α-玉米赤霉醇对小鼠骨髓间充质干细胞向成骨细胞分化过程中OPG和RANKL蛋白表达的影响

目的 探讨α-玉米赤霉醇(α-zearalanol,α-ZAL)对小鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, BMSCs)向成骨细胞增殖、分化的影响。方法 采用全骨髓培养差速贴壁法体外分离培养小鼠骨髓间充质干细胞,取第3代细胞分为空白对照组、雌二醇阳性对照组、低浓度α-ZAL组、中浓度α-ZAL组,高浓度α-ZAL组。镜下每天观察细胞形态变化,碱性磷酸酶染色鉴定成骨细胞纯度,MTT实验测定细胞增殖活性绘制细胞生长曲线,采用酶联免疫吸附法(ELISΑ)测定碱性磷酸酶(alkaline phosphatase,ALP)、骨形态发生蛋白2( Bone morphogenetic protein-2,BMP-2),运用蛋白免疫印迹法（Western blotting,WB）检测护钙素（Osteoprotegerin,OPG）和NF-KB受体活化配体（Receptor actiator of nuclear factor-KB ligand, RANKL）的蛋白水平表达变化。结果 不同浓度的α-玉米赤霉醇可显著的促进小鼠骨髓间充质干细胞向成骨细胞增殖、分化,增加细胞活性（P<0.05）,明显提高小鼠骨髓间充质干细胞向成骨细胞分化后ALP和BMP-2的表达（P<0.05）,并且显著的上调了成骨细胞内OPG/ RANKL的表达率（P<0.05）。结论 不同浓度的α-玉米赤霉醇可以促进骨髓间充质干细胞向成骨细胞增殖、分化,并且通过上调OPG/ RANKL的表达率抑制成骨细胞细胞向破骨细胞分化,有望成为临床骨折疏松症治疗的激素替代药物。     

破骨细胞骨吸收上清液对成骨活动影响的实验研究

目的 研究破骨细胞骨吸收活动中的分泌产物对成骨细胞增殖、分化及成骨功能的影响,以了解活化的破骨细胞在体外对成骨细胞的影响.方法 诱导小鼠RAW264.7细胞为破骨细胞,用抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色和破骨细胞特异性基因检测鉴定.破骨细胞与牛骨磨片共培养,甲苯胺蓝染色.收集共培养上清液作用于小鼠MC3T3-E1细胞,用甲基噻唑基四唑法(methyl thiazolyl tetrazolium,MTT)法、酶联免疫吸附测定法、茜素红染色及反转录聚合酶链反应检测细胞的增殖、骨钙素水平、矿化及成骨特征基因的表达.结果 TRAP 染色、破骨细胞特异性基因检测及甲苯胺蓝染色表明RAW264.7细胞可分化为具有骨吸收能力的破骨细胞;破骨细胞骨吸收上清液作用后,MC3T3-E1细胞增殖受抑制(A值在0.062±0.004和0.405±0.033之间,P＜0.05);骨钙素水平增高[第10天上清液组的骨钙素质量浓度为(2.965±0.047) μg/L],钙化结节增多,碱性磷酸酶和Runt相关转录因子2的转录增强.结论 RAW264.7破骨细胞骨吸收上清液具有抑制成骨样细胞增殖、促进分化和钙化成骨的作用.     

两种纳米形貌对MG63成骨细胞增殖、分化的影响

目的:评价钛表面纳米管和纳米颗粒形貌对MG63成骨细胞的增殖、分化能力的影响。方法:通过磁控溅射和阳极氧化技术制备纳米颗粒和纳米管形貌。采用扫描电镜和轮廓仪表征材料表面形貌以及粗糙度,并将MG63成骨细胞株与不同形貌的钛材料进行复合培养,检测1d、4d、7d的MTT值及4d、7d的碱性磷酸酶活性。结果:培养1d、4d、7d后,纳米管和纳米颗粒组MTT值高于对照组,7d时,纳米管组MTT值高于纳米颗粒组;培养7d后,纳米形貌实验组碱性磷酸酶活性明显高于对照组。结论:纳米管和纳米颗粒形貌均有利于成骨细胞的增殖和分化。相对于纳米颗粒,表面的纳米管形貌更有利于促进成骨细胞的增殖。     

成骨细胞与破骨细胞共培养及其应用研究进展

成骨细胞与破骨细胞并非孤立存在,两种细胞存在直接接触、分泌旁分泌因子、细胞与骨基质3种相互作用.成骨细胞与破骨细胞共培养技术有直接接触式、间接接触式2类方式,它能最大程度地模拟体内微环境,有利于研究两细胞间的相互作用,探讨两者失衡在骨质疏松症等骨代谢疾病形成中的作用;细胞共培养应根据病理状态下两细胞的比例选用种属来源一致的细胞.目前该技术主要用于探讨药物防治骨质疏松症的作用机制,已有淫羊藿、三七、补骨脂、雷奈酸锶等既促进成骨细胞骨形成,又抑制破骨细胞骨吸收活性的双重药理作用研究等报道.成骨细胞与破骨细胞共培养的一个新的应用方向,将是应用于软骨下骨重建功能活动的探讨以及中西药干预骨性关节炎的研究.     

In vivo monitoring of the bone healing process around different titanium alloy implant surfaces placed into fresh extraction sockets

Objectives

Increasing surface roughness and coating with tricalcium phosphate of titanium and titanium alloy implants has been proposed to provide better rates of osseointegration. However, how these changes in surface topography and chemistry influence the osseointegration process of immediate implants placed in fresh extraction sockets is unclear. This study investigated the influence of three clinically employed implant surfaces on the early bone healing events in vivo.

Methods

Machined smooth implants were milled from grade 5 Ti6Al4V titanium. Surfaces were moderately roughened by grit blasting, which were then coated with tricalcium phosphate. Implants were placed into freshly extracted incisor sockets of mandibles of normal Wistar rats and left for 1, 3 and 9 weeks. Healing bone tissue around the implants was examined by histochemistry and immunocytochemistry to localise PCNA proliferative cells, and osteoblast differentiation markers osteopontin and osteocalcin. Positive synthesising cells were counted using image analysis.

Results

Histology indicated no differences in the amount or pattern of bone formation within the healing tissue surrounding the different implant surfaces. Bone healing occurred predominantly on exposed bone surfaces (distance osteogenesis) and not on the implant surface (contact osteogenesis). No differences were observed in the number or timing of PCNA, osteopontin and osteocalcin positive cells within the bone healing tissue around each of the implant analysed.

Conclusion

For immediately placed implants, the surface modifications investigated appeared to have little influence on the activity of bone forming cells surrounding the implant, probably due to the high level of distance osteogenesis seen within this scenario.

Clinical significance

For immediate placement of implants into fresh extraction sockets, titanium implants with roughened surfaces and coating with tricalcium phosphate have negligible influence in accelerating the early bone healing events of osseointegration.     

骨母细胞性肿瘤的免疫组织化学研究

选择３２例骨母细胞性肿瘤，包括１４例良性骨母细胞瘤、２例侵袭性骨母细胞瘤和１６例骨母细胞型骨肉瘤，用ＡＢＣ法作多种抗血清标记（ｖｉｍｅｎｔｉｎ、Ｓ－１００、α－ＡＴ、ｌｙｓｏｚｙｍｅ、Ｌｅｕ－７、Ｋ１２和ＣＥＡ）。结果显示：３２例肿瘤性骨母细胞ｖｉｍｅｎｔｉｎ均呈不同程度的阳性反应；在６例骨母细胞型骨肉瘤和１例侵袭性骨母细胞瘤中散在的单个细胞Ｓ－１００蛋白呈阳性反应，表明肿瘤性的骨母细胞具有软骨分化的潜能１２４例肿瘤中的多核巨细胞α－ＡＴ和１５例肿瘤中的多核巨细胞ｌｙｓｏｚｙｍｅ均呈不同程度的阳性反应，进一步证实这些细胞可能是组织细胞起源；６例骨母细胞型骨肉瘤和４例良性骨母细胞瘤中的骨样基质Ｌｅｕ－７呈阳性反应。     

Effects of PEMF exposure at different pulses on osteogenesis of MC3T3-E1 cells

Objective

Pulsed electromagnetic fields (PEMFs) were considered to be a factor which may affect osteogenesis of osteoblasts, but the effects were diverse with different PEMF parameters. The aim of the current study is to explore the effects of exposure to PEMFs at different pulse number on osteogenesis of osteoblasts.

Design

The mouse osteoblast-like MC3T3-E1 cells were exposed to 0, 400 or 2800 pulses 400 kV/m PEMF and the proliferation, differentiation and mineralization of cells were observed after PEMF exposure by the methods of MTT, biochemical measurement, real-time PCR and Alizarin Red assay.

Results

Compared with 0 pulses groups, the growth curve, alkaline phosphatase (ALP) activity, mRNA level of osteocalcin (OCN) and mineralized nodule formation of MC3T3-E1 cells did not change after 400 pulses PEMF exposure, but decreased after 2800 pulses PEMF exposure. It suggested that under our experimental conditions, only 2800 pulses 400 kV/m PEMF exposure can suppress the proliferation, differentiation and mineralization of MC3T3-E1 cells, but 400 pulses 400 kV/m PEMF exposure cannot.

Conclusions

Pulse number is another involved parameter which may influence the effects of PEMF on osteogenesis of osteoblasts.     

Lipopolysaccharide derived from Aggregatibacter actinomycetemcomitans inhibits differentiation of osteoblasts

Background and objectiveAlveolar bone defects in aggressive periodontitis are generally caused by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) through osteoclast differentiation facilitated by the interaction between the host's immune system, stimulated by the bacterial outer components of A. actinomycetemcomitans, and osteoblasts. Recently, some reports on the direct effects of A. actinomycetemcomitans on osteoblast differentiation have been published. However, the mechanisms in detail have still remained unknown. We herein demonstrated that A. actinomycetemcomitans might inhibit differentiation of osteoblasts and identified a causative substance.Materials and methodsThe following culture supernatants of A. actinomycetemcomitans were added to mouse osteoblasts, MC3T3-E1: supernatants fractionated by the molecular weight, supernatant supplemented with proteinase K and then heated, and supernatant supplemented with polymyxin B. Subsequently, RNA was extracted to determine the expressions of bone differentiation markers, alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP). Additionally, lipopolysaccharide (LPS) was added to MC3T3-E1 culture to measure the markers.ResultsA. actinomycetemcomitans culture supernatant, added to the MC3T3-E1, inhibited the expressions of bone differentiation markers at all concentrations. Only the fraction with a molecular weight of >100 kDa inhibited the bone differentiation. Neither proteinase K nor heating had an effect. However, polymyxin B completely abrogated the differentiation inhibitory activity. A. actinomycetemcomitans LPS concentration dependently inhibited the expressions of the bone differentiation markers.ConclusionsA. actinomycetemcomitans LPS suppressed osteoblast differentiation. This suggests that suppressed bone differentiation is involved in the alveolar bone destruction.     

牙龈卟啉单胞菌脂多糖对成骨细胞EphA2表达的调控

目的：研究小鼠前成骨细胞系MC3T3-E1在成骨分化过程中,牙龈卟啉单胞菌脂多糖（Pg-LPS）对EphA2表达的影响。方法：使用终浓度1mg/L的Pg-LPS与MC3T3-E1共培养,分别在第3、7、14天3个时间点,采用RT-PCR和Western-Blot技术测定EphA2的表达和成骨相关基因的表达,并通过PNPP法测定碱性磷酸酶活性。结果：RT-PCR结果提示,在第3天和第7天,实验组比对照组EphA2基因表达分别增高4.5倍和1.3倍,两组之间存在显著差异（P〈0.05）。同时成骨标志物ALP和Sp7基因表达降低,与对照组相比差异有显著性,但Runx2和ColⅠ基因的表达则无明显差异。但是在第14天,实验组和对照组之间EphA2和成骨相关基因表达均无显著性差异。Western-Blot结果提示,在第7天实验组比对照组EphA2蛋白表达增加。结论：Pg-LPS对前成骨细胞系MC3T3-E1细胞,在成骨分化的早期和中期能够促进EphA2的表达,但是在成骨分化晚期对EphA2的表达则无明显作用。