suggest that HIF activation is a viable approach to increase vascularity and bone formation following skeletal trauma.

[The regulation of hypoxia inducible factor-1alpha on osteoblast function in postmenopausal osteoporosis].

OBJECTIVE: To study the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in postmenopausal osteoporosis. METHODS: From October 2004 to May 2006, Cre-Loxp recombinase was used to create mice which the HIF-1alpha gene in osteoblasts was conditional knock-out, 24 female wild-type (WT) mice and 24 female conditional knock-out (CKO) mice of 3 months old were operated on ovariotomy. At 0,4,8 weeks after operation, bone histomorphometry parameters were measured with computer image analysis in HE stain sections and in tetracycline bone double labeling fluorescence sections; Bone density and the trabecular bone architecture parameters were measured by Micro-CT; The mRNA expression of vascular endothelial growth factors (VEGF), RunX2, OC, ALP were detected with quantitative RT-PCR; The protein expression of VEGF and RunX2 were detected with Western-blotting. RESULTS: In CKO mice, the trabecular number, volume, thickness, bone density, mineral apposition rate (MAR), the expression of VEGF, RunX2, OC, ALP on mRNA level and the expression of VEGF, RunX2 on protein level decreased significantly compared with WT mice especially in 8 weeks after operation. CONCLUSIONS: The bone formation ability of osteoblasts in CKO mice was reduced compared with WT mice after ovariotomy. HIF-1alpha can regulate the bone formation ability of osteoblasts in postmenopausal osteoporosis.

[Regulation of hypoxia inducible factor-1alpha on osteoblast function in osteogenesis].

OBJECTIVE: To explore the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in osteogenesis. METHODS: Skull-cap bone of HIF-1alpha Loxp/Loxp and VHL Loxp/Loxp C57/BL6 mice were taken out and cultured so as to obtain osteoblasts which were infected with the recombinant adenovirus Ad-Cre so as to conditionally knock out the HIF-1alpha gene and its up-stream gene for von Hippel-Lindau disease (VHL) using Cre-Loxp recombinase technique. Then the osteoblasts were cultured under 2% O2 for 48 hours. Real-time PCR and Western-blotting were used to detect the mRNA and protein expression of vascular endothelial growth factor (VEGF), core binding factor al (RunX2), alkaline phosphatase (ALP) and osteocalcin (OC). Transgenic FVB mice mated with C57/BL6 mice with both HIF-1alpha and VHL alleles to obtain the mice with the osteoblasts with the HIF-1alpha and VHL genes conditionally knocked-out. At the age of 3 months the distal femurs of HIF-1alpha/VHL conditionally knocked-out mice and wild type mice were obtained to undergo hematoxylin-eosin staining and micro-CT to evaluate the bone histomorphometry and bone mineral density (BMD). RESULTS: The mRNA and protein expressions of VEGF, RunX2, ALP, and OC of the HIF-1alpha conditionally knocked-out osteoblasts were all decreased and the mRNA and protein expressions of VEGF, RunX2, ALP, and OC of the VHL conditionally knocked-out osteoblasts were all increased. The values of bone histomorphometry and bone mineral density (BMD) of the HIF-1alpha conditionally knocked-out mice were both significantly lower than those of the wild-type mice, whereas the values of bone histomorphometry and BMD of the VHL conditionally knocked-out mice were both significantly higher than those of the wild-type mice (all P < 0.01). CONCLUSION: Under both the physiological and pathological hypoxia environment in bone tissues HIF-1alpha can promote the bone formation ability of osteoblast.

[Experimental study of core binding factor a1 gene-modified rabbit skin fibroblasts enhance bone defect repair].

OBJECTIVE: To investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts. METHODS: Transfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test. RESULTS: In group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups. CONCLUSION: True bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.

[Expression of hypoxia inducible factor-1alpha in the fetal vertebra development: experiment with mice].

OBJECTIVE: To explore the expression pattern and effects of hypoxia inducible factor-1alpha (HIF-1alpha) in the fetal vertebra development. METHODS: Fetuses at different developmental stages were obtained from C57BL6 mice. The vertebrae of the fetuses were isolated and the development of vertebra was observed by stereoscopic and light microscope. The mRNA expression of HIF-1alpha at different time points was detected by using reverse transcription-polymerase chain reaction (RT-PCR) and the protein expression of HIF-1alpha was detected by immunohistochemistry. RT-PCR was used to detect the mRNA expression the gene of vascular endothelia growth factor (VEGF), the Col2a1, gene for the chondroblast marker collagen type 2, and the gene of the osteoblast marker, osteocalcin (OCN). RESULTS: The cartilaginous spine column began to form at E13.5. The primary ossification center was observed at E15.5, and then the osteogenesis expanded and extended to both sides. HIF-1alpha mRNA began to express at E13.5, and more significantly at E14.5 (P < 0.05), then decreased. The VEGF mRNA was expressed coincidently with HIF-1alpha. while the HIF-1alpha protein expression was observed at E14.5 and lasted a little longer till the time when the fetus was to be born. The Col2a1 mRNA expression was high at E13.3 - E15.5 and decreased at e16.5 - E18.5, however, the OCN mRNA expression was low at E13.5 - E15.5 and became high at E16.5 - 18.5. CONCLUSION: The vertebra development pattern appears to be an endochondral osteogenesis process. There exists a hypoxia microenvironment in the vertebra that may increase the mRNA and protein expression of HIF1alpha and the expression of VEGF, its downstream gene. It shows that HIF1alpha activates the downstream genes and initiates the cascade of endochondral osteogenesis.

[Experiment on induction of fibroblasts on 3-D cell-foam structures to express osteoblastic phenotype and its mechanism].

OBJECTIVE: To study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors. METHODS: The fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment. RESULTS: Fibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor. CONCLUSIONS: Fibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.

[Experimental study on the osseointegration of nanophase hydroxyapatite biograde-coated implants].

OBJECTIVE: To study the osseointegration of the nanophase hydroxyapatite biograde-coated implants and host bone. METHODS: Nanophase hydroxyapatite biograde-coated implants, hydroxyapatite biograde-coated implants and noncoated Ti-6Al-4V implants were inserted into both femoral of Beagle canines the tissue response, dynamic osteogensis and SEM were studied at 4, 8 and 12 weeks. RESULTS: The degradation of nanophase hydroxyapatite was slower than hydroxyapatite, dynamic osteogensis and the form of osteoblast were better than the control groups. CONCLUSION: The biological reaction of Nanophase hydroxyapatite biograde-coated implants is better than hydroxyapatite coated implant.

[Osteogenesis of rabbit skin fibroblast transfected with core binding factor a1/osteoblast specific transplanting factor-2 gene].

OBJECTIVE: To study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2). METHODS: Cbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope. RESULTS: Cbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells. CONCLUSIONS: New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.

[Experiment on fibroblast-PGA complexes cultured in rotary cell culture system].

OBJECTIVE: To investigate the effects of RCCS on cell seeding onto 3-D scaffold and cell-scaffold composite culture in vitro. METHODS: Rabbit skin fibroblasts of passage 2 were seeded at 2 x 10(6) cell per cm(3) onto/into polyglycolic acid (PGA) foams by static seeding (dropping a cell suspension onto foams) or dynamic seeding (rotating PGA foams and a cell suspension in RCCS). Attachment of cells in foams was observed by cell-counting after trypsin digestion. The effects of culture condition were next studied by culturing cell-PGA complexes in RCCS versus static culture condition. Distribution and proliferation of cells in foams were investigated with MTT, stereomicroscope and scan electron microscope. RESULTS: Numbers of cells adhering to polymers increased gradually during an initial period of 24 hours. Eight, 12 and 24 hours after seeding, the rates of adhering cells were significantly higher in the dynamic seeding group than in the static seeding group (46.70% + 2.16% vs. 31.50% +/- 3.54%; 56.36% +/- 3.18% vs. 34.28% +/- 3.16%; 66.32% +/- 4.60% vs. 37.38% +/- 4.66%; P < 0.01). The dynamic culture method as compared to the static method resulted in new tissues with a higher cellularity and more uniform cell distribution during a 3 period of weeks. CONCLUSIONS: RCCS has advantages of promoting cell attachment, uniform migration and proliferation in polymer scaffolds and can be used for construction of 3-D cell-polymer tissues in vitro.

[Study on estrogen receptor of adult human osteoblasts].

OBJECTIVE: To examine estrogen receptor (ER) in osteoblasts from adult human and to elucidate the mechanism of estrogen in modulating bone metabolism. METHODS: The cultured osteoblasts were harvested from bone chips by modified sequential digestive enzyme release and immunohistochemical assay of ER in osteoblasts were carried out in three groups of female adults: normal control (group 1), patients with moderate osteoporosis (group 2) and patients with serious osteoporosis (group 3). The percentages of ER-positive osteoblasts from the three groups were compared by t test. RESULTS: The brown marks that indicate ER were found in nuclei and plasma of the osteoblasts, and the percentages of ER-positive osteoblasts among three groups were significantly different. CONCLUSION: ERs exist in nuclei and plasma of the osteoblasts. Estrogen may modulate bone metabolism through binding ER in nuclei and plasma of the osteoblasts. The reduction of ER of osteoblasts may play an important role in the pathogenesis of postmenopausal osteoporosis.