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.     

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.     

Bisphosphonate Treatment Increases the Size of the Mandibular Condyle and Normalizes Growth of the Mandibular Ramus in Osteoprotegerin-Deficient Mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. OPG-deficient (OPG–/–) mice develop severe osteoporosis caused by significant enhancement of bone resorption by osteoclasts. We investigated the effect of administering bisphosphonate on mandibular growth and development in OPG–/– mice. Eight-week-old male OPG–/– mice and wild-type (WT) mice were administered bisphosphonate (1.25 mg/kg body weight) intraperitoneally once every 3 days for 30 days. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG–/– mice with bisphosphonate than in those without bisphosphonate. The volume of the whole condyle and the mandibular length in OPG–/– mice without bisphosphonate were significantly smaller than in WT mice without bisphosphonate. Bisphosphonate treatment of the OPG–/– mice resulted in an increase in the volume of the mandibular condyle and mandibular ramus length. In fact, the mandibular ramus length in OPG–/– mice with bisphosphonate was similar to the length in WT mice without bisphosphonate. Histologically, the surface irregularity of the mandibular condyle that was observed in the OPG–/– mice without bisphosphonate tended to be less marked in the OPG–/– mice with bisphosphonate, and the proportion of the area of the cartilage layer relative to the whole condyle was significantly larger in OPG–/– mice with bisphosphonate than in those without bisphosphonate. In conclusion, bisphosphonate treatment results in an increase in mandibular condylar dimensions and normalization of mandibular ramus growth.     

Mechanical force-induced midpalatal suture remodeling in mice

Mechanical stress is an important epigenetic factor for regulating skeletal remodeling, and application of force can lead to remodeling of both bone and cartilage. Chondrocytes, osteoblasts and osteoclasts all participate and interact with each other in this remodeling process. To study cellular responses to mechanical stimuli in a system that can be genetically manipulated, we used mouse midpalatal suture expansion in vivo. Six-week-old male C57BL/6 mice were subjected to palatal suture expansion by opening loops with an initial force of 0.56 N for the periods of 1, 3, 5, 7, 14 or 28 days. Periosteal cells in expanding sutures showed increased proliferation, with Ki67-positive cells representing 1.8 ± 0.1% to 4.5 ± 0.4% of total suture cells in control groups and 12.0 ± 2.6% to 19.9 ± 1.2% in experimental/expansion groups (p < 0.05). Starting at day 1, cells expressing alkaline phosphatase and type I collagen were seen. New cartilage and bone formation was observed at the oral edges of the palatal bones at day 7; at the nasal edges only bone formation without cartilage appeared to occur. An increase in osteoclast numbers suggested increased bone remodeling, ranging from 60 to 160% throughout the experimental period. Decreased Saffranin O staining after day 3 suggested decreased proteoglycan content in the secondary cartilage. Micro-CT showed a significant increase in maxillary width at days 14 and 28 (from 2334 ± 4 μm to 2485 ± 3 μm at day 14 and from 2383 ± 5 μm to 2574 ± 7 μm at day 28, p < 0.001). The suture width was increased at days 14 and 28, except in the oral third region at day 28 (from 48 ± 5 μm to 36 ± 4 μm, p < 0.05). Bone volume/total volume was significantly reduced at days 14 and 28 (50.2 ± 0.7% vs. 68.0 ± 3.7% and 56.5 ± 1.0% vs. 60.9 ± 1.3%, respectively, p < 0.05), indicative of increased bone marrow space. These findings demonstrate that expansion forces across the midpalatal suture promote bone resorption through activation of osteoclasts and bone and cartilage formation via increased proliferation and differentiation of periosteal cells. Mouse midpalatal suture expansion would be useful in further studies of the ability of mineralized tissues to respond to mechanical stimulation.     

氟离子植入猪骨源羟基磷灰石对 MC3T3-E1 细胞黏附、增殖与成骨分化的影响

目的 检测氟离子植入猪骨来源羟基磷灰石（PHA）对小鼠前成骨细胞（MC3T3-E1）黏附、 增殖及成骨分化的影响。方法 取猪骨松质用高温烧结的方法制备PHA,使用氟化钠浸泡结合二次高温 烧结进行氟离子植入,制备获得氟化猪骨源羟基磷灰石（FPHA）骨块,进行研磨获得颗粒材料后制备 压片,采用PHA颗粒制备的压片为对照组,采用FPHA颗粒制备的压片为实验组。在压片材料表面接种 小鼠MC3T3-E1前成骨细胞株,通过扫描电镜SEM观察不同时间细胞黏附形态,CCK-8法检测细胞增 殖情况,RT-PCR法检测细胞成骨相关基因碱性磷酸酶ALP、骨钙素BGLAP、骨桥蛋白OPN mRNA 的表达情况。结果 扫描电镜SEM观察可见,PHA与FPHA组细胞黏附生长良好;两组细胞接种1、7d 后OD值比较,差异无统计学意义（P＞0.05）;FPHA组接种3、5 d后OD值高于PHA组,差异有统计学 意义（P＜0.05）;RT-PCR结果显示,FPHA组细胞接种3、7 d后ALP、BGLAP mRNA表达水平均高于 PHA组,且两组细胞接种7 d后材料表面细胞ALP与BGLAP mRNA表达水平均高于细胞接种3 d后,差异 有统计学意义（P＜0.05）。结论 PHA与FPHA材料均具有良好的细胞相容性,氟离子植入猪骨羟基磷灰 石可促进小鼠前成骨细胞早期增殖与成骨分化。     

Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression

Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10 × 100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials.     

牙髓卟啉单胞菌脂多糖对小鼠成骨细胞表达MCP-1的影响

目的: 探讨牙髓卟啉单胞菌(Porphyromonas endodontalis,P.endodontalis)脂多糖对小鼠成骨细胞表达单核细胞趋化蛋白1(monocyte chemotactic protein-1, MCP-1)mRNA和蛋白的影响,以及是否有p38丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）信号通路和核因子κB（Nuclear Factor-κB, NF-κB）信号通路的参与。方法: 以不同质量浓度的P.endodontalis脂多糖(0~50 mg/L)刺激MC3T3-E1细胞24 h;以20 mg/L P.endodontalis脂多糖作用于细胞不同时间（0~48 h）后,采用实时反转录PCR和酶联免疫吸附测定检测MCP-1mRNA和蛋白的表达。采用p38MAPK抑制剂SB203580和NF-κB抑制剂BAY11-7082分别预处理细胞1 h,检测其对P.endodontalis脂多糖刺激MC3T3-E1细胞后MCP-1mRNA表达的影响。采用SPSS 13.0软件包对结果进行单因素方差分析和Dunnett t检验。结果: 不同质量浓度的P.endodontalis脂多糖（0~50 mg/L）刺激MC3T3-E1细胞后,MCP-1的mRNA表达和蛋白分泌呈剂量依赖性;观察时间内（0~48 h）,20 mg/L P.endodontalis脂多糖作用于MC3T3-E1细胞后,MCP-1 mRNA的表达和蛋白分泌呈时间依赖性;10 mol/L的SB203580和BAY11-7082分别预处理细胞1 h,可以降低P.endodontalis脂多糖诱导MCP-1 mRNA的表达,且SB203580的抑制作用强于BAY11-7082。结论: P.endodontalis脂多糖可能通过激活p38MAPK和NF-κB信号通路诱导成骨细胞表达MCP-1mRNA和蛋白。     

Temporal Pattern of Stimulation of Osteoblast-Associated Genes During Mechanically-Induced Osteogenesis In Vivo: Early Responses of Osteocalcin and Type I Collagen

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.