摘要
目的
通过建立钛颗粒局部干预抑制异位骨化形成的动物模型,初步探讨钛颗粒抑制异位骨形成的可能性和发生机制,为异位骨化的防治提供一个新的方法开拓新的思路。
方法
制备动物模型：36只昆明小鼠于双侧跟腱中点行跟腱切断术,跟腱切断部位左侧注射钛颗粒的悬浊液为实验组,右侧注射生理盐水为对照组。分别在跟腱切断后于第4、8、12周随机处死12只小鼠。对处死小鼠拍摄X片,并将切取的跟腱组织分成两份,分别进行HE染色和免疫组化分析。
结果
1.X片显示跟腱切断术后第8周对照组跟腱切断部位58.3%(7／12)出现异位骨化；12周后,对照组跟腱部位均出现异位骨化,实验组跟腱部位有1例出现异位骨化。
2.HE组织学表明,该方法形成的异位骨化为软骨内成骨。
3.跟腱部组织免疫组化结果显示：①BMP-2、TGF-β在各个时间点的表达实验组均少于对照组,但两组间无显著性差异(P>0.05)；②IL-1、TNF-a的表达随时间的延长逐步减少,各个时期实验组的表达明显高于对照组,两组间有显著性的差异(P<0.05)；③Runx2/Cbfal在各个时间点的表达实验组较对照组减少,并且在术后第4周、第8周时两组间有显著性差异(P<0.05)；④MMP-9在各个时间点的表达实验组明显高于对照组,并且在术后第8周、第12周两组间有显著性差异(P<0.05)。
结论
1.跟腱切断的方法可以有效诱导异位骨化,结果稳定可靠；
2.钛颗粒局部干预可以防止以软骨内成骨方式发生的异位骨化的形成,其机制可能为钛颗粒在部分抑制成骨的同时,增强了周围组织的破骨细胞活性产生溶骨效应。
目的
1.探讨钛微粒诱导巨噬细胞RAW264.7释放破骨性细胞因子的作用。
2.观察钛颗粒对破骨细胞分化的影响,探讨钛颗粒防治异位骨化形成的可能机制。
方法
1.巨噬细胞细胞RAW264.7分别与不同浓度的钛微粒联合培养,分别在24 h、48h、72h时点取细胞培养上清液,用酶联免疫吸附法(ELISA)技术检测肿瘤坏死因子-α(TNF-α)、白细胞介-1(IL-1)白细胞介素-6(IL-6)和前列腺素E2 (PGE2)的含量。
2.RT-PCR法和Western blot法分析钛颗粒干预后细胞内转录因子NFATc1在mRNA和蛋白水平的表达。
结果
1.钛颗粒组较对照组TNF-α、IL-1、IL-6和PGE2分泌量明显增高(P<0.05),且随浓度增高其分泌量增加;
2.钛颗粒刺激后NFATc1在mRNA和蛋白水平的表达显著增加(P<0.05)。
结论
钛颗粒具有诱导前破骨细胞RAW264.7分泌破骨性细胞因子的作用,并刺激其向破骨细胞分化。
目的
通过建立小鼠前成骨细胞MC3T3-E1和钛颗粒体外共同培养的细胞模型,研究钛颗粒对小鼠前成骨细胞MC3T3-E1增殖、分化、矿化及Runx2/Cbfα1、OPG、RANKL表达的影响,探讨钛颗粒通过作用于前成骨细胞进而影响骨代谢的可能机制。
方法：
1.MC3T3-E1细胞在含β2甘油磷酸钠、抗坏血酸的培养基中培养22 d。用钛颗粒干预小鼠前成骨细胞MC3T3-E1(颗粒数与细胞数之比为1 00：1),在细胞培养的不同时间(2,4,7,14 d),用四甲基偶氮唑盐检测各组细胞增殖活性;用p-NPP法测定碱性磷酸酶(ALP)活性;Van GieSon苦味酸酸性复红染色法染色细胞Ⅰ型胶原;茜素红染色观察矿化结节形成;
2.用RT-PCR和Western blot的方法检测不同时间细胞中Runx2/Cbfα1在mRNA水平和蛋白水平的表达情况。
3.用RT-PCR和Western blot的方法检测细胞在不同钛颗粒浓度下OPG、RANKL在mRNA和蛋白水平的表达情况。
结果
1.钛颗粒组与对照组相比各时间点细胞增殖均受抑制,且有显著性差异(P<0.05)；14 dⅠ型胶原染色可见钛颗粒组较对照组弱,碱性磷酸酶活性定量分析钛颗粒组显著低于对照组(P<0.01)；14,21 d茜素红染色可见钛颗粒组钙结节数量较对照组显著性降低(P<0.05)；
2.在不同时间点钛颗粒组MC3T3-E1细胞中Runx2/Cbfα1的mRNA和蛋白表达较单纯诱导组显著下调(P<0.05)；
3.钛颗粒能上调MC3T3-E1细胞OPG及RANKL的表达,且对后者作用强于前者。RANKL/OPG比率随着钛颗粒浓度的增高而增大。
结论
1.钛颗粒对MC3T3-E1细胞增殖、分化和矿化功能均具有抑制作用；
2.钛颗粒对MC3T3-E1细胞的分化抑制作用可能与其对Runx2/Cbfα1的表达调控有关；
3.钛颗粒对破骨细胞的影响可能与其对成骨细胞RANKL/OPG途径的调控有关。
Objective
To induce heterotopic ossification through traumatic animal model and to implant the titanium particles into the area where would form the heterotopic bone.To investigate the possible mechanism for this treatment method.
Methods
Thirty-six kunming strain mice were used to establish traumatic animal models by Achilles tenotomy and injecting 0.2 mL suspension of titanium particles into the left Achilles and the normal sodium to the other limb.X-ray, histological examination and immunohistochemistry were made at 4,8,12 weeks respectively.
Results
1. Under X-ray examination:by 8 weeks,new bone(radio-opaque zones)in the region of the Achilles tendon was found in 58.3% and in 100% by 12 weeks.
2.This heterotopic ossification occured through a process of endochondral ossification.
3.Immunohistoehemistry result:①The expression of BMP-2、TGF-βshow stronger in the control group than that in experimental group(P>0.05);②The expression of IL-1、TNF-a decreased with the time,and the experimental group was much higher than that in the contral group(P<0.05);③The expression of Runx2 in the experimental group was lower than that in the contral group;.④The expression of MMP-9 in the experimental group is significantly higher than that in contral group especially after 12 weeks(P<0.05).
Conclusion
1. Achilles tenotomy is a simple and feasible method to induce heterotopic ossification.
2. The local use of titanium particles is a effective way to inhibit the formation of endochondral ossification.
Objective
1.To clarify the role of titanium particles on inflammatory cytokines releasing in macrophage cells.
2.To explore the mechanism of titanium particles on the formation of osteoclast from osteoclast precursors (RAW264.7), and provide more evidences for titanium particles on the treatment of formation of hetero-topic ossification.
Methods
1.Macrophages were cocultured in vitro with cleaned titanium particles, TNF-α,IL-1, IL-6 and PGE2 in the supernatants were assayed after culturing for 24,48 and 72 hours.
2. RT-PCR and Western blotting were used to examine the mRNA and protein expression of NFATc1 in titanium particle-induced RAW264.7 undergoing differentiation into osteoclasts.
Results
1. In the experimental group,TNF-α、IL-1、IL-6和PGE2 content was gradually increased, compared with the control group, there is a significant difference (P<0.05)
2. The protein of NFATc1 as well as mRNA expressed by RAW264.7 was enhanced by stimulation of titanium particles.
Conclusion
1.The macrophages are sensitive to titanium particles to release inflammatory cytokines.
2.RAW264.7 was differentiate to osteoclast by stimulation of titani-um particles.
Objective
To evaluate the effect of titanium particles on the proliferation, differentiation, mineralization and Runx2/Cbfα1, OPG, RANKL in MC3T3-E1,then investigate the possible mechanism of titanium particles on bone metabolism.
Methods
1. MC3T3-E1 cells were cultured for 22 days,MTT test was used to assess the cell proliferation at 2,4,7,14 days; while alkaline phospha-tase(ALP) staining and ALP activity measurement were taken to assess the differentiation of osteoblasts at 7,14 days. The mineralization of osteoblasts was evaluated by Alizarin red staining at 14,21 days.
2. The expressions of Runx2/Cbfα1 mRNA and protein were tested using RT-PCR and Western blot respectively.
3. MC3T3-E1 cells were cultured with different dose of titanium particles,the expressions of OPG and RANKL were obtained with RT-PCR and Western blot method.
Results:
1.The osteoblastic proliferation had significant difference between the experimental group and the control group (P<0.05),the proliferation of experimental group is inhibited by titanium particles. However, the colleganⅠstaining was weaken in the experimental group than that of control group at 14 days, meanwhile,activity of ALP measurement in the experimental group was smaller than the control group (P<0.01), while the alizarin red staining demonstrated that the quantity of calcium nodules were markedly decreased in the experimental group at 21 days.
2. Titanium particles down-regulated the expression of Runx2/Cbfα1 at both mRNA and protein level at anytime.
3.The titanium particles induced a constant and varying increases in the OPG and RANKL, and the upregulated mRNA and protein level of RANKL was higher than that of OPG.The RANKL/OPG ratio is in accordance with dose increase of the titanium particles
Conclusion:
1.The proliferation,differentiation and mineralization of primary rat osteoblasts are decreased with exposure to titanium particles
2.The negative effect of titanium particles on differentiation of MC3T3-E1 cells may is related to its down-regulated the expression of Runx2/Cbfα1
3.The effects of titanium particles on osteoclast may is related to its regulating effect on the OPG/RANKL pathway of osteoblast.
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