Expression of bone sialoprotein (BSP) in developing human tissues

Summary Bone sialoprotein (BSP) and its messenger RNA were localized in developing human skeletal and nonskeletal tissues by means of immunohistochemistry andin situ hybridization. Both protein and mRNA were found in mature, bone-forming cells but not in their immature precursors. In addition, osteoclasts displayed positive immunostaining and high densities of autoradiographic grains byin situ hybridization experiments. BSP was expressed in fetal epiphyseal cartilage cells, particularly in hypertrophic chondrocytes of growth plates. Though neither the protein nor the mRNA were identified in a variety of other connective and nonconnective tissues, an unexpected finding was the expression of BSP in the trophoblast cells of placenta. These findings show that BSP is primarily an osteoblast-derived component of the bone matrix expressed at late stages of differentiation. We have also found that osteoclasts produce BSP, possibly as a mediator of cell attachment to bone.     

异黄酮类药Genistein对原代培养大鼠成骨细胞增殖、分化、钙含量及矿化功能的影响

目的：了解异黄酮类药Genistein对体外培养成骨细胞的作用。方法：应用MTT法、对硝基苯磷酸盐法，原子吸收分光光度法及茜素红染色方法观察Genistein对体外培养成骨细胞的增殖、ALP表达，基质钙含量及矿化结节形成的影响。结果：Genistein具有刺激成骨细胞增殖，提高ALP活性，细胞基质钙含量及矿化结节形成的数量的作用。结论：Genistein具有刺激体外培养成骨细胞增殖，分化成熟及促进矿化的作用。     

右归饮对体外成骨细胞增殖和分化影响的实验研究

目的在右归饮治疗激素性股骨头坏死的临床研究和动物实验研究基础上,进一步观察对体外成骨细胞增殖和分化的影响.方法采用血清药理学方法,对体外培养的大鼠成骨细胞作MTT法细胞增殖测定、PNPP法ALP活性测定、以及茜素红染色矿化结节计数.结果培养48、72h,10%和15%右归饮血清组的细胞增殖速度较对照组快(p＜0.05);培养72h,10%和15%右归饮血清组的ALP活性较对照组增加(p＜0.05);培养2w,矿化结节计数显示15%右归饮血清组多于对照组(p＜0.01).结论右归饮对体外大鼠成骨细胞的增殖和分化具有促进作用.     

盐酸二甲双胍对成骨细胞增殖、BMP-2及Cbfa-1基因表达的影响

目的初步研究二甲双胍（MF）在体外对小鼠颅盖骨成骨细胞增殖、骨形态发生蛋白一2（BMP一2）及核心结合因子（Cbfa一1）mRNA表达的影响,探讨二甲双胍对骨代谢的可能作用机制。方法（1）分离培养原代颅盖骨成骨细胞并对其进行鉴定。（2）以乳鼠成骨细胞为体外实验模型,不同浓度（0、50、100、200、400Ixmol／L）的MF干预体外培养的成骨细胞24h后,M1vr法检测成骨细胞的增殖能力,实时荧光定量PCR法检测成骨细胞BMP-2及Cbfa-1基因表达。结果二甲双胍干预成骨细胞24h后,可促进成骨细胞的增殖,在浓度400μxmol／L的OD值最大为0．298±0．047（P〈0．05）;可促进BMP-2及Cbfa-1mRNA的表达,呈剂量效应关系。结论二甲双胍可促进成骨细胞的增殖和分化,可能通过调节BMP-2及Cbfa-1的表达,从而促进骨的形成。     

Bone Histomorphometry in Male Idiopathic Osteoporosis

The pathogenesis of male osteoporosis at the cellular level is still elusive. We performed histomorphometric analysis of bone biopsy samples from 51 eugonadal men with idiopathic osteoporosis. Their median age was 54 (range 29–73) years. Eighty-two percent of the patients had a fracture history, and 57% had vertebral fractures. Bone volume, trabecular thickness, wall thickness, and osteoid thickness were significantly reduced in osteoporotic men compared with healthy men. Erosion depth was similar, as were the bone remodeling parameters such as bone formation rate, mineral apposition rate, and activation frequency. In the osteoporotic men, osteoid thickness was correlated to bone mineral density at the lumbar spine (R ² = 0.19, P < 0.01); together with wall thickness, the two parameters could explain 27% of the variation in lumbar spine bone mineral density. The osteoid thickness was correlated to anthropometric variables such as body weight (R ² = 0.24, P < 0.001) and body mass index (R ² = 0.14, P < 0.01), as well as to serum estradiol levels (R ² = 0.14, P < 0.01) and to the ratio insulin-like growth factor-1 (IGF-1) to IGF-binding protein-1 (IGFBP-1) (R ² = 0.12, P < 0.01). Regression analysis showed that 36% of the variation in osteoid thickness could be predicted by body weight and estradiol levels. In conclusion, bone histomorphometry in male idiopathic osteoporosis was characterized by thin bone structural units, which might suggest osteoblast dysfunction. Bone histomorphometry parameters were associated with low body weight, low estradiol levels, and increased levels of IGFBP-1, supporting the notion that estrogens and IGFs play regulatory roles in male bone turnover.     

Cbfa1基因调节骨形成与骨吸收的研究进展

骨的形成与吸收是一个涉及众多的基因和生长因子的复杂过程,凡可使骨形成下降和骨吸收增加的因素都会促进骨质疏松的发生.Cbfa1是成骨细胞分化和成熟的特异性转录因子,研究发现Cbfa1可通过促进骨形成和抑制骨吸收,对骨质疏松的发生、发展起到抑制作用,因此,我们期望随着Cbfa1基因研究的深入将为防治骨质疏松提供新思路.     

Docetaxel inhibits bone resorption through suppression of osteoclast formation and function in different manners

Osteoclasts are formed from the monocyte-macrophage lineage in response to receptor activator of nuclear factor κB ligand (RANKL) expressed by osteoblasts. Bone is the most common site of breast cancer metastasis, and osteoclasts play roles in the metastasis. The taxane-derived compounds paclitaxel and docetaxel are used for the treatment of malignant diseases, including breast cancer. Here we explored the effects of docetaxel on osteoclastic bone resorption in mouse culture systems. Osteoclasts were formed within 6 days in cocultures of osteoblasts and bone marrow cells treated with 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂. Docetaxel at 10⁻⁸ M inhibited osteoclast formation in the coculture when added for the entire culture period or for the first 3 days. Docetaxel, even at 10⁻⁶ M added for the final 3 days, failed to inhibit osteoclast formation. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited osteoclast formation when added for the final 3 days. Docetaxel at 10⁻⁸ M inhibited the proliferation of osteoblasts and bone marrow cells. RANKL mRNA expression induced by 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂ in osteoblasts was not affected by docetaxel even at 10⁻⁶ M. Docetaxel at 10⁻⁶ M, but not at 10⁻⁸ M, inhibited pit-forming activity of osteoclasts cultured on dentine. Actin ring formation and l-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10⁻⁶ M. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10⁻⁸ M and of osteoclast function at 10⁻⁶ M. These results suggest that taxanes have beneficial effects in the treatment of bone metastatic cancers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 μg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone.     

人健康和炎性牙槽骨成骨细胞COX-2、PGE2、OPG,RANKL的表达

目的：初步探讨COX-2、PGE2、OPG和RANKL在牙周炎发病机制中所起的作用及其相互关系。方法：采用组织块法对人健康和牙周炎性牙槽骨进行体外培养和传代,加入含100μg/LrhOPG的不含胎牛血清的DMEM2mL。严格按照ELISA试剂盒说明进行操作,获得各指标的检测数值。结果：牙周炎组RANKL、PGE2、COX-2的表达较健康组明显增高,OPG的表达较健康组明显降低。加入rhOPG后,牙周炎组RANKL、PGE2、COX-2的表达明显降低;OPG表达明显增加。健康组RANKL、PGE2、COX-2表达明显降低;OPG表达增加。结论：RANKL、PGE2、COX-2可促进牙周炎的发生、发展,而OPG对牙周炎的发生、发展可起抑制作用,同时表明人工重组OPG可能协同其内源性OPG来共同抑制RANKL、PGE2、COX-2的活性。     

Aminobisphosphonates Cause Osteoblast Apoptosis and Inhibit Bone Nodule Formation In Vitro

Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20-100 microM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ss-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.