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.     

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.     

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。     

Modulation by epidermal growth factor of the basal 1,25(OH)2D3 receptor level and the heterologous up-regulation of the 1,25(OH)2D3 receptor in clonal osteoblast-like cells

Summary The effects of epidermal growth factor (EGF) on basal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) receptor level and on parathyroid hormone (PTH)-induced 1,25-(OH)₂D₃ (OH)₂D₃ receptor up-regulation were studied in the phenotypically osteoblastic cell line UMR 106. EGF in concentrations exceeding 0.1 ng/ml reduced the number of 1,25(OH)₂D₃ binding sites without changing the binding affinity. Maximal reduction was 30% at about 1 ng/ml. This reduction was independent of a change in cAMP content. EGF dose-dependently attenuated both PTH-induced 1,25(OH)₂D₃ receptor up-regulation and PTH-stimulated cAMP production without and effect on the ED₅₀ of the PTH effects. For both PTH responses the IC₅₀ and the maximal effective dose were similar, 0.1 ng/ml an 1 ng/ml EGF, respectively. Reduction was first seen at 0.01 ng/ml EGF. At this concentration. EGF reduced PTH-stimulated 1,25-(OH)₂D₃ receptor binding without an inhibition of the cAMP response. Time-course studies with 1 ng/ml EGF revealed that at 2 h preincubation EGF reduced the heterologous up regulation by PTH, and maximal inhibition was seen after 4 h. In contrast, PTH-stimulated cAMP production was just significantly inhibited only after 6 h, with 60% inhibition after 24 h preincubation. The effects of prostaglandin E₂ and forskolin on both 1,25(OH)₂D₃ binding and cAMP production were inhibited in a similar fashion. On the other hand, dibutyryl cAMP- and 3-isobutyl-1-methylxanthinestimulated 1,25(OH)₂D₃ binding were not affected by EGF. Taken together, our results demonstrate that EGF reduces both the basal number of 1,25(OH)₂D₃ binding sites and the heterologous up-regulation of the 1,25(OH)₂D₃ receptor. The current data suggest that EGF reduces heterologous upregulation of the 1,25(OH)₂D₃ receptor independent of as well as dependent on the cAMP messenger system. The EGF effect is not primarily located at the PTH receptor, at cAMP phosphodiesterase, or at protein kinase A level.     

恒定磁场对骨组织中细胞影响的研究

目的阐明恒定磁场对成骨细胞和破骨细胞的生长和功能的影响和对骨髓基质细胞向成骨细胞方向分化的作用，从而在细胞水平较全面的解释磁场治疗骨质疏松的机理。方法利用体外原代培养的成骨细胞、破骨细胞和骨髓基质细胞，分别外加0．38T、0．48T恒定磁场处理后，观察恒定磁场对这三种细胞生长、分化和功能的影响。结果恒定磁场处理可促进骨髓基质细胞向成骨细胞方向分化，促进成骨细胞的增殖、分化及功能的表达，并且抑制破骨细胞的生长、分化和功能。结论恒定磁场促进骨组织的成骨作用，抑制骨分解，是其治疗骨质疏松良好效果的部分机制。     

Differentiation of periodontal ligament fibroblasts into osteoblasts during socket healing after tooth extraction in the rat

Background: The entire socket after tooth extraction is filled with new bone formed by osteoblasts (Obs), but the origin of these Obs remains unknown. Thus, the proliferation and migration of paravascular and endosteal fibroblastic cells and periodontal ligament (PDL) fibroblasts (Fbs) and their differentiation into Obs during socket healing after extraction of the first maxillary molars of the rat were investigated. Methods: The proliferative activity and migration of these cells in the sockets after tooth extraction were studied using radioautography and immunohistochemistry after injection of ³H-thymidine and 5-bromo-2′-deoxy-uridine (BrdU), respectively. Their morphological changes during differentiation was investigated by transmission electron microscopy. Results: One day after tooth extraction, PDL Fbs were the major cell type in the PDL remnant of the socket. Proliferation was low (labeling index (LI) = approximately 2%) until 16 h after tooth extraction but dramatically increased to a maximum level 1 day postextraction (LI = 23%). Between 1 and 2 days, numerous PDL Fbs in the PDL remnant actively migrated into the coagulum and continued to proliferate. On the basis of the high proliferative activity and small number of cellular organelles responsible for procollagen synthesis, these cells appear immature. At 3 days, Fbs contained more cellular organelles and deposited more collagen fibers as they replaced the coagulum with dense connective tissue and the LI declined. At 4 and 5 days, some of the Fbs began to differentiate into Obs, and the proliferation of Fbs dramatically decreased to baseline values. The migration of PDL Fbs and their differentiation into Obs were investigated by labeling with ³H-thymidine or BrdU 1 day after tooth extraction. Heavily labeled Fbs were observed in the PDL remnant at 1 day, in the coagulum at 2 days, and in the dense connective tissue at 3 days. Labeled Obs associated with new bone were seen 4 days after injection. Endosteal and paravascular Fbs also proliferated, but at a lower level and at later time periods than the PDL Fbs. Surprisingly, endosteal and paravascular Fbs contributed only a small population of Fbs to socket healing. Conclusions: These results indicate that PDL Fbs after tooth extraction actively proliferate, migrate into the coagulum, form dense connective tissue, and differentiate into Obs which form new bone during socket healing. © 1994 Wiley-Liss, Inc.     

Administration of colony stimulating factor-1 to toothless osteopetrotic rats normalizes osteoblast, but not osteoclast, gene expression

The toothless (tl) osteopetrotic mutation in the rat is characterized by generalized skeletal sclerosis, a severe reduction in the numbers of osteoclasts, monocytes, and macrophages, and absence of tooth eruption. Studies examining gene expression in bone-derived cells of tl rats and their normal littermates have shown that genes related to osteoblast function are aberrantly expressed in tl rats compared to normal littermates. We have previously shown that exogenous administration of colony stimulating factor-1 (CSF-1) to tl rats results in a dramatic reduction of the skeletal sclerosis and significant increases in the number of osteoclasts. Thus, we examined the effects of CSF-1 on osteoblast and osteoclast gene expression in tl rats as demonstrated by Northern blot analysis. While osteoblast-related gene expression as reflected by mRNA levels of alkaline phosphatase, osteocalcin, osteopontin, and type I collagen was normalized, osteoclastrelated gene expression, as reflected by mRNA levels of carbonic anhydrase II and tartrate-resistant adenosine triphosphatase, remained significantly lower in CSF-1-treated tl rats compared to untreated normal littermates. Since previous studies have not demonstrated the CSF-1 receptor on osteoblasts, these results suggest that osteoblast abnormalities in tl rats are an effect of the osteopetrotic condition rather than the cause of the disease.     

地塞米松对骨髓基质干细胞体外增殖的影响

目的 观察地塞米松对小鼠骨髓基质干细胞体外增殖的作用。方法 取雄性6周龄ICR小鼠股骨骨髓基质细胞进行体外培养，在细胞培养不同时间点加入10^-8mol／L地塞米松（实验组）或保持基础培养条件（对照组），用CellTiter方法分别检测两组的细胞增殖情况，并予比较。结果 在细胞培养不同阶段开始应用地塞米松的实验组中，细胞增殖能力较对照组降低，随着培养时间延长实验组细胞数量明显少于对照组。实验组中细胞形态较对照组更趋成熟。结论 地塞米松在促进骨髓干细胞定向分化早期抑制骨髓基质干细胞体外增殖。