成骨细胞与破骨细胞间接共培养研究进展

在骨代谢过程中,成骨细胞形成新骨,破骨细胞吸收旧骨,一旦成骨细胞介导的骨基质形成和破骨细胞介导的骨吸收失衡,则会导致骨质疏松症等危害人类健康的疾病产生。因此,不同研究者致力于开发模拟体内环境的成骨细胞与破骨细胞体外共培养模型,以进行骨代谢相关疾病的研究。间接式共培养是通过物理方式将成骨细胞与破骨细胞分隔,使二者可以进行细胞间的交流而不接触,可以针对单一的细胞进行分析,在药物筛选及研究方面,具有高通量和经济便捷等独特的优势,本文对成骨细胞和破骨细胞的间接共培养技术进行归纳和总结。     

共育体系中成骨细胞和破骨细胞生物学特性观察

目的建立成骨细胞和破骨细胞的体外共育体系,观察在此体系中成骨细胞和破骨细胞生物学特性的变化,探讨成骨细胞和破骨细胞间的相互作用。方法取髂骨松质骨,Ⅱ型胶原酶消化,分次获得成骨细胞和破骨细胞。建立培养上清相通但二者互不接触的成骨细胞-破骨细胞共育模型。以细胞增殖(MTT法)、碱性磷酸酶(ALP)活性代表成骨细胞的成骨活性,以抗酒石酸酸性磷酸酶(TRAP)活性、骨吸收陷窝面积代表破骨细胞的破骨能力,检测共育对成骨细胞和破骨细胞生物学特性的影响。结果成骨细胞呈饱满的梭形,ALP染色阳性;破骨细胞呈多核,TRAP染色阳性,可以吸收骨质形成骨陷窝。当成骨细胞与破骨细胞共育后,其MTT法OD值(0.60±0.08)较单独培养时(0.36±0.03)明显提高(P=0.000);其ALP活性(23.37±2.48)u/mg较单独培养时(18.33±0.34)u/mg明显提高(P=0.000)。破骨细胞与成骨细胞共育后,形成骨吸收陷窝的平均面积犤(6.55±0.34)×10-2犦μm2较单独培养时犤(5.15±0.17)×10-2犦μm2明显增大(P=0.000)。结论共育体系中成骨细胞和破骨细胞的功能相互促进,为骨组织代谢的体外研究提供了可靠的模型。     

血小板衍生生长因子-BB抑制成骨细胞-破骨细胞共育体系中破骨细胞的作用机制

目的　探讨血PDGF BB在共育体系中抑制破骨细胞 (OC)功能的机制。方法　将PDGF BB施加于两种培养体系。测定培养上清一氧化氮 (NO)产物的浓度 ;检测诱导型和内皮型一氧化氮合酶表达情况 ;检测共育体系内外骨保护素 (OPG )表达与PDGF BB浓度的关系 ;观察共育体系培养上清对OC吸附骨质的影响。结果　在PDGF BB刺激下 ,NO含量升高至 (2 5 .2 6±3 .2 4) μg/L (P <0 .0 1) ;加入抑制剂后 ,NO含量为 (9.84± 1.5 1) μg/L ;PDGF BB促进成骨细胞(OB)对eNOS和iNOS的表达 ,抑制剂使二者表达减弱 ;共育体系中OPG表达增强 ,而PDGF BB不能直接促进OPG的表达。共育体系培养上清使OC从骨质上脱离。结论　PDGF BB促进OB产生NO产物 ,后者抑制OC功能。     

健骨方对破骨细胞形成和成骨细胞增殖分化的影响

目的　探讨健骨方水提物对破骨细胞分化及成骨细胞增殖分化的影响。方法　制备健骨方水提物,通过MTT法测定药物对骨髓单核巨噬细胞（BMMs）细胞的毒性,采用核因子κB受体活化因子配体（RANKL）诱导BMMs分化形成破骨细胞,加入不同浓度药物进行干预,采用抗酒石酸酸性磷酸酶( TRACP) 染色法测定破骨细胞分化抑制作用,采用Western Blot 法测定RANKL诱导的NF-κB破骨细胞分化信号通路,运用RT-qPCR法测定信号通路下游破骨细胞分化关键基因NFATc1、C-FOS等的mRNA表达水平。以MC3T3-E1细胞作为前体成骨细胞,加入不同浓度药物进行干预,通过CCK8法测定细胞增殖能力、PNPP法检测碱性磷酸酶（ALP）活性、茜素红S染色法测定细胞矿化能力。结果　MTT法结果显示,健骨方细胞有毒性浓度大于500 μg/mL（P＜0.05）,破骨细胞分化抑制IC50为1.25 μg/mL。机制研究显示健骨方显著下调了RANKL-NF-κB信号通路中的p-P65、P53的蛋白表达（P＜0.05）,显著抑制了通路下游C-FOS、NFATc1等的mRNA表达水平（P＜0.01,P＜0.05）。此外,成骨细胞活性检测显示,健骨方能明显促进MC3T3-E1细胞增殖、提高ALP活性及增加成骨细胞钙化的能力。结论 健骨方具有抑制破骨细胞分化和促进成骨前体细胞增殖、分化、矿化的药效作用。其作用与抑制破骨细胞分化RANKL-NF-κB信号通路及其下游C-FOS、NFATc1等基因,上调成骨细胞分化促进因子CAL1A2、SPARC和FOSL1基因的表达有关。     

骨细胞对破骨细胞形成和功能的影响

破骨细胞的骨吸收作用和成骨细胞骨形成作用的交替进行维持了骨量的平衡。破骨细胞可以选择性吸收损伤部位的骨质,其激活和定位机制目前还未阐明。近年来的研究认为骨细胞是感知骨环境的基本单位,而且骨细胞还可以将所感知的信号传递给其它骨细胞,骨衬细胞,成骨细胞及破骨细胞等。对骨细胞和破骨细胞的研究中发现骨细胞可能在破骨细胞的激活和定位中起到了重要的作用,但是具体机制还有待研究。     

TZDs对骨髓破骨细胞生成作用和 成骨细胞生成作用的影响

[目的]体外观察TZDs类药物对小鼠原代骨髓细胞向成骨细胞和破骨细胞分化的影响,探讨其对糖尿病患者骨骼代谢的影响和作用机制.[方法]无菌条件下从小鼠股骨分离获取骨髓细胞,贴壁细胞进行成骨细胞生成实验研究,悬浮细胞进行破骨细胞生成实验研究.随后在1、2、5μmol/L罗格列酮干预下诱导成骨细胞分化培养3周,进行VonKossa染色观察成骨细胞矿化结节,并测定成骨细胞标记物ALP活性、BMP-2及TGF-β分泌的影响;观察罗格列酮对破骨细胞形成和成熟的影响及标志性基因表达.[结果]1、2、5μmol/L罗格列酮干预组与对照组相比,成骨细胞的钙结节形成比例显著降低(P＜0.05),ALP、BMP-2、TGF-β水平呈剂量依赖性地下降(均P＜0.05);同时剂量依赖性促进c-fos和RANK基因的表达,破骨细胞生成实验显示罗格列酮促进破骨细胞分化发育和成熟.[结论]罗格列酮可剂量依赖性地抑制骨髓细胞向成骨细胞分化,促进向破骨细胞分化.有助于理解Ⅱ型糖尿病患者在应用TZDs骨丢失的原因.     

破骨细胞参与时IGF-1对成骨细胞的促同化作用

目的探讨IGF-1对成骨细胞(osteoblast,OB)的促同化作用是否需要破骨细胞(Osteoclast,OC)的协同。方法体外培养MC3T3小鼠成骨细胞及RAW264.7小鼠单核巨噬细胞,RAW264.7细胞经核因子κB受体活化因子配基(Receptor activator of nuclear factor kappa-B ligand,RANKL)诱导分化为破骨细胞。以50 ng/ml重组人胰岛素样生长因子-1(rhIGF-1)分别干预成骨细胞及分化成熟的破骨细胞,Wstern blotting验证IGF-1受体的活化。以0、10、50、100 ng/ml的rhIGF-1分别干预成骨细胞、破骨细胞及共培养的成、破骨细胞。12 h后终止培养,收集破骨细胞经IGF-1干预后的条件培养基(OC conditioned medium after IGF-1 treatment,OCCM)对另一组新接种的成骨细胞干预12 h。ELISA检测3组成骨细胞培基中骨钙素(bone Gla protein,BGP)、RANKL的含量,Real-time PCR检测成骨细胞中Bgp基因的表达。结果 RANKL诱导培养8 d后RAW264.7细胞形成TRAP阳性,成熟的多核破骨细胞;Wstern blotting检测表明rhIGF-1可有效得使成、破骨细胞中的IGF-1受体发生磷酸化;ELISA与Real-time PCR测定结果显示,IGF-1直接干预OB时,Bgp基因表达水平和培养基中BGP及RANKL蛋白的含量均与无干预的对照组无明显区别;而OCCM干预及共培养的OB组,当IGF-1初始干预浓度为10 ng/ml时BGP、RANKL含量及Bgp基因的表达水平显著提高,共培养组与OCCM培养组间无明显区别。结论 IGF-1对OB的促同化作用依赖于OC的参与,两种细胞间的联系可能是通过可溶性的细胞因子来完成。     

脉冲电磁场对大鼠破骨细胞功能分子的影响

目的研究脉冲电磁场(puslsed electromagnetic fields,PEMFs)对大鼠破骨细胞的作用。方法取10w龄雌性SD大鼠随机分为三组:PEMFs组、Control组和Sham组;其中PEMFs组、Control组进行双侧卵巢切除手术,Sham组不切除卵巢。术后12w对PEMFs组大鼠进行PEMFs作用(70Hz,2mT),Control组和Sham组不进行PEMFs作用。作用结束后,取大鼠股骨骨髓,进行体外破骨细胞诱导培养。7天后分别进行破骨细胞的抗酒石酸酸性磷酸酶(Tartrate-resistant acid phosphatase,TRAP)检测和整合素αvβ3、NF-κB受体激活子(receptor activator of NF-κB,RANK)、组织蛋白酶K(cathepsin K)免疫荧光染色检测。结果经PEMFs作用后,TRAP检测显示破骨细胞形态;免疫荧光染色显示阳性破骨细胞也明显减少。结论 PEMFs对破骨细胞功能分子的表达有抑制作用,提示PEMFs作用可以抑制SD大鼠破骨细胞的骨吸收活性。     

雌激素通过调节骨髓来源的成骨细胞所产生的细胞因子抑制破骨细胞功能

目的：通过观察雌激素对骨髓来源的成骨细胞所产生的细胞因子的调节作用，探讨雌激素抑制破骨细胞功能的机制。方法：在诱导小鼠骨髓细胞分化为成骨细胞后，于成骨细胞培养中加入雌激素，应用抗体荧光免疫标记法检测成骨细胞培养液中IL－1、IL－6和TNF－α水平。结果：与对照组比较，经0．01－1nM雌激素处理后， 成骨细胞所产生的IL－1和IL－6水平呈浓度依赖性下降，TNF－α水平则无明显变化。结论：雌激素具有调节成骨细胞分泌细胞因子功能，提示雌激素抑制破骨细胞作用机制源自调控成骨细胞旁分泌。     

源于脾细胞的破骨细胞样细胞体外培养及其对成骨细胞的影响

目的探讨破骨细胞及其亚细胞结构对成骨细胞生长的影响。方法C57雌性小鼠,经尾静脉注射5-FU后,取其脾脏细胞,在白介素(IL)-3,6和粒细胞-巨噬细胞集落刺激因子(GM-CSF)、1α,25-(OH)2D3的诱导下获得大量的破骨样细胞(OLC)。将破骨样细胞、NaF、离心去除OLC的培养基及其亚细胞结构——细胞核、线粒体与成骨细胞共培养5d后,检测成骨细胞增殖率和碱性磷酸酶含量以及成骨细胞Cbfα1的表达活性。结果OLC及离心去除OLC的50%培养基均可使成骨细胞增殖率显著增加(P<0.05)。NaF、OLC、OLC细胞核和离心去除OLC的25%培养基均可使成骨细胞的碱性磷酸酶活性增高(P<0.05);离心去除OLC的培养基对成骨细胞的碱性磷酸酶比活性具有显著的促进作用(P<0.05)。NaF、OLC细胞质和离心去除OLC的50%培养基均可使成骨细胞的Cbfα1的表达明显加强(P<0.05)。结论OLC对成骨细胞的生长和功能均有促进作用。     

Effects of pulsed electromagnetic fields on benign prostate hyperplasia

Introduction  

Benign prostate hyperplasia (BPH) has been treated with various types of electromagnetic radiation methods such as transurethral needle ablation (TUNA), interstitial laser therapy (ILC), holmium laser resection (HoLRP). In the present study, the effects of a noninvasive method based on the exposure of patients with BPH to a pulsative EM Field at radiofrequencies have been investigated.     

Effects of pulsed electromagnetic fields on Steinberg ratings of femoral head osteonecrosis

Between 1979 and 1985, 95 patients with femoral head osteonecrosis met the protocol for treatment of 118 hips with selected pulsed electromagnetic fields (PEMFs). Etiologies included trauma (17), alcohol (9), steroid use (46), sickle cell disease (2), and idiopathy (44). The average age was 38 years, and the average follow-up period since the onset of symptoms was 5.3 years. PEMF treatment had been instituted an average of 4.1 years earlier. By the Steinberg quantitative staging method of roentgenographic analysis, none of the 15 hips in Stages 0-III showed progression, and grading improved in nine of 15. Eighteen of 79 hips (23%) with Stage IV lesions progressed and none improved. In the Stage V category, one of 21 hips (5%) worsened and none improved. Three Stage VI lesions were unchanged. The overall rate of quantified progression for the 118 hips, 87% of which had collapse present when entering the program, was 16%. This value represents a reversal of the percentage of progression reported recently by other investigators using conservative and selected surgical methods. PEMF patients also have experienced long-term improvements in symptoms and signs, together with a reduction in the need for early joint arthroplasty.     

Microcirculatory effects of pulsed electromagnetic fields

Purpose: Pulsed electromagnetic fields (PEMF) are used clinically to expedite healing of fracture non‐unions, however, the mechanism of action by which PEMF stimulation is effective is unknown. The current study examined the acute effects of PEMF stimulation on arteriolar microvessel diameters in the rat cremaster muscle. The study hypothesis was that PEMF would increase arteriolar diameters, a potential mechanism involved in the healing process. Methods: Local PEMF stimulation/sham stimulation of 2 or 60 min duration was delivered to the cremaster muscle of anesthetized rats. Arteriolar diameters were measured before and after stimulation/sham stimulation using intravital microscopy. Systemic hemodynamics also were monitored during PEMF stimulation. Results: Local PEMF stimulation produced significant (p < 0.001) vasodilation, compared to pre‐stimulation values, in cremasteric arterioles in anesthetized rats (n = 24). This dilation occurred after 2 min of stimulation (9% diameter increase) and after 1 h of stimulation (8.7% diameter increase). Rats receiving “sham” stimulation (n = 15) demonstrated no statistically significant change in arteriolar diameter following either “sham” stimulation period. PEMF stimulation of the cremaster (n = 4 rats) did not affect systemic arterial pressure or heart rate, nor was it associated with a change in tissue environmental temperature. Conclusions: These results support the hypothesis that local application of a specific PEMF waveform can elicit significant arteriolar vasodilation. Systemic hemodynamics and environmental temperature could not account for the observed microvascular responses. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.     

结合磁场对成骨细胞的促增殖作用

目的观察脉冲与静磁场结合的磁场对成骨细胞体外生长的影响.方法用胶原酶阶梯消化法体外分离、培养成骨细胞,随机取培养板2块,1块作为对照,在另1块培养板两侧放置脉冲与静磁场组合的磁场,静磁场强500 GS,脉冲峰值65 mV,频率75 Hz,作用24 h,用酶联免疫法检测磁场对成骨细胞增殖的影响.结果实验组A值为0.276 4±0.003 9;对照组A值为0.197 2±0.002 7,两者差异有非常显著性(P＜0.01).结论采用脉冲磁场与静磁场结合的方式,对成骨细胞有显著的促增殖作用.     

Effects of pulsed electromagnetic fields on cartilage apoptosis signalling pathways in ovariectomised rats

Purpose  

The purpose of this study was to determine the effect of exposure to pulsed electromagnetic fields (PEMF) on modulation of the cartilage apoptosis signalling pathway in ovariectomised rats by monitoring the expression of mRNA of X-linked inhibitor of apoptosis protein (XIAP) and Bax.     

脉冲电磁场对大鼠成骨细胞增殖影响的活细胞成像观察研究

目的应用活细胞成像技术观察脉冲电磁场作用下大鼠成骨细胞的生长、增殖过程,探讨不同脉冲电磁场强度对大鼠成骨细胞生长、增殖的影响。方法首先采用MTT法检测在15Hz,0.1、0.3、0.5、1.0mT脉冲电磁场连续刺激下,4种脉冲电磁场强度组与对照组的成骨细胞存活与增殖能力,筛选出最佳的脉冲电磁场刺激强度。根据筛选结果,选用频率15Hz、强度0.1mT的脉冲电磁场,应用活细胞成像技术,以Time-Lapse方式,对脉冲电磁场刺激组与对照组成骨细胞的生长、增殖过程,作多点平行对照记录,动态观察两组成骨细胞的贴壁速率与分裂速率。并采用MTT法同步检测两组细胞的增殖情况。结果原代培养3d的成骨细胞,在15Hz脉冲电磁场中连续刺激3d,经MTT法检测,1.0mT组细胞几乎全部死亡,0.5mT组与对照组比较细胞增殖能力无明显差别,0.3、0.1mT组与对照组比较细胞增殖能力增强(P0.05、P0.01)。活细胞成像Time-Lapse平行对照记录,动态观察分析表明,15Hz、0.1mT脉冲电磁场组观察野的细胞贴壁速率与对照组比较无明显差别,分裂速率较对照组观察野增快(P0.05),MTT法同步检测也证明0.1mT脉冲电磁场刺激组的细胞增殖能力较对照组增强。结论 15Hz、0.1mT脉冲电磁场对体外培养的大鼠成骨细胞增殖具有促进作用,但强度超过0.5mT的脉冲电磁场对成骨细胞增殖无明显促进作用,如果强度≥1mT反而会导致成骨细胞的死亡。     

Prevention of osteoporosis by pulsed electromagnetic fields

Using an animal model, we examined the use of pulsed electromagnetic fields, induced at a physiological frequency and intensity, to prevent the osteoporosis that is concomitant with disuse. By protecting the left ulnae of turkeys from functional loading, we noted a loss of bone of 13.0 per cent compared with the intact contralateral control ulnae over an eight-week experimental period. Using a treatment regimen of one hour per day of pulsed electromagnetic fields, we observed an osteogenic dose-response to induced electrical power, with a maximum osteogenic effect between 0.01 and 0.04 tesla per second. Pulse power levels of more or less than these levels were less effective. The maximum osteogenic response was obtained by a decrease in the level of intracortical remodeling, inhibition of endosteal resorption, and stimulation of both periosteal and endosteal new-bone formation. These data suggest that short daily periods of exposure to appropriate electromagnetic fields can beneficially influence the behavior of the cell populations that are responsible for bone-remodeling, and that there is an effective window of induced electrical power in which bone mass can be controlled in the absence of mechanical loading.     

Role of pulsed electromagnetic fields after joint replacements

Although the rate of patients reporting satisfaction is generally high after joint replacement surgery, up to 23% after total hip replacement and 34% after total knee arthroplasty of treated subjects report discomfort or pain 1 year after surgery. Moreover, chronic or subacute inflammation is reported in some cases even a long time after surgery. Another open and debated issue in prosthetic surgery is implant survivorship, especially when related to good prosthesis bone ingrowth. Pulsed Electro Magnetic Fields(PEMFs) treatment, although initially recommended after total joint replacement to promote bone ingrowth and to reduce inflammation and pain, is not currently part of usual clinical practice. The purpose of this review was to analyze existing literature on PEMFs effects in joint replacement surgery and to report results of clinical studies and current indications. We selected all currently available prospective studies or RCT on the use of PEMFs in total joint replacement with the purpose of investigating effects of PEMFs on recovery, pain relief and patients' satisfaction following hip, knee or shoulder arthroplasty. All the studies analyzed reported no adverse effects, and good patient compliance to the treatment. The available literature shows that early control of joint inflammation process in the first days after surgery through the use of PEMFs should be considered an effective completion of the surgical procedure to improve the patient's functional recovery.