Effects of erythropoietin on osteoblast proliferation and function

The purposes of this study were to investigate the effects of erythropoietin (EPO) on the proliferation and function of human osteoblast cells (hFOB 1.19) cultured in vitro and to explore the underlying molecular mechanisms to provide a theoretical foundation for clinical applications of EPO in oral implant and restoration therapies. Cultured hFOB 1.19 cells were treated with high and low doses of EPO. Changes in cell viability after 24 and 48 h of treatment were evaluated with the Mosmann tetrazolium assay. Changes in cell proliferation after 48 h of EPO treatment were measured by bromodeoxyuridine (BrdU) labeling, and changes in alkaline phosphatase (ALP) activity were determined by a specific assay. The effects of EPO on osteocalcin secretion were determined with the enzyme-linked immunosorbent assay, and changes in the protein expression of osteoprotegerin (OPG), osteopontin (OPN) and receptor activator of NF-κB ligand (RANKL) were assayed by western blot. The effects of EPO treatment on the levels of the EPO receptor (EPOR), phosphorylated Jak2 (P-Jak2) and phosphorylated Stat3 (P-Stat3) in hFOB 1.19 cells were evaluated in conjunction with a Jak2/Stat3 inhibitor. After 24 h of EPO treatment, hFOB 1.19 cells showed increased cell viability compared with the blank control group (p < 0.05). After 48 h, cell viability and growth were further improved relative to controls, with a significant increase observed for viability (p < 0.05). A significant increase in the proportion of BrdU-labeled proliferating cells was observed in the high-dose EPO group (p < 0.05), and EPO-treated cells also showed enhanced ALP activity (p < 0.05). There were no statistically significant differences in osteocalcin secretion between groups after 48 h of EPO treatment (p > 0.05); however, increased secretion was observed in EPO-treated cells after 96 h of treatment (p < 0.05). EPO treatment significantly promoted OPG and OPN expression (p < 0.05) while significantly inhibiting RANKL expression (p < 0.01). EPO treatment also significantly upregulated the levels of EPOR, P-Jak2 and P-Stat3 in hFOB 1.19 cells (p < 0.01); these effects were abrogated by co-treatment with a Jak2/Stat3 inhibitor (AG490) (p < 0.01). EPO significantly stimulated osteoblast proliferation and differentiation. The underlying molecular mechanism is associated with the ability of EPO to promote ALP activity, osteocalcin secretion and OPG and OPN expression and to inhibit RANKL expression in osteoblasts. This mechanism appears to be mediated by the Jak2/Stat3 pathway.     

Bone morphogenetic proteins 5 and 6 stimulate osteoclast generation

Bone regeneration is required for fracture-healing, and different procedures have been used to promote osteogenesis. Recently, BMP-2 has been shown to induce bone formation in vivo and has been tested in clinical trials. A recent in vitro study evaluated the osteogenic activity of 14 BMPs on osteoblastic progenitor cells with an osteogenic hierarchical model in which BMP-2 and BMP-6 may play an important role in inducing osteoblast differentiation. Although the relative osteoinductive activity of each BMP is important, bone regeneration is a process consisting of bone formation and bone resorption. Therefore, it remains unclear which effects BMP-5 and -6 have on the generation of osteoclasts and by which mechanism osteoclastogenesis is stimulated. To compare osteoclastic potency of each BMP, primary murine bone marrow cells were treated with human recombinant BMP-2, BMP-5, or BMP-6 and 1,25-(OH)2 vitamin D3 and stained for the TRAP enzyme. Osteogenic activity of BMP-5 was determined by measuring induction of ALP-activity and proliferation after incubation with primary murine osteoblasts. For elucidating the molecular mechanism, primary bone marrow cells with various concentrations of OPG were added to the TRAP assay and mRNA levels of RANKL and OPG were measured after stimulation with BMP-5. The presented data show that BMP-5 and BMP-6, unlike BMP-2, enhanced the formation of murine TRAP+/MNCs in a biphasic curve. BMP-5 and -6 were less potent in stimulating osteoclastogenesis compared to BMP-2. Concerning the effects of BMP-5 on osteoblasts, there was a dose-dependent increase of ALP activity and proliferation up to a maximum dose of 300 ng/mL. At the mRNA level, BMP-5 increased the RANKL/OPG ratio. In conclusion, this study demonstrates that in contrast to BMP-2, BMP-5 and -6 influences the generation of osteoclasts in a biphasic mode. Both proteins might be very important regulators of bone homeostasis, and therefore, potent candidates for future treatment strategies of bone regeneration.     

下调骨细胞TGF-β/Smad4信号可抑制小鼠BMSCs成骨及破骨分化

目的检测骨细胞TGF-β/Smad4信号通路对骨髓间充质干细胞(BMSCs)成骨和破骨分化的作用,并初步探讨其相关机制。方法用条件性基因敲减Cre/loxp技术特异性敲减骨细胞Smad4,获得下调骨细胞TGF-β/Smad4信号通路的小鼠;体外分离骨细胞并与野生型小鼠骨髓间充质细胞(BMSCs)共培养;碱性磷酸酶(ALP)染色、茜素红(alizarin red)染色检测早期成骨分化和晚期钙盐沉积,酸性磷酸酶(TRAP)染色检测破骨细胞;real-time PCR检测成骨分化特异标志物Runx2、Osterix(OSX)、ALP、osteocalcin和破骨分化特异标志物RANKL和OPG的mRNA表达水平。Western blot检测成骨分化特异标志物Runx2和osteocalcin和破骨分化特异标志物RANK蛋白表达水平。结果下调骨细胞TGF-β/Smad4信号能够抑制BMSCs成骨转录因子Runx2、Osterix(P0.01)、成骨分化特异标志物ALP和osteocalcin(P0.01)以及破骨分化特异标志物RANK(P0.01)的表达;增加破骨分化抑制物OPG的表达(P0.05);而RANKL的表达无明显变化;最终下调了RANKL/OPG的比值(P0.05)。结论终末分化的骨细胞调控骨的代谢,下调其TGF-β/Smad4信号可抑制BMSCs成骨和破骨细胞的分化。     

木通皂苷D通过丝裂原活化蛋白激酶信号通路促进大鼠骨髓间充质干细胞分化为成骨细胞

目的:探讨木通皂苷D(ASD)是否促进大鼠骨髓间充质干细胞(BMSCs)分化为成骨细胞及其机制。方法:分离培养大鼠BMSCs;观察ASD对其向成骨细胞分化的影响以及p38丝裂原激活蛋白激酶(p38MAPK)抑制剂SB203580和细胞外信号调节激酶(ERK)抑制剂PD098059的干预作用;检测BMSCs分化过程中碱性磷酸酶(ALP)活性和骨钙素(OC)含量;实时荧光定量PCR检测护骨素(OPG)和核因子κB受体活化因子配体(RANKL)mRNA的表达;Westernblotting法检测p38MAPK和ERK活性水平。结果:ASD处理后第9d,成骨性分化标志物OPGmRNA表达量明显增高,RANKLmRNA的表达量明显降低,同时显著提高BMSCs分化为成骨细胞的ALP活性和OC的表达,而且p38MAPK和ERK活性也显著增加。SB203580和PD098059则显著抑制ASD的成骨作用。结论:ASD在体外具有促进大鼠BMSCs向成骨细胞分化的作用,这一作用与MAPK途径的p38MAPK和ERK蛋白有关。     

A comparative analysis of the osteogenic capacity of osteoblasts from newborn and two-week-old rats

AimTo compare the proliferation and osteogenic differentiation of osteoblasts between newborn rats (1d group) and two-week-old rats (14d group) and to clarify the mechanism underlying these effects.MethodThe endogenous expression of osteogenic marker genes was detected by qPCR, including ALP, OCN, Col1a1, and Runx2. The osteoblasts proliferation was evaluated by EdU assay and Western Blotting [PCNA and Cyclin D1]. ALP activities in osteoblasts were detected using a PNPP kit, ALP staining and qPCR. Mineralized nodule formation and intracellular calcium levels were assessed by Alizarin Red staining and calcium colorimetric assay respectively while OCN, Col1a1 and Runx2 levels in osteoblasts were analyzed by immunostaining. Osteogenesis-associated pathways including Wnt/β-Catenin, Akt/PPAR and Smad were analyzed via Western Blotting.ResultEndogenous ALP, OCN, Col1a1, and Runx2 expression levels were significantly higher in osteoblasts from 14d group than those from 1d group. After treatment with osteogenic induction medium, osteoblast proliferation, ALP activity, mineralized nodule formation, and intracellular calcium levels were markedly increased in osteoblasts from 1d group, with similar results also being observed for the expression of OCN, Col1a1, and Runx2. Wnt3a, β-catenin, p-Akt, p-Smad1/5/8, and p-Smad5 protein levels were also higher in osteoblasts from 1d group relative to those from 14d group, while the expression of PPARγ was lower.ConclusionThe superior osteogenic differentiation capacity in osteoblasts was associated with the higher activation levels of Wnt/β-Catenin, Akt/PPAR and Smad signaling pathways, and the enhanced proliferative activity in osteoblasts from 1d group.     

三七总皂苷干预股骨头缺血性坏死兔成骨细胞护骨素基因的表达

背景：前期研究已明确三七总皂苷能抑制乙醇诱导的兔骨髓基质干细胞成脂分化。 目的：进一步观察三七总皂苷对兔成骨细胞的增殖和分化以及骨保护素、细胞核因子κB受体活化因子配体mRNA表达的影响。 方法：白酒灌胃4周制备新西兰兔股骨头缺血坏死模型,随机分为生理盐水组、复方骨肽组和三七总皂苷组。通过组织块培养法提取各组兔成骨细胞,检测细胞的分化,骨保护素、细胞核因子κB受体活化因子配体基因表达情况,检测各目的基因杂交信号的强弱。 结果与结论：三七总皂苷组兔碱性磷酸酶活性、钙结节计数及骨保护素、细胞核因子κB受体活化因子配体的表达强度、骨保护素mRNA/细胞核因子κB受体活化因子配体mRNA均高于生理盐水组(P < 0.01);与复方骨肽组效果接近。证实三七总皂苷能够促进兔成骨细胞的增殖和分化,并能提高成骨细胞的骨保护素mRNA的相对表达量而对其细胞核因子κB受体活化因子配体mRNA有抑制作用。     

Osteoprotegerin (OPG) Production by Cells in the Osteoblast Lineage is Regulated by Pulsed Electromagnetic Fields in Cultures Grown on Calcium Phosphate Substrates

Pulsed electromagnetic fields (PEMF) used clinically to stimulate bone formation enhance the osteogenic effects of BMP-2 on human mesenchymal stem cells (MSCs) if the MSCs are grown in osteogenic medium and are cultured on calcium phosphate (CaP) surfaces rather than tissue culture polystyrene plastic (TCPS). This study tested if PEMF’s effects on cells in the osteoblast lineage are substrate dependent and if factors produced by osteoblasts that regulate osteoclastic bone resorption, might also be regulated by PEMF. Human MSCs treated with BMP-2 and human osteoblast-like cells (normal human osteoblasts [NHOst cells], MG63 cells, SaOS-2 cells) were cultured on CaP or TCPS and their response to PEMF (4.5 ms bursts of 20 pulses repeating at 15 Hz for 8 h/day) determined as a function of decoy receptor osteoprotegerin (OPG) and RANK ligand (RANKL) production, both of which are associated with regulation of osteoclast differentiation. The results showed that when osteoblast-like cells were cultured on CaP, PEMF decreased cell number and increased production of paracrine factors associated with reduced bone resorption like OPG. RANKL was unaffected, indicating that the OPG/RANKL ratio was increased, further supporting a surface-dependent osteogenic effect of PEMF. Moreover, effects of estrogen were surface dependent and enhanced by PEMF, demonstrating that PEMF can modulate osteogenic responses to anabolic regulators of osteoblast function. These effects of PEMF would not be evident in models examining cells in traditional culture on plastic.     

低强度高频率振动对成骨细胞生物学特性的影响

目的　研究低强度高频率振动（low-magnitude high-frequency vibration, LMHFV）对成骨细胞生物学特性的影响。 方法　建立LMHFV加载MC3T3-E1细胞模型,观察不同频率LMHFV对MC3T3-E1细胞OPG/RANKL浓度比的影响,获得OPG/RANKL浓度比最高的频率（F）为后续研究频率;以0 Hz为对照,观察LMHFV对MC3T3-E1细胞碱性磷酸酶 (ALP)、骨钙素(OCN) mRNA和蛋白活性,及钙化结节形成的影响;LMHFV加载形成的条件培养液（CMF）孵育RAW264.7细胞,观察CMF对破骨细胞抗酒石酸酸性磷酸酶（TRAP）染色、多核破骨细胞形成、TRAP mRNA及蛋白活性的影响;观察LMHFV对MC3T3-E1细胞环氧化酶2(COX-2)蛋白水平的表达及COX-2抑制剂NS-398对LMHFV影响MC3T3-E1细胞分化的作用。 结果　30 Hz LMHFV获得OPG/RANKL浓度比最高,促进ALP、OCN mRNA及蛋白活性增加,增加钙化结节形成。30 Hz LMHFV形成的CM抑制RAW264.7细胞向多核破骨细胞分化,抑制TRAP mRNA及活性;LMHFV可诱导COX-2蛋白水平增加,NS-398能抑制LMHFV促进成骨细胞分化。 结论　30 Hz的LMHFV对MC3T3-E1细胞OPG/RANKL浓度比及成骨分化具有积极的影响,通过调控成骨细胞OPG/RANKL浓度比间接抑制骨吸收,COX-2通路参与了LMHFV对成骨细胞生物学特性的调节作用。     

淫羊藿苷对大鼠血管平滑肌细胞钙化的抑制作用

目的:研究淫羊藿苷(Icariin)对大鼠血管平滑肌细胞(VSMCs)钙化的抑制作用。方法:组织贴壁法培养大鼠胸主动脉VSMCs,取5-8代细胞分为对照组、钙化模型组和淫羊藿苷干预A、B、C组。对照组用DMEM培养基培养;钙化模型组用含10mmol/Lβ-甘油磷酸盐(β-GP)的DMEM培养基培养;干预A、B、C组均以钙化模型组为基础分别与10-6 mol/L、10-5 mol/L和10-4 mol/L Icariin的DMEM培养基共培养,茜素红-S染色法鉴定各组细胞钙化情况,硝基苯酚法测定各组细胞碱性磷酸酶(ALP)活性,RT-PCR检测各组细胞内骨保护素(OPG)和核因子κB(NF-κB)受体活化因子配体(RANKL)的mRNA水平,用Western blotting检测各组细胞OPG和RANKL的蛋白表达。结果:与对照组比较,钙化模型组VSMCs发生细胞钙化,并形成红色结节,ALP活性显著升高(P0.01),OPG、RANKL mRNA和蛋白表达均增加(P0.01);与钙化模型组比较,干预A、B、C组细胞钙化减少,ALP活性减低(P0.01),OPG、RANKL mRNA和蛋白表达均下调(P0.01),尤以干预B组效果最显著。结论:淫羊藿苷可能通过降低ALP活性及OPG、RANKL表达来抑制动脉钙化。     

雷奈酸锶通过上调骨形态发生蛋白2的表达促进骨髓间充质干细胞分化为成骨细胞

目的: 探讨雷奈酸锶(strontium ranelate,Sr)是否可以通过上调骨形态发生蛋白 2(bone morphogenetic protein 2,BMP-2)的表达而促进大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分化为成骨细胞。方法: 对大鼠BMSCs进行分离、纯化、培养及向成骨细胞的定向诱导分化,并在诱导培养时,根据实验目的加入不同浓度Sr以及BMP-2的拮抗剂noggin。用酶标法检测成骨细胞分化和功能成熟的早期标志物——碱性磷酸酶(alkaline phosphatase,ALP)活性的变化,用茜素红染色检测细胞钙化水平,用Western blotting法检测BMP-2蛋白的表达水平。结果: 应用0.1~7 mmol/L Sr处理细胞7 d均可使细胞ALP活性显著增加,其中浓度为3 mmol/L时效果最显著;应用3 mmol/L Sr处理细胞21 d可使细胞钙化结节明显增多;应用0.1~7 mmol/L Sr处理细胞7 d后细胞内BMP-2蛋白水平明显增高;在Sr处理BMSCs前,应用BMP-2拮抗剂noggin预处理细胞2 h不仅抑制Sr对BMP-2表达的上调作用,还拮抗Sr对ALP活性及钙化结节形成的促进作用。结论: 上调BMP-2的表达可能是Sr促进大鼠BMSCs分化为成骨细胞的作用机制之一。     

脂肪间充质干细胞成骨分化条件培养基联合骨形态发生蛋白2治疗去卵巢大鼠骨质疏松症

文题释义： 骨质疏松症：是由多种原因引起的老年性骨病。通常由于骨量下降、骨的细微结构发生改变,导致骨脆性增加,易发生骨折,好发于胸、腰椎椎体、桡骨远端及股骨上端等。骨质疏松可发生在任何年龄和任何性别,但通常多发生于绝经后妇女和老年男性。 RANKL/OPG：骨重建需要骨形成和骨吸收之间的精确平衡,RANKL/OPG在骨重建中起着至关重要的作用。RANKL是一种细胞因子,可诱导祖细胞分化为成熟的破骨细胞。OPG通过与RANKL结合而成为诱骗受体。因此,RANKL/OPG的比例是维持骨形成和骨吸收之间平衡的关键。 背景：脂肪间充质干细胞分泌各种骨重塑需要的细胞因子和生长因子,被认为是骨再生的优良候选细胞。骨形态发生蛋白2与脂肪间充质干细胞对骨再生有协同作用,并能显著增强脂肪间充质干细胞的成骨分化作用。 目的：探讨脂肪间充质干细胞成骨分化条件培养基与骨形态发生蛋白2联合应用对大鼠绝经后骨质疏松症的影响。 方法：8-10月龄雌性SD大鼠75只,随机取60只采用卵巢切除方法建立绝经后骨质疏松症大鼠模型,余15只进行假手术,未切除卵巢。将60只造模成功大鼠随机分为4组：骨质疏松症组、条件培养基组、骨形态发生蛋白2组、联合治疗组,通过尾静脉分别注射DMEM培养基、脂肪间充质干细胞成骨分化条件培养基、骨形态发生蛋白2、脂肪间充质干细胞成骨分化条件培养基联合骨形态发生蛋白2。治疗12周,取各组大鼠股骨和血清,组织学观察骨小梁数量和结构以及骨小梁间距,ELISA检测血清P1NP、ALP、TRAP、OPG、RANKL水平,Western blot、Realtime PCR检测RANKL、OPG蛋白和mRNA水平,细胞因子芯片分析脂肪间充质干细胞成骨分化条件培养基中细胞因子水平。 结果与结论：①与假手术组相比,骨质疏松症组大鼠的骨小梁间距扩大,骨小梁数量明显减少,骨小梁失去正常结构并且不连续。与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组大鼠骨小梁间距扩大较少,骨小梁结构更完整、更连续;②与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组大鼠血清P1NP和ALP水平显著升高(P < 0.05),血清TRAP水平显著降低(P < 0.05);③与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组RANKL/OPG比值显著降低(P < 0.01),从而促进更多的骨形成;④脂肪间充质干细胞成骨分化条件培养基含有多种与骨形成密不可分的细胞因子,包括骨形态发生蛋白4和7、白血病抑制因子、脑源性神经营养因子、骨保护素、胰岛素样生长因子1等;⑤结果表明,脂肪间充质干细胞成骨分化条件培养基与骨形态发生蛋白2联合应用可减轻卵巢切除大鼠骨质疏松,可能成为治疗绝经后骨质疏松症的新方案。 ORCID: 0000-0003-0552-4818(杨九杰) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

假体磨损颗粒钴铬离子影响成骨细胞增殖及RANKL、骨保护素的表达

背景：金属-金属假体置入体内后可以发生腐蚀或磨损,释放镍、钴、铬、钛等金属离子,诱导局部炎性因子的释放。 目的：观察Co²⁺、Cr³⁺对小鼠成骨细胞增殖的影响,以及成骨细胞暴露在Co²⁺ 、Cr³⁺条件下RANKL、骨保护素基因的表达。 方法：体外培养成骨细胞,实验分两组,对照组给予生理盐水,离子组给予钴、铬离子干预。 结果与结论：显微镜直接计数法显示干预后1~6 d对照组随时间推移细胞数目显著增加,离子组则增加不明显。共培养24,48 h后,RT-PCR结果显示离子组RANKL、骨保护素基因表达较对照组均增加,以RANKL增加更显著(P < 0.05),RANKL/OPG mRNA的比率也明显增加。提示金属离子对成骨细胞的增殖有显著抑制作用,且可刺激成骨细胞RANKL、骨保护素mRNA的表达。     

The control of bone growth by parathyroid hormone,leptin, & statins

There is a need for anabolic drugs that can stimulate bone growth, improve bone microarchitecture, accelerate fracture healing and thus restore bone strength to oteoporotics. The anabolic agents currently leading the way to the clinic are the parathroid hormone (PTH) and some of its adenylyl cyclase-stimulating fragments. Here we discuss what is known about the genes and their products that are stimulated by PTHR1 receptor signals and in four ways cause a large accumulation of bone-building osteoblasts. We will also discuss the currently controversial anabolic activity of the cholesterol-lowering statins and outline a possible mechanism by which they might stimulate BMP-2 expession and bone growth. Finally, we will present the growing evidence for the body's "fat-o-stat" cytokine-leptin-indirectly restraining bone growth via a hypothalamic factor and at the same serving as a local autocrine/paracrine stimulator of osteoblast activity via IGF-I and an inhibitor of osteoclast generation by stimulating osteoblastic cells' antiosteoclast OPG (osteoprotegerin) expression and reducing their proosteoclast RANKL expression.     

抗菌钛合金Ti6Al4V-6Cu表面构建成骨细胞膜片的实验研究

目的:探讨大鼠颌骨成骨细胞在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片的可行性。方法:体外培养大鼠颌骨成骨细胞,采用富含维生素C培养基在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片（细胞膜片组）,并以单纯培养基作对照（对照组）,检测膜片形成过程中碱性磷酸酶（ALP）和成骨相关基因ALP、I型胶原（Col-1）、骨形成蛋白2（BMP-2）的表达情况。结果:采用富含维生素C的培养基连续培养可在抗菌钛合金Ti6Al4V-6Cu表面成功构建成骨细胞膜片,该细胞膜片由多层细胞构成,富含胞外基质。相对于对照组,细胞膜片组的膜片形成过程中成骨细胞ALP活性及成骨相关基因ALP、Col-1、BMP-2的表达均显著增高。结论:在抗菌钛合金Ti6Al4V-6Cu表面可成功构建成骨细胞膜片,有望与Ti6Al4V-6Cu联合应用于引导性骨再生术。     

A role for the immunomodulatory molecules CD200 and CD200R in regulating bone formation

Altered osteoprotogerin (OPG) and OPG ligand (RANKL) ratios are known to regulate bone metabolism. We investigated whether CD200:CD200R interaction would alter OPG:RANKL ratios, and thus modulate bone differentiation in cultures derived from neonatal calvariae, a source of osteoblast precursors (OBp), or bone marrow-derived myeloid cells as a source of osteoclast precursors (OCp). We characterized cells in cultures using real-time PCR to measure expression of a number of mRNAs characteristic of cells differentiating towards the osteoblast or osteoclast lineage, and enumerated bone nodule formation and osteoclasts directly. CD200Fc or anti-CD200 mAbs were included as modulating agents. In addition, calvariae from transgenic mice overexpressing CD200 under control of a doxycycline-inducible promoter were used as a source of OBp endogenously overexpressing CD200. Our data show that increased endogenous expression of CD200 on OBp, or addition of CD200Fc into cultures, led to increased OPG:RANKL ratios and increased bone nodule growth, while anti-CD200 abolished this effect.     

鼠骨髓基质干细胞的特性及向成骨细胞的分化

目的分离纯化成年小鼠骨髓基质干细胞并进行定向诱导。方法利用Percoll液分离成年小鼠骨髓基质干细胞,Ter119、CD45磁珠纯化细胞,培养2-3代的细胞用矿化液进行定向诱导。对诱导前后的细胞进行免疫组化染色。结果 镜下见原代细胞呈多种形态。Ter119、CD45磁珠筛选可获得纯度达57.95％以上骨髓基质干细胞。碱性磷酸酶、Von Kossa染色阳性,油红染色弱阳性,矿化液诱导2周后,可形成钙结节。结论利用Percoll液结合磁珠分离法是离体条件下获得骨髓基质干细胞的一个简便有效的方法,纯化后定向诱导可得到较纯的成骨细胞。     

枸橼酸铁铵通过提高ROS水平激活MAPK通路并诱导成骨细胞凋亡

 目的：探讨铁超载提高人成骨细胞(hFOB1.19)活性氧（ROS）水平在激活丝裂原活化蛋白激酶（MAPK）通路和诱导细胞凋亡中的作用。方法：采用细胞贴壁法培养成骨细胞hFOB1.19,将不同浓度枸橼酸铁铵(100、300、500 μmol/L)加入细胞培养基,用MTT法检测成骨细胞增殖活性;DCFH-DA荧光探针检测成骨细胞ROS水平;Annexin V-FITC/PI法检测细胞凋亡;Western blotting检测MAPK通路相关蛋白。结果：枸橼酸铁铵处理成骨细胞后,细胞增殖活性明显降低,早期凋亡和总死亡率显著增加;不同浓度的枸橼酸铁铵处理成骨细胞后,其ROS水平分别增高至(35.73±2.52)%、(62.89±4.24)%和(76.06±3.55)%,MAPK通路相关蛋白的表达也明显增加并呈剂量效应关系,抗氧化剂N-乙酰半胱氨酸可以在降低ROS水平及MAPK通路相关蛋白表达的同时抑制枸橼酸铁铵所致的细胞凋亡。结论：枸橼酸铁铵能使成骨细胞增殖受到抑制,并且通过增加细胞内ROS水平,激活MAPK通路,诱导成骨细胞凋亡。     

Sol-gel bioactive glasses support both osteoblast and osteoclast formation from human bone marrow cells

We have examined the ability of bioactive sol-gel glass ceramics to support both osteoblast and osteoclast differentiation from human bone marrow cells (HBMC). Nucleated cells from human bone marrow were cultured on tissue culture plastic and on two sol-gel coatings: A2 glass-ceramic containing 54 mol % CaO/40 mol % SiO(2) and S2 glass-ceramic containing 16 mol % CaO/80 mol % SiO(2). Osteoblast differentiation was followed by measuring alkaline phosphatase (ALP) activity, mRNA levels for ALP, osteopontin, RANK ligand (RANKL), and immunofluorescent co-localization of ALP and RANKL. Osteoclasts were identified by morphology and positive staining for tartrate-resistant acid phosphatase (TRAP). ALP activity and mRNA levels were similar for cells on A2 coatings and on tissue culture plastic, but mRNA levels of osteopontin and RANKL were tenfold higher on A2 than on plastic. Cultures on A2 coatings also contained multinucleated osteoclasts staining positively for TRAP. In contrast, cells cultured on S2 coatings had the characteristics of more differentiated osteoblasts as measured by higher ALP expression. However, the levels of osteopontin and RANKL mRNA on S2 glass were lower than on A2 glass and there were fewer, weakly staining TRAP-positive multinucleate cells. Thus, sol-gel glass-ceramic materials differing in CaO/SiO(2) ratios can produce markedly different effects on the osteoblast and osteoclast differentiation from HBMC.