Iron Overload Inhibits Osteoblast Biological Activity Through Oxidative Stress

Iron overload has recently been connected with bone mineral density in osteoporosis. However, to date, the effect of iron overload on osteoblasts remains poorly understood. The purpose of this study is to examine osteoblast biological activity under iron overload. The osteoblast cells (hFOB1.19) were cultured in a medium supplemented with different concentrations (50, 100, and 200 μM) of ferric ammonium citrate as a donor of ferric ion. Intracellular iron was measured with a confocal laser scanning microscope. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescin diacetate fluorophotometry. Osteoblast biological activities were evaluated by measuring the activity of alkaline phosphatase (ALP) and mineralization function. Results indicated that iron overload could consequently increase intracellular iron concentration and intracellular ROS levels in a concentration-dependent manner. Additionally, ALP activity was suppressed, and a decline in the number of mineralized nodules was observed in in vitro cultured osteoblast cells. According to these results, it seems that iron overload probably inhibits osteoblast function through higher oxidative stress following increased intracellular iron concentrations.     

染料木黄酮对成骨细胞增殖分化的影响

目的：研究染料木黄酮对体外培养乳鼠颅盖骨成骨细胞增殖分化的影响。方法：取乳鼠颅盖骨,采用胶原-胰蛋白酶消化法,进行颅骨成骨细胞培养,取第二代成骨细胞,添加10^-5～10^-7mol／L染料木黄酮,在CO2孵箱中培养48h和72h后MTT比色法测定细胞增殖,培养72h采用^3H-TdR和^H-Pro掺入实验测定DNA和胶原合成。用试剂盒检测细胞裂解液碱性磷酸酶(ALP)活性。结果：染料木黄酮明显增加成骨细胞MTT的吸光度值、^3H-TdR和^3H-Pro的掺入,增加成骨细胞碱性磷酸酶活性。结论：染料木黄酮促进体外培养的乳鼠颅盖骨成骨细胞DNA和胶原的合成,促进增殖和分化。     

Ferrous and ferric differentially deteriorate proliferation and differentiation of osteoblast-like UMR-106 cells

The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.     

高浓度地塞米松通过下调LMP-1表达抑制大鼠成骨细胞的分化

为探讨地塞米松（dexamethasone,DEX)抑制成骨细胞分化的机制,观察了不同浓度DEX对体外培养大鼠成骨细胞的碱性磷酸酶活性,骨钙素（osteocalcin,OC)合成,I型胶原蛋白表达的影响。并用RT-PCR方法检测了成骨细胞中LIM矿化蛋白1mRNA的表达量,结果显示：低浓度（10^-9mol/L）的DEX能增强碱性磷酸酶的活性、OC的分泌和I型胶原蛋白的表达;而高浓度（10^-7mol/L)的DEX对它们则起抑制作用,并下调成骨细胞正调节因子LMP-1mRNA的表达,上述结果表明,低浓度的DEX促进成骨细胞的分化;高浓度的DEX则抑制成骨细胞的分化,其抑制作用可能是通过下调LMP-1mRNA的表达而实现的。     

Shared oxidative pathways in response to gravity-dependent loading and gamma-irradiation of bone marrow-derived skeletal cell progenitors

Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.     

Facilitation of human osteoblast apoptosis by sulindac and indomethacin under hypoxic injury

Hypoxic–ischemia injury occurs after trauma causes consequential bone necrosis. Non‐steroid anti‐inflammatory drugs (NSAIDs) are frequently used in orthopedic clinics for pain relief. However, the underlying mechanism and outcome for usage of NSAIDs is poorly understood. To investigate the damage and loss of osteoblast function in hypoxia, two hypoxia mimetics, cobalt chloride (CoCl₂) and desferrioxamine (DFO), were used to create an in vitro hypoxic microenvironment. The cell damage was observed by decreases of cell viability and increases in cyclooxygenase‐2 and cleaved poly(ADP‐ribose) polymerase (PARP). Cell apoptosis was confirmed by WST‐1 cytotoxic assays and flow cytometry. The functional expression of osteoblast in alkaline phosphatase (ALP) activity was significantly decreased by CoCl₂ and inhibited when treated with DFO. To simulate the use of NSAID after hypoxic injury, four types of anti‐inflammatory drugs, sulindac sulfide (SUL), indomethacin (IND), aspirin (Asp), and sodium salicylate (NaS), were applied to osteoblasts after 1 h of hypoxia mimetic treatment. SUL and IND further enhanced cell death after hypoxia. ALP activity was totally abolished in hypoxic osteoblasts under IND treatment. Facilitation of osteoblast apoptosis occurred regardless of IND dosage under hypoxic conditions. To investigate osteoblast in vivo, local hypoxia was created by fracture of tibia and then treated the injured mice with IND by oral feeding. IND‐induced osteoblast apoptosis was confirmed by positive staining of TUNEL assay in fractured mice. Significant delay of fracture healing in bone tissue was also observed with the treatment of IND. These results provide information pertaining to choosing appropriate anti‐inflammatory drugs for orthopedic patients. J. Cell. Biochem. 113: 148–155, 2012. © 2011 Wiley Periodicals, Inc.     

补骨脂素对TCP磨损颗粒所致大鼠成骨细胞损伤的干预作用及其机制

目的: 探讨补骨脂素(psoralen)对TCP磨损颗粒诱导成骨细胞损伤的影响及其分子机制。方法: 通过消化法从SD大鼠乳鼠颅骨中获取原代的成骨细胞,应用碱性磷酸酶(ALP)染色鉴定成骨细胞。TCP磨损颗粒(0.1 mg/ml)与成骨细胞共孵育48 h构建成骨细胞损伤的体外实验模型,实验随机分为正常对照组(Control)、模型(TCP)组和psoralen(10^-7 mol/L、10^-6 mol/L和10^-5 mol/L)组。WST法和流式细胞术分别检测各组成骨细胞活性变化和凋亡情况;化学比色法检测成骨细胞中ALP活性;各组成骨细胞培养14 d后应用茜素红S染色观察矿化结节形成。Western blot法检测各组成骨细胞中葡萄糖调节蛋白78/94(GRP78/94)、肌醇依赖酶1α(IRE1α)、剪切型X盒结合蛋白1(XBP1s)和磷酸化c-Jun氨基末端激酶(p-JNK)等蛋白质的表达。结果: 与Control组比较,TCP组成骨细胞活性、ALP活性和矿化结节的生成显著降低(P＜0.05),细胞凋亡率、GRP78/94、IRE1α、XBP1s和p-JNK等蛋白质表达明显增加(P＜0.05);与TCP组比较,补骨脂素各组成骨细胞损伤情况明显减轻,细胞凋亡率显著减少(P＜0.05),GRP78/94、IRE1α、XBP1s和p-JNK等蛋白质表达也明显下降(P＜0.05)。结论: 补骨脂素可抑制TCP磨损颗粒诱导的IRE1α-XBP1s-JNK信号通路的激活,阻止TCP颗粒所致的成骨细胞损伤及凋亡。     

葛根素对体外培养的人老年女性骨质疏松症成骨细胞分化的初步研究

目的:观察葛根素对体外培养老年女性骨质疏松症骨折患者体外分离培养的成骨细胞分化的影响。方法:采集老年女性骨质疏松症股骨颈骨折行人工股骨头置换术中取下的股骨颈松质骨,采用骨组织块法原代培养成骨细胞。观察不同浓度(0、0.01、0.1、1μmol/L)葛根素对成骨细胞表达碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OC)的影响,并比较各组成骨细胞钙化能力的大小。结果:0.01~1.00μmol/L的葛根素随着浓度增加,成骨细胞表达ALP、OC活性呈增强趋势,各浓度组两两比较差异均有统计学意义(P0.05);随葛根素浓度增高,成骨细胞钙化结节数量也明显增加,组间比较差异有统计学意义(P0.05)。结论:葛根素呈浓度依赖性促进成骨细胞表达ALP、OC并能提高成骨细胞的钙化能力。     

镁离子对成骨细胞活力和分化的促进作用及其机制研究

目的:研究不同浓度镁离子对成骨细胞活力和分化的影响,并探讨镁基生物材料促进骨再生的机制。方法:分离培养大鼠乳鼠颅骨成骨细胞,之后将细胞分别在DMEM培养基(含有0.8 m M镁离子;对照组)和含有6 m M、10 m M、18 m M镁离子(实验组)的培养基中进行培养,通过MTT法测定细胞活力,ALP活力、茜素红染色法测定成骨细胞的分化,通过western blot法测定不同浓度镁离子组中PI3K/Akt信号通路的表达情况。结果:6 m M、10 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平较对照组明显增加(P0.05),18 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平对照组明显降低(P0.05)。在10 m M镁离子组加入wortmannin后,上述增强的结果受到抑制。结论:6-10 m M镁离子促进成骨细胞的活力和分化,而过高浓度镁离子(18 m M)对成骨细胞的活力和分化具有抑制作用。10 m M镁离子通过激活PI3K/Akt信号通路促进成骨细胞的活力和分化。这项研究为医用镁基生物材料的进一步研究提供了很好的参考作用。     

Bisphosphonates modulate the effect of macrophage-like cells on osteoblast

Macrophages (MPs) are present in many tissues and have been implicated in the excessive bone resorption seen in patients with skeletal disorders. Our previous studies showed that macrophage-like cells influenced osteoblasts (OB) in co-culture, as number and activity of osteoblasts were decreased in co-cultures compared with controls. Macrophages are probable precursors of osteoblasts which have been shown to be inhibited by bisphosphonates (BPs). Bisphosphonates also modulate macrophage and osteoblasts activity. This study investigated whether addition of bisphosphonates to co-cultures of osteoblast and macrophages could reduce or block the adverse effects of macrophages on osteoblasts. The results showed that, compared to controls, fewer osteoblasts were present over time in macrophage/osteoblast co-cultures (at day 12, 15.5 x 10(4) and 8.8 x 10(4); P<0.0001) and that addition of bisphosphonates (10(-9)-10(-5)M) to the co-cultures prevented this reduction (P<0.001). Bisphosphonates also elicited an increase in numbers of osteoblast (82%) and restored alkaline phosphatase (ALP) activity, which was reduced by 15% (P approximately equal to 0.05) compared to control levels. The number of macrophages in co-cultures was reduced when bisphosphonates were added (P<0.001) and release of lactate dehydrogenase (LDH) was seen, which was not detectable in control cultures. It therefore, appears that bisphosphonates initiated macrophage death. These results demonstrated that the inhibitory effect of macrophages on osteoblasts in vitro could be overcome by the action of bisphosphonates. These findings have implications for the treatment of skeletal conditions where macrophage-derived cytokines are important, such as arthritis and implant loosening, although it is clearly important to distinguish between those bisphosphonates which enhance synthesis of pro-inflammatory cytokines and those which inhibit such synthesis.     

Prostaglandin E₂ increases both osteoblastic and osteoclastic activity in the scales and participates in calcium metabolism in goldfish

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E? (PGE?) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE?(10?? and 10?? M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE? (10?? to 10?? M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE? (10(-7) to 10?? M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE? treated-scales. Thus, PGE? acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE?-injencted goldfish increased significantly. We conclude that, in teleosts, PGE? activates both osteoblasts and osteoclasts and participates in calcium metabolism.     

Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2

BackgroundSome microRNAs (miRNAs) are involved in osteogenic differentiation. In recent years, increasing evidences have revealed that exosomes contain specific miRNAs. However, the effect and mechanism of miR-23a-5p-containing exosomes in osteoblast remain largely unclear.MethodsWe extracted exosomes from RANKL-induced RAW 264.7 cells, and identified exosomes via transmission electron microscopy, western blot and flow cytometry analysis. In addition, exosome secretion was inhibited by GW4869 and Rab27a siRNAs. miR-23a-5p expression was analyzed by qRT-PCR, and the related protein levels were examined by western blot assay. Furthermore, the number and distribution of osteoclasts were detected by TRAP staining, and early osteogenesis was evaluated by ALP staining. Combination of YAP1 and Runx2 was verified by Co-IP assay, and the regulation of miR-23a-5p and Runx2 was measured by dual luciferase reporter assay.ResultsWe successfully extracted exosomes from RANKL-induced RAW 264.7 cells, and successfully verified exosomes morphology. We also indicated that miR-23a-5p was highly expressed in exosomes from RANKL-induced RAW 264.7 cells, and osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity, while its inhibition weakened osteoclasts. In mechanism, we demonstrated that Runx2 was a target gene of miR-23a-5p, YAP interacted with Runx2, and YAP or Runx2 inhibited MT1DP expression. In addition, we proved that knockdown of MT1DP facilitated osteogenic differentiation by regulating FoxA1 and Runx2.ConclusionsWe demonstrated that osteoclast-derived miR-23a-5p-containing exosomes could efficiently suppress osteogenic differentiation by inhibiting Runx2 and promoting YAP1-mediated MT1DP. Therefore, we suggested miR-23a-5p in exosomes might provide a novel mechanism for osteoblast function.     

甲状旁腺素和胰岛素样生长因子促成骨样细胞ROS 17/2.8增殖分化中的协同作用

 间歇性小剂量地给予甲状旁腺素 (parathyroid hormone,PTH)可促进成骨 .胰岛素样生长因子 - I(insulin- like growth factor- I,IGF- I)由成骨细胞所产生并贮存于骨基质中 ,可促进成骨细胞的增殖分化 .为进一步了解向钙性激素和骨源性生长因子对骨生长的影响 ,利用成骨样细胞 ROS1 7/ 2 .8进行体外实验 ,观察了 PTH和 IGF- I这两种在骨生长和代谢中有重要作用的激素和因子相互作用的效果 ,并对其相互作用机制作出初步探讨 .结果显示 :联合使用 IGF- I及 PTH(间歇性给药 )时 ,(1 ) SRB(sodium rhodamine B,SRB)染色显示经 PTH(1 0 -9mol/ L,间歇给药 )和 IGF- I(1 0 -9mol/ L)联合处理的细胞 ,其数目明显增加 ,且明显高于单独处理组 ;(2 ) 3H- Td R参入增加 ,也明显高于单独处理组 ;(3)与增殖相关的原癌基因 (c- fos,c- jun,c- ki- ras)的表达增强 ,明显高于单独处理组 ;(4)骨钙素 (osteocalcin)基因 m RNA表达增强 ,明显高于单独处理组 ;(5) IGF- I(1 0 -8mol/L,1 0 -9mol/ L)可使 PTH受体基因 m RNA表达增强 .这些结果提示 PTH和 IGF- I在成骨样细胞ROS 1 7/ 2 .8增殖分化中具有协同作用 ,原癌基因的表达增强可能是其作用的一个环节 .此外 ,IGF- I可能通过增强 PTH受体表达 ,使细胞对 PTH的反应性增强     

Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements

Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.     

In vitro and in vivo effects of the overexpression of osteopontin on osteoblast differentiation using a recombinant adenoviral vector

Osteopontin (OPN) is a highly acidic secreted phosphoprotein that binds to cells via an RGD (arginine-glycine-aspartic acid) cell adhesion sequence that recognizes the alphaVbeta3 integrin. OPN may regulate the formation and remodeling of bone. To elucidate the function of OPN in bone tissue, we examined the overexpression of OPN in osteoblasts in vitro and in vivo using an adenoviral vector carrying an OPN cDNA (Adv-OPN). Rat bone marrow-derived osteoblasts infected with Adv-OPN were examined by Western blotting, immunofluorescence, nodule formation measurements, assay of alkaline phosphatase (ALP) activity, and Northern blotting. The results suggested that not only osteoblast differentiation markers such as osteocalcin and ALP, but nodule formation and ALP activity are markedly enhanced by OPN overexpression in the case of viral infection. On the contrary, when Adv-OPN and uninfected osteoblasts were implanted into subcutaneous sites with a porous ceramic scaffold, the ALP activity and calcium content of the OPN-infected composite were higher than in uninfected composites, however, the differences were smaller than expected from the in vitro experiments. We speculate that the difference in the result of in vitro and in vivo experiments originates from the inhibitory effect of secreted OPN on the crystal growth of apatite in vivo, which competes with the induced activity of osteoblasts.