双膦酸盐对骨形成蛋白诱导骨骨吸收抑制作用的研究

目的研究双膦酸盐(bisphosphonate YM 175)对骨形成蛋白(bone morphogenetic protein．简称BMP)诱导骨骨吸收的抑制作用。方法 42只大白鼠背部植入BMP,诱导出异位骨后,将大白鼠分成2组,即投药组和对照组。投药组在BMP植入后第3w至第7w,双膦酸盐(YM 175)每周投药3次,剂量1(μg／kg．d)。对照组按同样的方式给予等量的生理盐水。在BMP植入第3w、 4w、7w和10w,将BMP诱导骨取出,采用TRAP和cathepsin K染色方法来观察双膦酸盐对破骨细胞的作用。结果 BMP诱导骨3w时(即未投双膦酸盐药前),在BMP植入体周边形成编织骨 (woven bone),大量破骨细胞出现在新生骨组织表面。在4 w时,双膦酸盐投药组和对照组,新生骨组织均向BMP植入体内生长,但双膦酸盐投药组的破骨细胞较对照组有减少。在10 w时,双膦酸盐投药组和对照组均可观察到骨细胞变小并且有规律地排列的板层状骨(lamellar bone)的特征。而双膦酸盐投药组,破骨细胞死亡,与对照组比较,破骨细胞数目明显减少。结论双膦酸盐对破骨细胞性骨吸收有明显的抑制作用。     

Fatty Acids and Bone

Nutritional status is an important determinant of skeletal health. Increased fat mass favorably influences bone mineral density and reduces fracture risk. The mechanisms by which adiposity influences skeletal health include mechanical skeletal loading, the effects of adipocyte and pancreatic β-cell-derived hormones that act on bone, and neuroendocrine outputs from the hypothalamus that respond to peripheral nutritional signals. A growing body of evidence, including the recognition that specific fatty acid receptors are expressed in skeletal tissue, also suggests that fatty acids affect skeletal health. These effects include indirect actions of dietary and circulating fatty acids, mediated by hormonal signals derived from the intestine and pancreas, and direct effects of some fatty acid species on bone cells. Emerging evidence suggests that marrow adipocyte-derived fatty acids might affect skeletal health via paracrine mechanisms. The existing evidence suggests that there is potential for both positive and negative effects of fatty acids on the skeleton, such that the net effect may be context-specific. Careful laboratory and clinical investigation is required to increase our understanding of a potentially important area of skeletal health.     

Osteoclasts and Biomaterials

Abstract There is a growing market of biomaterials for orthopedic applications. As soon as these materials are surgically introduced into the constantly remodeling bone of the patient, they start to interact with the local cells: osteoblasts and osteoclasts. At the first glance, the bone building osteoblasts seem to be the more important cells for osseointegration of implants. However, it is mainly the bone resorbing action of osteclasts that determines the longevity of the implant. In this paper, we give a short overview over the current understanding of osteoclast biology; we review the interaction between biomaterials, biomaterial particles and osteoclasts, and the effects of treatment with antiosteoclastic agents like bisphosphonates on biomaterial implant healing.     

1,25(OH)2维生素D3诱导小鼠混合培养细胞白细胞介素1α mRNA的表达

目的本研究旨在探讨破骨细胞形成过程中１，２５（ＯＨ）２维生素Ｄ３［１，２５（ＯＨ）２Ｄ３］和白细胞介素１α（ＩＬ１α）两种生物因子间的相互作用关系，以期进一步了解正畸牙齿移动过程中牙周组织改建的生物学机理。方法ＲＴＰＣＲ将ＩＬ１αｍＲＮＡ表达信号放大后，采用Ｓｏｕｔｈｅｒｎ法检测混合培养细胞在１，２５（ＯＨ）２Ｄ３作用下，ＩＬ１αｍＲＮＡ的表达。结果发现ＩＬ１αｍＲＮＡ的表达强度与１，２５（ＯＨ）２Ｄ３浓度呈正相关关系，在时间顺序上ＩＬαｍＲＮＡ在第２天表达最强，第４天、第６天相对减弱。结论证明ＩＬ１参与１，２５（ＯＨ）２Ｄ３对破骨细胞生成的诱导作用。推测前４天促进破骨细胞前体增殖，后２天促进其分化。     

Release of the lymphokine osteoclast activating factor requires cyclic AMP accumulation

Summary The bone resorbing lymphokine osteoclast activating factor (OAF) is released by lymphocytes activated by phytohemagglutinin (PHA). In this study we have found that release of OAF by activated lymphocytes is dependent on cyclic AMP (cAMP). OAF release is preceded by cAMP accumulation in the lymphocytes, and when cAMP accumulation is impaired by treatment of activated leukocytes with indomethacin, OAF is not released. OAF release in these cultures is restored by the addition of agents that mimic or increase lymphocyte cAMP content by different mechanisms. When activated purified lymphocytes that do not release OAF by themselves were cultured with these agents, OAF appeared in the culture media. These results suggest that cyclic AMP accumulation in lymphocytes is required for OAF to be released after activation.     

Volumes of chick and rat osteoclasts cultured on glass

We have examined the relationship between the number of nuclei of an osteoclast and its volume. Chick and rat cells were released from long bones by chopping the shafts and flushing the fragments in Eagle's Minimum Essential Medium with added 10% fetal calf serum. The bone cell suspension was seeded onto glass coverslips. In Experiment 1, rat and chick cells were allowed to settle for 15 minutes, more medium was then added, and the cells were cultured in 5% CO₂ at 37°C for 4 hours. In Experiment 2, only rat cells were used, and the cells were cultured in the presence or absence of 10^-6 M 3-amino-1-hydroxypropylidene-1, 1-bisphosphonate (APD) in the medium for 4 or 6 hours. The coverslips were washed in 37°C phosphatebuffered saline and fixed for 24 hours in 2.5% glutaraldehyde in isotonic cacodylate buffer (initially 37°C). The chick cells were critical point dried (CPD) or freeze dried (FD); all rat cells were FD. After drying, cells were coated with gold by vacuum evaporation. The volumes and areas of osteoclasts were measured using a video-rate, line-confocal reflection laser scanning microscope and the number of nuclei in each cell was counted. The volumes and volumes per nucleus of the FD cells were larger than those of the CPD cells but there was no significant difference in plan-areas. Rat osteoclasts were larger than chick cells in all the measured parameters except the mean number of nuclei/cell. The correlation coefficients for the areas, volumes, and the numbers of nuclei for rat and chick cells were all high (r>0.725). The volumes and volumes per nucleus, but not the areas or areas per nucleus, of the osteoclasts cultured with APD were significantly smaller than control cells. We conclude that FD causes less shrinkage than CPD; chick osteoclasts are about two-thirds the size of rat osteoclasts; and 10^-6 m APD caused a reduction of rat osteoclast volume and volume per nucleus of 21%.     

Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis

In order to investigate the ultrastructure of bone resorptive cells in the two types of adult benign human osteopetrosis, iliac crest biopsies were obtained from 11 patients and 10 normal males, who served as a control group. Six patients had the radiological type I (4 women, 2 men, aged 23–58 years, MEAN = 36.5 years), and 5 type II disease (5 men, aged 20–48 years, MEAN = 29.8 years). The normal controls (aged 23–48 years, mean 34.1 years) were recruited from the medical staff. The biopsies were immediately divided. From each patient, half was embedded in paraffin for histochemistry and light microscopy, and half in epon for transmission electron microscopy.

The osteoclasts were markedly reduced in number and size hi Type I disease (0.2 ± .7 cells vs. 2.9 ± 1.0 cells per 2.7 mm² of bone area, p < 0.01) compared to controls, and stained only weakly for tartrate-resistant acid phosphatase (TRAP). At the ultrastructural level, no signs of active bone resorption were identified, whereas numerous mononuclear cells were observed at the bone surfaces.

In type II disease, the osteoclasts were large and highly multi-nucleated, with an increased number (8.3 ± 2.3 cells vs. 2.9 ± 1.0. cells per 2.7 mm² of bone area, p < 0.01) compared to controls. In all patients with this type, but never in type I or in the controls, a smooth, TRAP-positive substance was seen between the osteoclasts and the bone surface. Ultrastructurally, this substance was amorphous, with a condensation along the cell membrane. Neither ruffled borders nor clear zones were identified. Nuclear inclusions resembling tubular structures were observed in some osteoclasts in all patients with type II disease.

It is concluded that characteristic differences exist between the two types of adult human osteopetrosis at the ultrastructural level. Type I is morphologically similar to some murine mutations characterized by defective maturation of bone resorptive cells. In type II, a defect in the resorptive capacity of their giant osteoclasts is proposed. The pathogenetical significance of nuclear inclusions in type II osteoclasts is unknown.     

Cytoplasmic modifications at the contact zone of osteoclasts and calcified tissue in the diaphyseal growing plate of foetal guinea-pig tibia

Longitudinal sections of foetal guinea-pig tibia prepared from decalcified and undecalcified samples were examined in the electron microscope. Osteoclasts in contact with calcified tissue in the growing plate of the diaphysis showed a modified oval-shaped cytoplasmic zone, situated at the brim of the cup-like depression containing the ruffled border, with dense material arranged either in parallel bands or in a loose network according to the section observed. This modified zone, interpreted as roughly ring-shaped around the edge of the resorption region, probably participates in the development of the ruffled border and thus contributes to the extension of the surface of active resorption.     

Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function

Summary Organ cultures of newborn mouse calvaria were used to test the hypothesis that tartrate-resistant acid phosphatase might serve as a biochemical marker for osteoclast function. When bone resorption was stimulatedin vitro with either parathyroid hormone or 1,25(OH)₂D₃, there was a significant increase in both tartrate-resistant and tartrate-sensitive acid phosphatase activity in the medium relative to cultured controls. Tartrate-resistant activity was localized histochemically primarily over the osteoclast and appeared as three distinct activity bands when electrophoresed on polyacrylamide gels. The tartrate-sensitive activity was found primarily associated with bone cells other than the osteoclast using histochemical techniques, and was resolved into five bands on polyacrylamide gels. The results obtained from biochemical assays, histochemical observations, and polyacrylamide gel electrophoresis suggest that bone resorptionin vitro results in the release of tartrate-resistant acid phosphatase from osteoclasts and tartrate-sensitive acid phosphatase from other bone cells as well as osteoclasts. Tartrate-resistant acid phosphatases of bone may be suitable biochemical probes for osteoclast function, but it will be necessary to achieve further purification in order to develop analytical methods with sufficient sensitivity and specificity (e.g., immunochemical) to ensure precise localization and quantitation.     

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.