Compression and hypoxia play independent roles while having combinative effects in the osteoclastogenesis induced by periodontal ligament cells

Objective:To investigate the isolated and combined effects of compression and hypoxia on the osteoclastogenesis induced by periodontal ligament cells (PDLCs).Materials and Methods:A periodontal ligament tissue model (PDLtm) was established by 3-D culturing human PDLCs on a thin sheet of poly lactic-co-glycolic acid scaffold. The PDLtm was treated with hypoxia and/or compression for 6, 24, or 72 hours. After that, a real-time polymerase chain reaction was used for gene expression analysis. The conditioned media were used for the coculture of osteoblast and osteoclast (OC) precursors; tartrate-resistant acid phosphatase staining was done to examine OC formation.Results:Either compression or hypoxia alone significantly up-regulated the gene expression of pro-osteoclastogenic cytokines in the PDLtm and enhanced osteoclastogenesis in the cocultures, and the combination of the two had significantly stronger effects than either stimulation alone. In addition, comparing the two stimulants, we found that the osteoclastogenic property of the PDLCs peaked earlier (at 6 hours) in the compression group than in the hypoxia group (at 24 hours).Conclusions:Both compressive force and hypoxia may take part in initiating osteoclastogenesis in orthodontic tooth movement and may have combinatory effects, which could update our concepts of the mechanisms involved in the initiation of bone resorption on the pressure side of the tooth in question.     

n-Butanol extracts of Panax notoginseng suppress LPS-induced MMP-2 expression in periodontal ligament fibroblasts and inhibit osteoclastogenesis by suppressing MAPK in LPS-activated RAW264.7 cells

Objective

Periodontitis is a group of inflammatory diseases that affect connective tissue attachments and the supporting bone that surround the teeth. Osteoclasts are responsible for skeletal modeling and remodeling but may also destroy bone in several bone diseases, including osteoporosis and periodontitis. This study examined the anti-inflammatory effects of Panax notoginseng (PN) on periodontal ligament fibroblasts (PDLFs) and RAW264.7 cells under lipopolysaccharide (LPS) induced inflammatory conditions.

Design

The effects of PN on PDLFs were determined by measuring the cell viability and mRNA expression of tissue-destructive proteins. The effects of PN on osteoclasts were examined by measuring the following: (1) the cell viability, (2) the formation of Tartrate-resistant acid phosphatase (TRAP)(+) multinucleated cells, (3) MAPK signaling pathways, (4) mRNA expression of inflammatory-related proteins and (5) nitric oxide (NO) production.

Results

The n-butanol extracts of PN (bPN) increased the cell proliferation of the PDLFs and decreased the mRNA expression of matrix metalloproteinase (MMP)-2 in the PDLFs. bPN inhibited the formation of LPS-stimulated TRAP(+) multinucleated cells. bPN also inhibited the LPS-stimulated activation of JNK and ERK signaling, and inhibited the LPS-stimulated degradation of I_KB in the RAW264.7 cells. In addition, bPN decreased the mRNA expression of MMP-9 and iNOS, which are involved in the range of pathophysiological processes, such as inflammation in the RAW264.7 cells. NO production was also decreased via the inhibition of iNOS.

Conclusions

These findings suggest that bPN has therapeutic effects on bone-destructive processes, such as those that occur in periodontal diseases.     

髁突手术对其压缩力学性能及骨密度的影响

目的 探讨不同术式后髁突力学性能的变化及与材料特性之间的关系。方法 采用压缩力学性能测试技术和双能Ｘ线吸收法,定量分析１２例正常、６例髁突高位切削术及６侧关节重建术的成年杂种犬髁突的压缩力学性能和骨矿含量。结果 髁突的载荷与位称呈非线性关系。其弹性极限负荷及位移、最大负荷及位移以正常组为最高,而刚度及骨矿含量则以切削组为最高,减径组各项指标均最低。３组髁突弹性极限负荷、最大负荷及刚度与骨密度之间均     

不同静压力对新生SD大鼠髁突软骨细胞增殖与凋亡的影响

目的：探讨不同静压力对髁突软骨细胞增殖与凋亡的影响。方法：对培养到第3代新生SD大鼠髁突软骨细胞加载0、12、24、36kPa静压力1h后立即收集样本，用流式细胞仪检测细胞凋亡与增殖指数的变化。结果：随着压力的增加（0、12、24、36kPa），除24kPa外，细胞增殖指数和凋亡指数在加力结束时（0h）均减少（P〈0．05），其中，细胞增殖指数在36kPa加力结束时减幅最大，细胞凋亡指数则在12kPa加力结束时减幅最大。结论：在0、12、24、36kPa力值范围内，软骨细胞增殖、凋亡与应力值存在一定的关系，但这并非是简单的线性关系。     

压应力对小鼠单核细胞RAW264.7 DNAX活化蛋白12、抗酒石酸酸性磷酸酶表达的影响

目的 探讨压应力对小鼠单核细胞RAW264.7 DNAX活化蛋白12（DAP12）、抗酒石酸酸性磷酸酶（TRAP）表达的影响。方法 以小鼠单核细胞RAW264.7为研究对象,采用四点弯曲体外细胞加载装置加载压应力0、3、6、12 h,分别以逆转录聚合酶链反应（RT-PCR）、蛋白免疫印迹法检测DAP12、TRAP mRNA和DAP12蛋白的表达情况。结果 小鼠单核细胞RAW264.7经破骨细胞培养液培养后,体积变大,核数目增多,TRAP染色阳性。受压应力刺激后,DAP12、TRAP mRNA及DAP12蛋白表达随加力时间延长而增加（P<0.05）。结论 小鼠单核细胞RAW264.7经破骨细胞培养液培养后成功向破骨细胞转化,转化细胞受压应力刺激活化过程中存在DAP12高表达。     

体外周期性单轴压应力下髁突软骨细胞Raf激酶抑制蛋白(RKIP)的动态变化研究

目的:探讨体外周期性单轴压应力对大鼠髁突软骨细胞的Raf激酶抑制蛋白(RKIP)及其mRNA变化的早期影响,以助于阐明力学引起细胞应答反应的分子机制。方法:利用四点弯曲细胞力学加载仪对第3代大鼠髁突软骨细胞进行体外周期性单轴压应力加载,力值为4000μstrain,时间为0、15、30、60、120、240min。采用实时荧光定量PCR技术和Western blot免疫印迹技术研究大鼠髁突软骨细胞在周期性单轴压应力作用下RKIP及其mRNA变化。结果:大鼠髁突软骨细胞RKIP mRNA和蛋白表达呈现几乎相反的趋势。RKIP mRNA的表达于30min上升(P<0.001),60min达到高峰后迅速回落,低于细胞的正常基态水平;而RKIP的蛋白表达30min开始下调(P<0.001),持续至120、240min时回升。结论:RKIP在髁突软骨细胞的早期力学应答机制中可能扮演着相当重要的作用,其蛋白和mRNA水平表达变化不同,转录水平的调控最终要影响蛋白水平的表达。     

Effect of vascular endothelial growth factor on RANK gene expression in osteoclast precursors and on osteoclastogenesis

OBJECTIVE: The aim of this study was to determine if vascular endothelial growth factor (VEGF) can upregulate the gene expression of receptor activator of nuclear factor kappa B (RANK) in osteoclast precursors, as does CSF-1. A secondary aim was to determine if VEGF can promote osteoclastogenesis in vitro comparable to CSF-1. DESIGN: Osteoclast precursors (mononuclear cells) were incubated with different concentrations of VEGF, CSF-1, or a combination of the two, and the gene expression of RANK was determined by RT-PCR. A TRAP assay also was conducted to determine their effect on osteoclastogenesis. An Alamar blue assay was done to analyse the effect of the molecules on proliferation of the osteoclast precursors. RESULTS: VEGF upregulated RANK expression in osteoclast precursors as effectively as CSF-1. VEGF did not promote osteoclastogenesis, as did CSF-1. A combination of the two did. CSF-1 enhanced proliferation of the osteoclast precursors but VEGF did not. However, VEGF in combination with CSF-1 did increase proliferation. CONCLUSIONS: At the time of the secondary burst of osteoclastogenesis prior to tooth eruption, VEGF expression in the dental follicle is high but the expression of CSF-1 is low. This study demonstrates that VEGF can fully substitute for CSF-1 to upregulate the RANK expression in osteoclast precursors that is needed for osteoclastogenesis. However, VEGF alone neither can promote osteoclastogenesis nor stimulate proliferation of the osteoclast precursors in vitro. For proliferation and osteoclastogenesis, a low dose of CSF-1 in combination with VEGF is needed.     

压应力对体外培养兔髁突软骨细胞CTGF基因表达的影响

目的:观察不同压应力状态下,兔髁突软骨细胞结缔组织生长因子(CTGF)mRNA表达水平的变化.方法:半定量RT-PCR方法检测兔髁突软骨细胞在压应力作用下CTGF mRNA的表达变化.结果:在一定压应力范围内,兔髁突软骨细胞CTGF的表达,随着压应力增大而显著增强(P相似文献   

IL-17联合IFN-γ体外诱导RAW264.7细胞增殖和凋亡的影响

目的研究白细胞介素-17(IL-17)联合干扰素-γ(IFN-γ)在小鼠破骨前体细胞系RAW264.7分化为成熟破骨细胞过程中,对细胞增殖和凋亡的影响。方法将核因子κB受体活化因子配体(RANKL)和巨噬细胞集落刺激因子(M-CSF)诱导小鼠破骨前体细胞系RAW264.7建立破骨细胞体外诱导分化研究模型,被诱导的RAW264.7细胞培养24 h后,分为IL-17组、IFN-γ组、IL-17+IFN-γ联合组(IL-17+IFN-γ等量组、IL-17固定+IFN-γ梯度组)进行干预。抗酒石酸酸性磷酸酶(TRAP)染色对破骨细胞成熟进行鉴定,利用CCK-8细胞增殖试验检测各组细胞增殖情况,AnnexinⅤ细胞凋亡实验评价各组细胞凋亡的差异,实时荧光定量PCR检测凋亡相关基因mRNA表达差异。结果 50 ng/m L IL-17与不同浓度的IFN-γ联合,随着IFN-γ浓度升高,IL-17对RAW264.7细胞生长抑制呈浓度依赖性。IL-17和IFN-γ的联合作用比单独应用IL-17,凋亡率更高;与单独应用IFN-γ相比,凋亡相关基因Fas L表达明显增加。结论试在RAW264.7细胞向破骨细胞的分化过程中,IL-17和IFN-γ联合能抑制破骨前体RAW264.7细胞向破骨细胞的增殖,并促进RAW264.7细胞的凋亡。     

Expression of hypoxia-induced semaphorin 7A correlates with the severity of inflammation and osteoclastogenesis in experimentally induced periapical lesions

ObjectiveWhile hypoxia and inflammation are intimately linked, the effects of inflammatory hypoxia on the pathogenesis of periapical lesions remain largely unknown. The aim of this study was to examine hypoxia during the progression of experimentally induced rat periapical lesions, and to derive correlations between hypoxia-induced Semaphorin 7A (Sema7a) expression, severity of inflammation, and osteoclastogenesis in the lesions.DesignPeriapical lesions were developed after mandibular first molar pulp exposure in forty Sprague-Dawley rats. The animals were randomly divided into four groups and sacrificed at 0, 7, 14, and 28 days after pulpal exposure. The bilateral mandibles containing the first molar were obtained and routinely prepared for histological, immunohistochemical, enzyme histochemical analyses and quantitative polymerase chain reaction detecting Sema7a mRNA expression. Data were analysed by one-way analysis of variance and the Pearson’s correlation and linear tendency test.ResultsPeriapical tissues become hypoxic during the development of experimentally induced periapical lesions, with steadily increasing numbers of HIF-1α-positive cells that positively correlate with the expression of Sema7a mRNA in the lesions. Furthermore, significant positive correlates were derived for the expression of Sema7a and the degree of inflammatory infiltration and osteoclast number, respectively.ConclusionsHypoxia-induced Sema7a participates in the pathogenesis of periapical lesions, providing a novel therapeutic target for the treatment of this inflammatory disease in the future.     

周期性单轴压力对大鼠髁突软骨细胞结缔组织生长因子表达的影响

目的 探讨在髁突软骨细胞受到周期性单轴压力后,结缔组织生长因子(connective tissuegrowth factor, CTGF)表达的影响,为正畸治疗中髁突软骨受力后的改建提供生物学依据。方法 选取1周龄SD大鼠,提取并培养髁突软骨细胞,免疫组化鉴定。利用四点弯曲细胞力学加载仪对第3代细胞进行力值为2000u strain、0.5Hz的体外周期性单轴压力加载,分别在加力0min、30min、60min和120min后继续培养24h,应用蛋白印迹法检测在不同加力时间CTGF蛋白表达的变化。应用SPSS18.0软件对数据进行统计学分析。结果 CTGF的相对蛋白量在加力0min、30min、60min和120min后,分别为0、1.59、2.34和3.16,随着加载时间的增加,表达呈逐渐上升趋势。且组间差异具有统计学意义(P<0.05) 结论 周期性单轴压力可刺激大鼠髁突软骨细胞CTGF的表达。     

机械应力对人牙周膜成纤维细胞整合素β1mRNA表达的调节

目的观察机械力刺激对人牙周膜成纤维细胞整合素β1 mRNA表达的影响。方法用Forcel四点弯曲加载装置通过对体外培养的人牙周膜成纤维细胞分别施加不同动态张、压应力（强度为1 000、2 000、4 000 μstrain，加力时间为0、0.5、1、4、8、12 h），采用实时荧光定量PCR法研究机械力对人牙周膜成纤维细胞整合素β1 mRNA表达的影响。结果施加动态的张、压应力后，人牙周膜成纤维细胞整合素β1 mRNA表达量下调，这种下调变化与所施加应力的性质、大小和作用持续时间相关。人牙周膜成纤维细胞整合素β1对张、压应力刺激的感应不完全一致，机械力刺激越强，整合素β1表达越明显。结论动态张、压应力刺激在本实验提供的微应力范围内可以促进目的基因mRNA表达改变，不同的机械力对细胞整合素β1效应不同。     

Egr-1对机械力诱导人牙周膜细胞炎症反应的作用及机制研究

目的:研究早期生长转录因子(early growth responsive gene-1,Egr-1)对机械应力(mechanical stress,MS)介导人牙周膜细胞(human periodontal membrane cells,hPDLs)炎症因子分泌和表达的影响及可能机制.方法:以实时定量PCR和ELISA法检测MS加载不同时间(6、12、24和48 h)和不同形变率(3％、6％、12％和15％)时炎症因子IL-13、IL-6、IL-8和IL-11的分泌和表达水平.MS对Egr-1表达的影响采用实时定量PCR和Western印迹法检测,沉默Egr-1对炎症因子的作用采用实时定量PCR和ELISA检测.应用Western印迹法检测Egr-1下调对PTEN/PI3K/Akt信号通路相关蛋白表达的影响.进一步采用20 μmol/L PI3K/Akt抑制剂LY294002预处理hPDLs 30 min,研究Egr-1沉默在MS介导炎症因子表达中的可能机制.采用SPSS 11.0软件包对数据进行统计学分析.结果:IL-1[β、IL-6、IL-8和IL-11的分泌和mRNA表达随着作用时间和形变率的增加而升高,12％的MS作用24 h后,其分泌程度和表达水平最高.12％的MS加载24 h显著上调Egr-1表达.沉默Egr-1显著抑制MS诱导炎症因子表达.Egr-1沉默下调PTEN表达,上调p-PI3K和p-Akt蛋白表达水平,LY294002预处理可部分阻断Egr-1沉默对炎症因子分泌及表达的抑制作用.结论:沉默Egr-1通过PETN/PI3K/Akt信号通路抑制MS诱导的炎症因子分泌和表达.     

Effects of vibration stimulus on lower-jaw-position sensation in patients with cerebral palsy during inhalation of laughing gas

To clarify the effects of a vibration stimulus applied during sedation with nitrous oxide (hereafter referred to as laughing gas) on the ability of muscles attached to the lower jaw to sense lower-jaw position and on the sensation of muscle spindles attached to the lower jaw in patients with cerebral palsy (CP) using healthy adult subjects without functional abnormalities of the jaws and oral cavities as control subjects (hereafter referred to as healthy subjects). Experiments were performed under the following conditions: for each subject, before the application of the vibration stimulus (referred to as S_pre) and after the application of the vibration stimulus (S_post); before the inhalation of laughing gas (LG) and oxygen (air-inhalation condition: referred to as without LG inhalation) and during the inhalation of LG and oxygen (inhalation condition of LG and oxygen under LG-induced sedation: referred to as during LG inhalation). Subjects in the experiments were eight CP patients and eight healthy people as controls. The ability to discriminate lower-jaw position was estimated by asking the subjects to determine whether the diameter of a test stick was larger or smaller than that of a reference stick after performing the following tasks: a) holding a reference stick between the central teeth of their upper and lower jaws for 5 s, and b) replacing the reference stick with a test stick and holding it at the same position for 5 s, and the test stick was then removed. The following findings were obtained.

• 1)In comparing the ability of healthy subjects to discriminate between S_pre and S_post during LG inhalation using different test sticks, when the test stick diameter was 9.5 mm (smaller than the reference stick diameter), the rate of mis-estimation (RME) for Spost was significantly larger than that for Spre (P < 0.05). No significant differences were observed for any other test sticks.
• 2)In comparing the ability of CP patients to discriminate between S_pre and S_post during LG inhalation using different test sticks, when the test stick diameter was 9.5 mm (smaller than the reference stick diameter), the RME for S_post was significantly smaller than that for S_pre (P < 0.05). No significant differences were observed for any other test sticks.

These results suggest the following: the combination of LG for sedation with vibration stimulus further inhibits neuronal functions at the upper level of the central nervous system in CP patients, compared with cases in which each variable is applied separately, and the combination also inhibits the sustained increase in muscle tonus, which is characteristic of CP patients. LG reduces the activity of γ-motor neurons via the upper level of the central nervous system. In addition, tonic vibration reflex (TVR) develops due to the vibration stimulus, which increases the threshold value of sensitive muscle sensation and decreases the activity of γ-motor neurons, and furthermore decreases the activity of muscle spindles attached to the lower jaw. Consequently, a tendency toward increased ability to discriminate lower-jaw position is observed.     

周期性单轴压应力对大鼠髁突软骨细胞Stress70/GRP75的早期影响

目的:探讨体外周期性单轴压应力对大鼠髁突软骨细胞糖调控蛋白Stress70/GRP75的早期动态变化影响。方法:利用四点弯曲细胞力学加载仪,对第3代大鼠髁突软骨细胞进行体外周期性单轴压应力加载,力值为4000μstrain,时间分别为0、15、30、60、120、240min;采用Western免疫印迹技术和图像分析技术,研究大鼠髁突软骨细胞糖调控蛋白Stress70/GRP75的动态变化,对结果进行单因素方差分析。结果:4000μstrain周期性单轴压应力作用下,大鼠髁突软骨细胞糖调控蛋白Stress70/GRP75的表达发生变化,0min条带灰度值为114.2±5.08;30min时降至最低,为86.1±5.09(P<0.001);60min后有所回升,至104.0±4.41(P<0.01),但仍表达下调;120min后表达开始增强,灰度值为134.5±3.74(P<0.001)。结论:4000μstrain压应力刺激,对大鼠髁突软骨细胞糖调控蛋白Stress70/GRP75存在时间效应性,初期表达下调;随着应力加载时间延长,其表达反馈增强。     

机械牵张力对人牙周膜细胞成骨样细胞功能的影响

目的 观察机械力作用下牙周膜细胞成骨样细胞特性的改变，以深入探讨正畸牙齿移动的机理。方法 利用自行研制的细胞加力装置对体外培养的人牙周膜细胞施加间歇性机械牵张力，检测其成骨样细胞表型蛋白碱性磷酸酶（alkaline phosphotase,ALP)、骨桥蛋白（osteopontin,OPN)和骨钙素（osteocalcin,OCN)表达的改变。结果 人牙周膜细胞在受到机械牵引力刺激后表现为ALP分泌活性的影响，而且24h的时段内波动，2、4h和24h出现2个显著增高期（P＜0．01）；OCN受机械牵张力影响出现较缓慢和晚期的表达增强，加力4h后缓慢增高，在12h最为明显（P＜0．05）；非分泌型ALP、OPN随观察时间延长蛋白表达水平，如ALP、OCN和OPN都增强，具有时序性。提示人牙周膜细胞在机械力诱导下向成骨样细胞分化成熟，从而可能在机械力介导的骨改建中起作用。