Effects of retinoic acid on bone formation and resorption in cultured mouse calvaria

1. The effects of retinoids on bone metabolism were examined in newborn mouse calvaria. 2. Incubation of calvaria with 0.01-1 microM retinoic acid for 4 days decreased their alkaline phosphatase (ALP) activity, mineral content and collagen content in a concentration-dependent fashion. 3. With treatment for 2 days, retinoic acid (1 microM) decreased the ALP activity and collagen content, but not the mineral content. 4. All these inhibitory effects were observed in calvaria from 0-day-old mice, but no inhibition of ALP activity was observed in calvaria from 14-day-old mice. 5. 1-Hydroxyethylidene-1,1-bisphosphonate (HEBP, 1 mM), which inhibits bone resorption, prevented the effect of retinoic acid (1 microM) on the bone mineral content, but not the effects on ALP and collagen (synthesized by osteoblasts). HEBP (1 mM) alone had no effect on the calvarial mineral and collagen contents. 6. These findings indicate that retinoic acid both stimulates bone resorption and inhibits osteoblastic activity by different mechanisms, and that stimulation of bone resorption by retinoic acid is inhibited by HEBP.     

Effects of methylprednisolone on bone formation and resorption in rats

Excessive glucocorticoids induce osteoporosis. However, there is some controversy regarding the mechanism of action, and even the endpoint result. The present study was carried out to obtain further insight into the action of glucocorticoids on bone formation and resorption in rats. Growing rats were injected subcutaneously with methylprednisolone (mPSL) at doses of 0, 2.5, 5, 10 or 20 mg/kg per day for 4 weeks. Bone mineral density (BMD), enchondral and periosteal bone formation, collagen synthetic activities of osteoblasts, numbers of osteoblasts and osteoclasts, and serum markers to assess bone turnover were determined. Administration of mPSL dose-dependently increased the BMD in the tibial metaphysis, while it dose-dependently decreased the BMD in the diaphysis. Both enchondral and periosteal bone formation were decreased in a dose-dependent fashion. The incorporation and secretion of (3)H-proline by osteoblasts were both decreased in trabecular and cortical bones. The number of osteoclasts, together with the number of osteoblasts, in the tibial metaphysis was drastically decreased. Serum alkaline phosphatase and osteocalcin were decreased at higher doses. These results support the recent notion that glucocorticoids inhibit both bone formation and resorption. In addition, BMD as an endpoint result might differ from site to site in bone due to a different balance between bone formation and resorption.     

Genistein prevents bone resorption diseases by inhibiting bone resorption and stimulating bone formation

Genistein, a soybean-derived isoflavone, has been shown to suppress osteoclastic bone resorption. To clarify the mechanisms underlying this action, we investigated the effects of genistein on the differentiation, cytoskeleton and function in mice osteoclasts in vitro and bone metabolism in ovariectomized rats. Study design: Primary OCs were isolated from 3 week-old mice and induced by 1,25(OH)(2)D(3). Then OCs were exposed to genistein at various concentration of 0 M, 10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M, and 10(-5) M. The number of TRAP+ cells were counted as well as the surface area of bone resorption on bone slice. F-actin change was observed by Confocal. In vivo, forty 12 week-old female SD rats were randomly assigned to four groups: (1) sham operated (Sham); (2) (OVX); (3) ovariectomized and treated with estradiol (OVX-E); (4) ovariectomized and received genistein (OVX-G). After 12 weeks, BMD, body weight, serum level of alkaline phosphatase (ALP), acid phosphatase (ACP), osteocalcin (OC), IL-1beta, TNFalpha, IL-6 and calcitonin (CT) were evaluated. Femur were sectioned. In addition, the serum estradiol, the weight of uteri and histological behavior were also examined to indicate the side effect of genistein to the uteri. Results: In vitro, the number of TRAP+ cells decreased depending on the concentration of genistein as well as the area of bone resorption. F-actin became disorder under Confocal. In vivo, after treated with genistein, BMD and the serum level of ALP, ACP, osteocalcin increased significantly, while the serum level of IL-1beta and TNFalpha decreased. Especially, the increase of ALP and osteocalcin of OVX-G was higher than that of OVX-E. Histologically, the pachy-trabecula were observed as well as the more mineral deposition lines. Additionally, the uterus weight index and the serum estradiol in OVX-G rats were lower significantly than those of OVX-E. The epithelia of uteri gland in OVX-G appeared cubic while those of OVX-E became squamous. Conclusions: Genistein can prevent bone resorption diseases by the promotion of bone formation and the prevention of bone resorption with slight side effect.     

Inhibitory effects of gamma-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant gamma-interferon (gamma-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and prostaglandin E2 (PGE2) have been studied using cultures of neonatal calvarial bones and analyzing the release of 45Ca from prelabelled bones as a parameter of bone resorption. In addition, the effects of gamma-IFN and indomethacin on formation of PGE2 in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mumol/l) totally abolished bradykinin (1 mumol/l) induced 45Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE2 (1 mumol/l). gamma-IFN (1000 U/ml) almost totally inhibited 45Ca release stimulated by bradykinin (1 mumol/l), but the inhibitory effect could only be partially overcome by PGE2. gamma-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg9-bradykinin (1 mumol/l) on 45Ca release. The stimulatory effects of PGE2 (1 mumol/l) on radioactive calcium mobilization was partially inhibited by gamma-IFN (1000 U/ml), whereas indomethacin (1 mumol/l) was without effect. The inhibitory effect of gamma-IFN on 45Ca release stimulated by bradykinin and PGE2 was dose-dependent with threshold for action at 3-30 U/ml. Comparative dose-response curves showed that gamma-IFN was most potent as inhibitor of bradykinin induced 45Ca release. Bradykinin (1 mumol/l) significantly stimulated PGE2 formation by a mechanism that was completely inhibited by indomethacin (1 mumol/l). gamma-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE2 formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

GDF8 inhibits bone formation and promotes bone resorption in mice

Growth Differentiation Factor 8 (GDF8), also called myostatin, is a member of the transforming growth factor (TGF)‐β super‐family. As a negative regulator of skeletal muscle growth, GDF8 is also associated with bone metabolism. However, the function of GDF8 in bone metabolism is not fully understood. Our study aimed to investigate the role of GDF8 in bone metabolism, both in vitro and in vivo. Our results showed that GDF8 had a negative regulatory effect on primary mouse osteoblasts, and promoted receptor activator of nuclear factor κB ligand (RANKL)‐induced osteoclastogenesis in vitro. Intraperitoneal injection of recombinant GDF8 repressed bone formation and accelerated bone resorption in mice. Furthermore, treatment of aged mice with a GDF8 neutralizing antibody stimulated new bone formation and prevented bone resorption. Thus, our study showed that GDF8 plays a significant regulatory role in bone formation and bone resorption, thus providing a potential therapeutic pathway for osteoporosis.     

蛇床子素对体外培养破骨细胞骨吸收及细胞凋亡的影响

研究蛇床子素对破骨细胞骨吸收的影响及其分子机制。采用体外分离、培养兔破骨细胞, 与盖玻片及骨磨片共同培养, 使用1×10⁻⁵ mol·L⁻¹蛇床子素刺激破骨细胞, 观察活体细胞并依据HE、TRAP、骨陷窝甲苯胺蓝染色鉴定破骨细胞; 进行骨吸收陷窝和面积定量分析, 吖啶橙染色统计凋亡细胞; real time PCR及Western blotting法检测相关基因和蛋白。与空白对照组比较, 1×10⁻⁵ mol·L⁻¹蛇床子素能够明显提高破骨细胞凋亡率并通过抑制RANKL和TRAP等相关基因及JNK1/2磷酸化水平抑制其骨吸收。结果提示, 蛇床子素可以通过RANK+RANKL/ TRAF6/Mkk/JNK途径刺激破骨细胞凋亡并抑制骨吸收。     

Regulation of bone resorption and formation by purines and pyrimidines

Growing evidence suggests that extracellular nucleotides, signalling through P2 receptors, might play important roles in the regulation of bone and cartilage metabolism. ATP and other nucleotides can exert impressive stimulatory effects on the formation and activity of osteoclasts (bone-resorbing cells) in addition to inhibiting bone formation by osteoblasts. In this review, the current understanding of the actions of nucleotides on skeletal cells and the probable receptor subtypes involved are discussed.     

Effect of strontium chloride on bone resorption induced by prostaglandin E2 in cultured bone

We examined whether the bone resorption induced by PGE₂ was inhibited by SrCl₂ using⁴⁵Ca-labelled calvaria of CD-strain mice in tissue culture. It was found that Sr salts inhibited physiological bone resorption in a dose-dependent manner (0.1–5.0 mM) and did not act via PGE₂. Accordingly, it was suggested that Sr salts did not inhibit bone resorption induced by exogenous PGE₂.Abbreviations PGE₂ Prostaglandin E₂ - PTH Parathyroid hormone - CT Calcitonin - Vit. D₃ Vitamin D₃ - HSDM₁ Harvard School of Dental Medicine 1     

烧伤小鼠血清对离体培养的骨髓基质细胞的影响

目的　研究小鼠烧伤血清对离体培养的骨髓基质细胞的影响。方法　利用流式细胞仪、HE染色等技术观察小鼠烧伤后不同时间的血清对小鼠骨髓基质细胞 (BMSC)贴壁率和基质祖细胞 (CFU F)集落形成的作用及对不同剂量的6 0 Coγ射线体外照射后的BMSC的影响。结果　小鼠烧伤后 1～ 11d的血清对基质细胞贴壁率和CFU F的形成主要表现为抑制作用 ,13～ 15d的血清主要表现为刺激作用。小鼠烧伤后 15d的血清可明显减少 5 0、70Gy照射后BMSC的凋亡和坏死。 结论　小鼠烧伤后不同时间的血清对BMSC的作用不同 ,可能与血清内细胞因子的变化有关。     

人参皂苷Rg_1对培养猪骨髓基质细胞增殖的影响

人参为中医传统“补气生血”药物 ,人参皂苷单体Ｒｇ1是人参三醇组皂苷的一个代表性单体 ,被认为是其主要药物成分 ,对心血管、神经系统及免疫功能等具有广泛的药理作用[1,2 ] 。既往的研究表明 ,人参总苷对体外培养的骨髓造血干细胞及祖细胞有增殖作用 ,对骨髓造血因子具有促分泌作用[3 ,4] ,但对骨髓基质细胞 (ｂｏｎｅｍａｒｒｏｗｓｔｒｏｍａｌｃｅｌｌｓ,ＢＭ ＳＣ)的增殖作用研究较少。为此 ,应用ＣＦＵ Ｆ形成法、[3 Ｈ]ＴｄＲ参入法和ＭＴＴ法研究人参皂苷Ｒｇ1对ＢＭＳＣ体外生长及其增殖的影响。1　材料与方法1.1　动物　实验用…     

Stimulative effects of cadmium on bone resorption in neonatal parietal bone resorption.

Effects of cadmium on bone resorption were investigated using neonatal mouse parietal bone culture system. Cadmium at 0.5 microM and above stimulated hydroxyproline release as well as 45Ca release. As cadmium-stimulated bone resorption was inhibited by calcitonin, bone resorption induced by cadmium is osteoclast-mediated bone resorption. CI-1, collagenase inhibitor, depressed cadmium-stimulated bone resorption in a dose-dependent manner. Osteoblasts are also involved in cadmium-induced bone resorption. Indomethacin-inhibited cadmium-stimulated bone resorption and cadmium-treated bones released prostaglandin E2 to a greater extent than untreated bones. Cadmium-stimulated bone resorption was shown to be dependent on the production of prostaglandin E2. 3-Isobutyl-1-methylxanthine potentiated cadmium-stimulated bone resorption and verapamil depressed it. It is possible that an increase in levels of cAMP and calcium ion in bone cells is involved in cadmium-induced bone resorption. From these results, cadmium was found to stimulate osteoclast-mediated bone resorption which is dependent on prostaglandin E2. Second messengers in cadmium-induced bone resorption may be cAMP and calcium ion.     

葫芦素脂肪乳剂成型性影响因素考察

目的了解影响葫芦素脂肪乳剂成型性的因素.方法以葫芦素脂肪乳的外观、粒径、药物含量及稳定性为指标,分别考察渗透压、高压乳匀、pH、空气置换剂及灭菌等因素对乳剂成型性的影响.结果等渗调节剂甘油对乳剂粒径没有影响;高压乳匀可降低乳剂粒径,适当压力下多次乳匀可获得粒径小、均匀性好的乳剂;pH可影响乳剂中药物含量;使用空气置换剂的乳剂在37℃放置3个月后稳定性良好,而充空气的乳剂发生破裂;高压灭菌时,升温速度对乳剂的稳定性亦有影响.结论控制和掌握好高压乳匀、pH、空气置换剂及灭菌等因素,就可获得粒径大小适宜、粒度均匀、性质稳定、供静脉注射的葫芦素脂肪乳剂.     

Flufenamic acid inhibits osteoclast formation and bone resorption and act against estrogen-dependent bone loss in mice

Postmenopausal osteoporosis is one of the most common types of osteoporosis resulting from estrogen deficiency in elderly women. Nonsteroidal anti-inflammatory drugs (NSAIDs) are important drugs for pain relief in patients with osteoporosis. In this study, we report for the first time that flufenamic acid, a clinically approved and widely used NSAID, not only has analgesic properties but also shows a significant effect in terms of preventing postmenopausal osteoporosis. Quantitative RT-PCR analysis showed that treatment with flufenamic acid significantly downregulated the genes associated with osteoclast differentiation. Meanwhile, RNA-sequencing and western blot analyses suggested that flufenamic acid could inhibit the bone resorption by suppressing the phosphorylation of MAPK pathways. Moreover, an ovariectomy (OVX)-induced bone-loss mouse model indicated that flufenamic acid might be a potent drug for preventing osteoporotic fractures, as verified by micro-CT scanning and histological analysis. Therefore, this study proposes an attractive and potent drug with analgesic properties for the prevention of postmenopausal osteoporosis.     

Subacute nicotine exposure in cultured cerebellar cells increased the release and uptake of glutamate

Cerebellar granule and glial cells prepared from 7 day-old rat pups were used to investigate the effects of sub-acute nicotine exposure on the glutamatergic nervous system. These cells were exposed to nicotine in various concentrations for 2 to 10 days in situ. Nicotine-exposure did not result in any changes in cerebellar granule and glial cell viability at concentrations of up to 500 microM. In cerebellar granule cells, the basal extracellular levels of glutamate, aspartate and glycine were enhanced in the nicotine-exposed granule cells. In addition, the responses of N-methyl-D-aspartate (NMDA)-induced glutamate release were enhanced at low NMDA concentrations in the nicotine-exposed granule cells. However, this decreased at higher NMDA concentrations. The glutaminase activity was increased after nicotine exposure. In cerebellar glial cells, glutamate uptake in the nicotine-exposed glial cells were either increased at low nicotine exposure levels or decreased at higher levels. The inhibition of glutamate uptake by L-trans-pyrollidine-2,4-dicarboxylic acid (PDC) was lower in glial cells exposed to 50 microM nicotine. Glutamine synthetase activity was lower in glial cells exposed to 100 or 500 microM of nicotine. These results indicate that the properties of cerebellar granule and glial cells may alter after subacute nicotine exposure. Furthermore, they suggest that nicotine exposure during development may modulate glutamatergic nervous activity.     

The in vitro and in vivo effects of nicotine on bone,bone cells and fracture repair

Introduction: Cigarette smoke has negative effects on bone metabolism and fracture repair. However, no study has reviewed effects of nicotine on bone and fracture repair independent of other constituents of cigarette smoke. The authors review the existing evidence of the effect of nicotine on ‘bone' and ‘bone cells' and fracture repair, drawing conclusions relevant to clinical practice and future research.

Areas covered: A literature review was conducted using PRISMA guidelines and PubMed, Cochrane, MEDLINE/OVID, EMBASE, NHS Evidence and Google scholar databases. Articles were included if they specifically investigated the effects of nicotine on ‘bone' or fracture repair in animal or human models or in vitro effects on ‘bone cells'. A total of 64 papers were included in this review, of which 15 were human in vitro studies and 49 animal studies wherein 9 were in vitro and 40 in vivo. In vivo studies of the effects of nicotine in animals demonstrated widespread effects on bone including osteoneogenesis, osseointegration, steady-state skeletal bone and genes and cytokines relevant to bone cell physiology and bone homeostasis. In these studies, nicotine's effects are predominately negative, inhibiting bone cell metabolism and fracture repair, whereas most in vitro studies reported biphasic responses in all bone cells except osteoclastic cells.

Expert opinion: The review suggests that nicotine has effects on osteoneogenesis, osseointegration and steady-state skeletal bone in animal in vivo models, as well as effects on all ‘bone cells', via several mechanisms in both animal and human cell in vitro studies. The effect of nicotine is dose-dependent, with higher concentrations having predominantly negative effects, whereas at low concentrations a stimulatory effect is seen. Stimulatory effects on certain cells may indicate a possible, limited therapeutic role; advice regarding smoking cessation perioperatively should remain due to the other harmful components of cigarette smoke, but there may be scope for allowing the use of nicotine patches instead of complete abstention. Further research into clinical outcomes is required before the exact response of bone and fracture repair in humans to nicotine is known.     

Effects of nicotine on the number and activity of circulating endothelial progenitor cells

Recently, some studies have shown that nicotine increased neovascularization, which involves endothelial progenitor cells (EPCs). The effects of nicotine on EPCs are still unclear at present. Therefore, the authors investigated whether nicotine had influences on EPC number and activity. The EPCs were stimulated with nicotine (to make a series of final concentrations: 10(-12) mol/L, 10(-10) mol/L, 10(-8) mol/L, 10(-6) mol/L, 10(-4) mol/L) or vehicle control for the respective time points(12, 18, 24, 32, and 48 hours). The EPCs were characterized as adherent cells double positive for DiLDL uptake and lectin binding by direct fluorescent staining under a laser-scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR-2, and AC133 with flow cytometry. The EPC proliferation, migration, and in vitro vasculogenesis activity were assayed with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay; the modified Boyden chamber assay; and the in vitro vasculogenesis kit, respectively. The EPC adhesion assay was performed by replating those on fibronectin-coated dishes and then counting the adherent cells. As a result, nicotine dose dependently increased the EPC number and the proliferative, migratory, adhesive, and in vitro vasculogenesis capacity at nicotine concentrations of 10(-12) to 10(-8) mol/L. The peak effects on EPCs were observed at concentrations of nicotine 10(-8) mol/L, similar to those in the blood of smokers. In addition, nicotine (10(-8) mol/L) time dependently increased the EPC number and activity. However, cytotoxicity was seen at higher nicotine concentrations (> 10(-6) mol/L). In conclusion, nicotine had complex effects on EPCs: nicotine might induce the augmentation of EPCs with enhanced functional activity at relatively low concentrations. However, cytotoxicity was seen at higher nicotine concentrations.     

维生素K对成骨细胞骨形成和破骨细胞骨吸收的影响

目的 比较维生素K₁(VK₁)、维生素K₂(MK₄)、维生素K₂(MK₇)和维生素K₃(VK₃)促进骨形成和抑制骨吸收的作用。方法 采用新生大鼠颅盖骨分离成骨细胞和以核因子κB受体活化因子配体(RANKL)诱导骨髓单核细胞的破骨细胞为模型,用磷酸苯二钠法检测成骨碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(TRAP)活性;用CellTilter试剂盒检测破骨细胞代谢活力;用Z-FR-MCA荧光底物和胶原底物降解检测组织蛋白酶K(CTSK)抑制作用。结果 MK₄和MK₇在0.1~1 μmol/L时显著促进成骨细胞增殖(P<0.05),1 μmol/L时显著提高ALP活性和骨结节形成面积,VK₃抑制了骨结节形成(P<0.05)。VK₁、VK₃、MK₄和MK₇在1 μmol/L时对破骨细胞代谢活力均无影响,MK₄和MK₇在0.1~1 μmol/L时显著抑制TRAP活性(P<0.05),而VK₁和VK₃无抑制作用。MK₄在25 μmol/L可对CTSK与Z-FR-MCA底物结合的抑制率达58.9%,在100 μmol/L对CTSK胶原降解的抑制率达73.2%。结论 相较于VK₁和VK₃,MK₇和MK₄有显著促进成骨细胞活性和抑制破骨细胞骨吸收的作用,MK₄能显著抑制破骨细胞CTSK酶活性。