α7nAChR在尼古丁相关性牙周炎中的作用及其信号转导机制研究
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摘要
牙周炎是口腔常见病、高发病,可引起牙周组织降解、牙周袋形成、进行性附着丧失和牙槽骨吸收,最终导致牙齿松动脱落或被拔除,是成人牙齿丧失的首要原因。吸烟者牙周炎的患病率高、病情重、治疗效果和预后差,其具体机制却不清楚。现已证实,尼古丁作为烟草中的主要毒性物质,在吸烟对牙周组织的破坏过程中发挥重要作用。然而,尼古丁究竟通过何种途径对牙周组织产生破坏作用,目前尚不明了。
课题组前期研究表明,体外培养人牙周膜细胞中功能性表达α7 nAChR;本人前期研究发现大鼠牙周组织中存在尼古丁靶受体:α7型尼古丁乙酰胆碱受体(alphaα7 nicotinic acetylcholine receptor,α7 nAChR)的mRNA和蛋白表达,尼古丁可明显上调该受体的表达,且α7 nAChR表达调节与大鼠牙周组织破坏、牙槽骨吸收程度密切相关。然而,尼古丁激活α7 nAChR后如何对牙周组织产生破坏作用?其分子机制是什么?目前尚未见相关报道。
目的:本课题采用尼古丁作用下人牙周膜细胞和实验性牙周炎大鼠为模型,观察α7 nAChR对体外培养人牙周膜细胞增殖、贴附与迁移等生物学特性的调控,以及对牙周膜细胞和牙周组织分泌牙周炎相关炎性介质的影响,并进一步筛选和探明α7 nAChR激活后下游关键分子信号通路,以期阐明α7 nAChR在吸烟促进牙周炎发生发展中所扮演的角色及其作用机制。
材料与方法:体外研究,培养人牙周膜细胞,并分别给予α7 nAChR激动剂尼古丁(0.1-10μM)和/或拮抗剂-银环蛇毒素( -bungarotoxin, -BTX, 10nM);及ERK1/2信号通路抑制剂U0126(10μM)、P38信号通路抑制剂SB203580 (10μM), JNK信号通路抑制剂SP600125(10μM)和PI3K信号通路抑制剂LY-294,002(10-5μM)处理。分别采用WST-1、贴附实验和划痕修复实验测定细胞增殖、贴附与迁移能力改变;采用实时定量PCR、免疫荧光染色和Western blot等实验检测各组细胞α7 nAChR、IL-1?、IL-6、TNF-、RANKL和OPG等基因和蛋白表达,及ERK1/2和p-ERK1/2等磷酸化信号通路蛋白表达情况。
体内研究,24只SD大鼠采用两次完全随机分组方法分为4组,每组6只;采用3-0丝线结扎上颌左侧或右侧第二磨牙颈部,对侧不予结扎,作为自身对照;次日起分别给予生理盐水、尼古丁1.62 mg/kg/d、-BTX 10μg/kg/d、-BTX(10μg/kg/d)+30分钟后尼古丁(1.62 mg/kg/d)腹腔注射,常规饲养。全部大鼠于结扎前行麻醉下活体显微CT头部扫描,并分别于给药后α7、14、21和28天行活体显微CT头部扫描;采用Tri-Bon影像分析系统,重建各组大鼠颅颌面及牙体牙周组织影像;采用严格的标准化定位方法,创建一种基于“四根定位”的新型牙槽骨定量分析策略;定量分析各组大鼠牙槽骨骨密度、骨小梁厚度、骨体积分数等参数的动态改变;各组大鼠于给药后28天取材,解剖取出双侧上颌磨牙区组织,制作牙体牙周联合切片;HE染色进行病理学观察;免疫组织化学检测各组大鼠牙周组织α7 nAChR、IL-1?、IL-6、TNF-等蛋白表达情况。
结果:体外研究,实时定量PCR结果表明,10μM尼古丁作用下人牙周膜细胞α7 nAChR基因表达明显上调,约为对照组的4.α7倍(P < 0.001);-BTX +尼古丁处理组α7 nAChR基因表达水平与对照组近似,差异无统计学意义;免疫荧光染色和Western blot结果证实,体外培养正常人牙周膜细胞中α7 nAChR蛋白呈阳性表达;与对照组相比,尼古丁处理组α7 nAChR表达增强, -BTX预处理可拮抗尼古丁对α7 nAChR表达的上调作用;α7 nAChR激活可抑制细胞增殖和贴附,而促进其迁移。
体内研究,尼古丁作用下大鼠α7 nAChR表达明显上调,与之相对应的是,牙槽骨吸收亦明显加重,而-BTX预处理可拮抗尼古丁的作用;尼古丁单独作用下,大鼠牙槽骨显微结构呈现慢性破坏,其特征为首先出现骨小梁厚度降低且骨小梁分离度升高,后期出现骨体积分数和骨小梁数目降低;尼古丁给药结合丝线结扎可造成大鼠牙槽骨急剧丧失。
体内和体外研究均显示,尼古丁作用下人牙周膜细胞或大鼠牙周组织中α7 nAChR表达上调的同时,IL-1?、IL-6、TNF-和RANKL表达亦明显增强且OPG表达减弱;而ERK1/2和PI3K信号通路抑制剂可显著降低IL-1?、IL-6和TNF-的表达;尼古丁作用下人牙周膜细胞ERK1/2磷酸化水平明显增高。
结论:本研究证实α7 nAChR在体外培养人牙周膜细胞中存在功能性表达;发现尼古丁可通过作用于α7 nAChR调节牙周膜细胞增殖、贴附和迁移等生物学特性;人牙周膜细胞α7 nAChR激活后可通过作用于下游ERK1/2和PI3K信号通路影响IL-1?、IL-6和TNF-等炎性细胞因子分泌而参与牙周组织的炎症反应和牙槽骨吸收过程;大鼠牙周组织α7 nAChR激活可通过特定模式动态调控牙槽骨的显微结构改变。本研究表明尼古丁可通过作用于α7 nAChR而激活下游ERK1/2和PI3K信号转导通路,从而参与吸烟相关性牙周炎的发生和发展。
Background: Periodontitis, which causing periodontium destruction, pocket formation, alveolar bone resorption, and eventually teeth exfoliation, is one of the most common chronic diseases in adults. Numerous data demonstrated that tobacco smoking is one of the major risk factors for periodontitis. In addition, nicotine, the major component in tobacco smoke, has been proven to play an important role in smoking- associated periodontitis. However, the mechanisms behind the effects of nicotine on periodontal destruction are still not well understood.
Our previous studies demonstrated the functional expression of lphaα7 subunit of nicotinic acetylcholine receptors (α7 nAChR) in rat periodontal tissues. What’s more, nicotine acted directly and specifically throughα7 nAChR to affect periodontitis activity. However, initial data regarding how theα7 nAChR modulates nicotine’s effects on periodontitis, such as regulating cells behavior and pro-inflammatory cytokines expression of PDL cells in vitro, or affecting periodontal tissue destruction in vivo, are still lacking.
Objective: The aim of this study was to investigate how theα7 nAChR regulates nicotine’s effects on PDL cells behavior, inflammatory related cytokines expression and periodontal tissue destruction, and to explore the signalling pathway downstream ofα7 nAChR after its activation.
Materials and methods: In vitro, human PDL cells were cultured with or without different concentration of nicotine (Concentration ranging from 0.1μM to 10μM), alpha-bungarotoxin ( -Btx, inhibitor ofα7 nAChR, 10nM), U0126 (inhibitor of ERK1/2, 10μM), SB203580 (inhibitor of P38, 10μM), SP600125 (inhibitor of JNK, 10μM) and LY-294,002 (inhibitor of PI3K, 10μM). Cell proliferation, adhesion and migration were measured by WST-1 assay, attachment assay and wound healing assay, respectively. The expression ofα7 nAChR, IL-1?, IL-6, TNF- , RANKL and OPG in PDL cells were explored by Real Time-PCR, Immunofluorescence and Western blot assay.
In vivo, twenty-four male Sprague-Dawley rats were anesthetized and scanned by an in vivo micro-CT system (day 0, baseline). They were then tied by silk ligatures around cervixes of the second maxillary molar while the contra lateral one was left untreated. The rats were randomly divided into four groups, and received intraperitoneal injections of saline, nicotine (1.62 mg/kg) and/or -Btx (10μg/kg), respectively. All the animals were then scanned at dayα7, 14, 21, and 28 after ligatures placement. The alveolar bone loss below the roof of the furcation, and above the root apex of the second maxillary mola was three-dimensionally selected and analyzed. All rats were sacrificed at day 28. The maxillas were harvested and paraffin serial sections were cut. Hematoxylin and eosin examination, and immunohistochemical staining ofα7 nAChR, IL-1β, IL-6, TNF-α, RANKL, OPG and TRAP were performed.
Results: In vitro, The expression ofα7 nAChR was significantly up-regulated (about 4.α7-fold) by the administration of nicotine at the concentration of 10μM, and this effect could be partially suppressed by pretreatment with -Btx. Nicotine significantly induced the migration, but inhibited the proliferation and adhesion of cultured PDL cells in a dose dependent manner. Moreover, whileα7 nAChR expression by PDL cells was down-regulated by pretreatment of -Btx, abrogated functions of cell behaviors were observed.
In vivo, compared with the control group, nicotine administration up-regulated the number of TRAP positive cells in periodontal tissues, of which the periodontal destruction was correlated. However, the changes were insignificant when pretreated with -Btx. Nicotine alone caused moderate bone microstructure deteriorations begin with decreasing Tb.Th and increasing Tb.Sp, and latter the BVF and Tb.N loss. Nicotine combined with ligature induced robust bone loss that occurred earlier and last over time. The expression ofα7 nAChR in periodontal tissue was up-regulated by nicotine administration, whereas -Btx treatment partially suppressed this effect.
Both in vitro and in vivo, induced IL-1β, IL-6, TNF-α, RANKL and reduced OPG expression were also related with nicotine administration and partially suppressed byα-Btx pretreatment, which were correlated withα7 nAChR expression. In addition, ERK1/2 and PI3K inhibition significantly diminished nicotine induced IL-1β, IL-6 and TNF- expression in vitro.
Conclusion: The present studies demonstrate that nicotine inhibits human PDL cells attachment, proliferation and enhances PDL cells migration through activation ofα7 nAChR. In addition, Nicotine itself causes moderate alveolar bone microstructure deteriorations, and nicotine combined with ligature induces robust bone loss that occurred earlier and last over time. Furthermore, the nicotine induced IL-1β, IL-6, TNF- and RANKL expression, and reduced OPG expression were regulated by periodontalα7 nAChR, partially through ERK1/2 and PI3K pathway. All those effects are critical for the inflammatory reaction, periodontal destruction and alveolar bone loss in the process of periodontitis.
Our work indicates that nicotine may affect periodontal tissue destruction and inflammatory cytokines expression through regulation ofα7 nAChR in smoking-associated periodontitis, which may be pertinent to a better understanding of the relationships among smoking, nicotine, and periodontitis.
课题组前期研究表明,体外培养人牙周膜细胞中功能性表达α7 nAChR;本人前期研究发现大鼠牙周组织中存在尼古丁靶受体:α7型尼古丁乙酰胆碱受体(alphaα7 nicotinic acetylcholine receptor,α7 nAChR)的mRNA和蛋白表达,尼古丁可明显上调该受体的表达,且α7 nAChR表达调节与大鼠牙周组织破坏、牙槽骨吸收程度密切相关。然而,尼古丁激活α7 nAChR后如何对牙周组织产生破坏作用?其分子机制是什么?目前尚未见相关报道。
目的:本课题采用尼古丁作用下人牙周膜细胞和实验性牙周炎大鼠为模型,观察α7 nAChR对体外培养人牙周膜细胞增殖、贴附与迁移等生物学特性的调控,以及对牙周膜细胞和牙周组织分泌牙周炎相关炎性介质的影响,并进一步筛选和探明α7 nAChR激活后下游关键分子信号通路,以期阐明α7 nAChR在吸烟促进牙周炎发生发展中所扮演的角色及其作用机制。
材料与方法:体外研究,培养人牙周膜细胞,并分别给予α7 nAChR激动剂尼古丁(0.1-10μM)和/或拮抗剂-银环蛇毒素( -bungarotoxin, -BTX, 10nM);及ERK1/2信号通路抑制剂U0126(10μM)、P38信号通路抑制剂SB203580 (10μM), JNK信号通路抑制剂SP600125(10μM)和PI3K信号通路抑制剂LY-294,002(10-5μM)处理。分别采用WST-1、贴附实验和划痕修复实验测定细胞增殖、贴附与迁移能力改变;采用实时定量PCR、免疫荧光染色和Western blot等实验检测各组细胞α7 nAChR、IL-1?、IL-6、TNF-、RANKL和OPG等基因和蛋白表达,及ERK1/2和p-ERK1/2等磷酸化信号通路蛋白表达情况。
体内研究,24只SD大鼠采用两次完全随机分组方法分为4组,每组6只;采用3-0丝线结扎上颌左侧或右侧第二磨牙颈部,对侧不予结扎,作为自身对照;次日起分别给予生理盐水、尼古丁1.62 mg/kg/d、-BTX 10μg/kg/d、-BTX(10μg/kg/d)+30分钟后尼古丁(1.62 mg/kg/d)腹腔注射,常规饲养。全部大鼠于结扎前行麻醉下活体显微CT头部扫描,并分别于给药后α7、14、21和28天行活体显微CT头部扫描;采用Tri-Bon影像分析系统,重建各组大鼠颅颌面及牙体牙周组织影像;采用严格的标准化定位方法,创建一种基于“四根定位”的新型牙槽骨定量分析策略;定量分析各组大鼠牙槽骨骨密度、骨小梁厚度、骨体积分数等参数的动态改变;各组大鼠于给药后28天取材,解剖取出双侧上颌磨牙区组织,制作牙体牙周联合切片;HE染色进行病理学观察;免疫组织化学检测各组大鼠牙周组织α7 nAChR、IL-1?、IL-6、TNF-等蛋白表达情况。
结果:体外研究,实时定量PCR结果表明,10μM尼古丁作用下人牙周膜细胞α7 nAChR基因表达明显上调,约为对照组的4.α7倍(P < 0.001);-BTX +尼古丁处理组α7 nAChR基因表达水平与对照组近似,差异无统计学意义;免疫荧光染色和Western blot结果证实,体外培养正常人牙周膜细胞中α7 nAChR蛋白呈阳性表达;与对照组相比,尼古丁处理组α7 nAChR表达增强, -BTX预处理可拮抗尼古丁对α7 nAChR表达的上调作用;α7 nAChR激活可抑制细胞增殖和贴附,而促进其迁移。
体内研究,尼古丁作用下大鼠α7 nAChR表达明显上调,与之相对应的是,牙槽骨吸收亦明显加重,而-BTX预处理可拮抗尼古丁的作用;尼古丁单独作用下,大鼠牙槽骨显微结构呈现慢性破坏,其特征为首先出现骨小梁厚度降低且骨小梁分离度升高,后期出现骨体积分数和骨小梁数目降低;尼古丁给药结合丝线结扎可造成大鼠牙槽骨急剧丧失。
体内和体外研究均显示,尼古丁作用下人牙周膜细胞或大鼠牙周组织中α7 nAChR表达上调的同时,IL-1?、IL-6、TNF-和RANKL表达亦明显增强且OPG表达减弱;而ERK1/2和PI3K信号通路抑制剂可显著降低IL-1?、IL-6和TNF-的表达;尼古丁作用下人牙周膜细胞ERK1/2磷酸化水平明显增高。
结论:本研究证实α7 nAChR在体外培养人牙周膜细胞中存在功能性表达;发现尼古丁可通过作用于α7 nAChR调节牙周膜细胞增殖、贴附和迁移等生物学特性;人牙周膜细胞α7 nAChR激活后可通过作用于下游ERK1/2和PI3K信号通路影响IL-1?、IL-6和TNF-等炎性细胞因子分泌而参与牙周组织的炎症反应和牙槽骨吸收过程;大鼠牙周组织α7 nAChR激活可通过特定模式动态调控牙槽骨的显微结构改变。本研究表明尼古丁可通过作用于α7 nAChR而激活下游ERK1/2和PI3K信号转导通路,从而参与吸烟相关性牙周炎的发生和发展。
Background: Periodontitis, which causing periodontium destruction, pocket formation, alveolar bone resorption, and eventually teeth exfoliation, is one of the most common chronic diseases in adults. Numerous data demonstrated that tobacco smoking is one of the major risk factors for periodontitis. In addition, nicotine, the major component in tobacco smoke, has been proven to play an important role in smoking- associated periodontitis. However, the mechanisms behind the effects of nicotine on periodontal destruction are still not well understood.
Our previous studies demonstrated the functional expression of lphaα7 subunit of nicotinic acetylcholine receptors (α7 nAChR) in rat periodontal tissues. What’s more, nicotine acted directly and specifically throughα7 nAChR to affect periodontitis activity. However, initial data regarding how theα7 nAChR modulates nicotine’s effects on periodontitis, such as regulating cells behavior and pro-inflammatory cytokines expression of PDL cells in vitro, or affecting periodontal tissue destruction in vivo, are still lacking.
Objective: The aim of this study was to investigate how theα7 nAChR regulates nicotine’s effects on PDL cells behavior, inflammatory related cytokines expression and periodontal tissue destruction, and to explore the signalling pathway downstream ofα7 nAChR after its activation.
Materials and methods: In vitro, human PDL cells were cultured with or without different concentration of nicotine (Concentration ranging from 0.1μM to 10μM), alpha-bungarotoxin ( -Btx, inhibitor ofα7 nAChR, 10nM), U0126 (inhibitor of ERK1/2, 10μM), SB203580 (inhibitor of P38, 10μM), SP600125 (inhibitor of JNK, 10μM) and LY-294,002 (inhibitor of PI3K, 10μM). Cell proliferation, adhesion and migration were measured by WST-1 assay, attachment assay and wound healing assay, respectively. The expression ofα7 nAChR, IL-1?, IL-6, TNF- , RANKL and OPG in PDL cells were explored by Real Time-PCR, Immunofluorescence and Western blot assay.
In vivo, twenty-four male Sprague-Dawley rats were anesthetized and scanned by an in vivo micro-CT system (day 0, baseline). They were then tied by silk ligatures around cervixes of the second maxillary molar while the contra lateral one was left untreated. The rats were randomly divided into four groups, and received intraperitoneal injections of saline, nicotine (1.62 mg/kg) and/or -Btx (10μg/kg), respectively. All the animals were then scanned at dayα7, 14, 21, and 28 after ligatures placement. The alveolar bone loss below the roof of the furcation, and above the root apex of the second maxillary mola was three-dimensionally selected and analyzed. All rats were sacrificed at day 28. The maxillas were harvested and paraffin serial sections were cut. Hematoxylin and eosin examination, and immunohistochemical staining ofα7 nAChR, IL-1β, IL-6, TNF-α, RANKL, OPG and TRAP were performed.
Results: In vitro, The expression ofα7 nAChR was significantly up-regulated (about 4.α7-fold) by the administration of nicotine at the concentration of 10μM, and this effect could be partially suppressed by pretreatment with -Btx. Nicotine significantly induced the migration, but inhibited the proliferation and adhesion of cultured PDL cells in a dose dependent manner. Moreover, whileα7 nAChR expression by PDL cells was down-regulated by pretreatment of -Btx, abrogated functions of cell behaviors were observed.
In vivo, compared with the control group, nicotine administration up-regulated the number of TRAP positive cells in periodontal tissues, of which the periodontal destruction was correlated. However, the changes were insignificant when pretreated with -Btx. Nicotine alone caused moderate bone microstructure deteriorations begin with decreasing Tb.Th and increasing Tb.Sp, and latter the BVF and Tb.N loss. Nicotine combined with ligature induced robust bone loss that occurred earlier and last over time. The expression ofα7 nAChR in periodontal tissue was up-regulated by nicotine administration, whereas -Btx treatment partially suppressed this effect.
Both in vitro and in vivo, induced IL-1β, IL-6, TNF-α, RANKL and reduced OPG expression were also related with nicotine administration and partially suppressed byα-Btx pretreatment, which were correlated withα7 nAChR expression. In addition, ERK1/2 and PI3K inhibition significantly diminished nicotine induced IL-1β, IL-6 and TNF- expression in vitro.
Conclusion: The present studies demonstrate that nicotine inhibits human PDL cells attachment, proliferation and enhances PDL cells migration through activation ofα7 nAChR. In addition, Nicotine itself causes moderate alveolar bone microstructure deteriorations, and nicotine combined with ligature induces robust bone loss that occurred earlier and last over time. Furthermore, the nicotine induced IL-1β, IL-6, TNF- and RANKL expression, and reduced OPG expression were regulated by periodontalα7 nAChR, partially through ERK1/2 and PI3K pathway. All those effects are critical for the inflammatory reaction, periodontal destruction and alveolar bone loss in the process of periodontitis.
Our work indicates that nicotine may affect periodontal tissue destruction and inflammatory cytokines expression through regulation ofα7 nAChR in smoking-associated periodontitis, which may be pertinent to a better understanding of the relationships among smoking, nicotine, and periodontitis.
引文
1 Bergstrom J. Tobacco smoking and chronic destructive periodontal disease. Odontology 2004; 92: 1-8.
2 Bosco AF, Bonfante S, de Almeida JM, Luize DS, Nagata MJ, Garcia VG. A histologic and histometric assessment of the influence of nicotine on alveolar bone loss in rats. J Periodontol 2007; 78: 527-532.
3 Nogueira-Filho GR, Froes Neto EB, Casati MZ, Reis SR, Tunes RS, Tunes UR, Sallum EA, Nociti FH, Jr., Sallum AW. Nicotine effects on alveolar bone changes induced by occlusal trauma: a histometric study in rats. J Periodontol 2004; 75: 348-352.
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5 Saldanha JB, Pimentel SP, Casati MZ, Sallum EA, Barbieri D, Moreno HJ, Nociti FH. Guided bone regeneration may be negatively influenced by nicotine administration: a histologic study in dogs. J Periodontol 2004; 75: 565-571.
6 Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. Receptor- mediated tobacco toxicity: cooperation of the Ras/Raf- 1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of alpha7 nicotinic receptor in oral keratinocytes. FASEB J 2006; 20: 2093-2101.
7 Chernyavsky AI, Arredondo J, Karlsson E, Wessler I, Grando SA. TheRas/Raf-1/MEK1/ERK signaling pathway coupled to integrin expression mediates cholinergic regulation of keratinocyte directional migration. J Biol Chem 2005; 280: 39220-39228.
8 Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. Receptor-mediated tobacco toxicity: alterations of the NF-kappaB expression and activity downstream of alpha7 nicotinic receptor in oral keratinocytes. Life Sci 2007; 80: 2191-2194.
9全国牙病防治指导组.第3次全国口腔健康流行病学抽样调查北京:人民卫生出版社,2005
10 Vered Y, Livny A, Zini A, Sgan-Cohen HD. Periodontal health status and smoking among young adults. J Clin Periodontol 2008; 35: 768-772.
11 Bergstrom J. Periodontitis and smoking: an evidence-based appraisal. J Evid Based Dent Pract 2006; 6: 33-41.
12 Johnson GK, Hill M. Cigarette smoking and the periodontal patient. J Periodontol 2004; 75: 196-209.
13 Shimazaki Y, Saito T, Kiyohara Y, Kato I, Kubo M, Iida M, Yamashita Y. The influence of current and former smoking on gingival bleeding: the Hisayama study. J Periodontol 2006; 77: 1430-1435.
14 Dietrich T, Bernimoulin JP, Glynn RJ. The effect of cigarette smoking on gingival bleeding. J Periodontol 2004; 75: 16-22.
15 Morozumi T, Kubota T, Sato T, Okuda K, Yoshie H. Smoking cessation increases gingival blood flow and gingival crevicular fluid. J Clin Periodontol 2004; 31: 267-272.
16 Nair P, Sutherland G, Palmer RM, Wilson RF, Scott DA. Gingival bleeding on probing increases after quitting smoking. J Clin Periodontol 2003; 30: 435-437.
17 Kurtis B, Tuter G, Serdar M, Akdemir P, Uygur C, Firatli E, Bal B. Gingival crevicular fluid levels of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in patients with chronic and aggressive periodontitis. J Periodontol 2005; 76: 1849-1855.
18 Hou LT, Liu CM, Liu BY, Lin SJ, Liao CS, Rossomando EF. Interleukin-1beta, clinical parameters and matched cellular-histopathologic changes of biopsied gingival tissue from periodontitis patients. J Periodontal Res 2003; 38: 247-254.
19 Rawlinson A, Grummitt JM, Walsh TF, Ian Douglas CW. Interleukin 1 and receptor antagonist levels in gingival crevicular fluid in heavy smokers versus non-smokers. J Clin Periodontol 2003; 30: 42-48.
20 Tomar SL, Asma S. Smoking-attributable periodontitis in the United States: findings from NHANES III. National Health and Nutrition Examination Survey. J Periodontol 2000; 71: 743-751.
21 Susin C, Dalla Vecchia CF, Oppermann RV, Haugejorden O, Albandar JM. Periodontal attachment loss in an urban population of Brazilian adults: effect of demographic, behavioral, and environmental risk indicators. J Periodontol 2004; 75: 1033-1041.
22 Lappin DF, Sherrabeh S, Jenkins WM, Macpherson LM. Effect of smoking on serum RANKL and OPG in sex, age and clinically matched supportive-therapy periodontitis patients. J Clin Periodontol 2007; 34: 271-277.
23 Tang TH, Fitzsimmons TR, Bartold PM. Effect of smoking on concentrations of receptor activator of nuclear factor kappa B ligand and osteoprotegerin in human gingival crevicular fluid. J Clin Periodontol 2009; 36: 713-718.
24 Buduneli N, Baylas H, Buduneli E, Turkoglu O, Dahlen G. Evaluation of therelationship between smoking during pregnancy and subgingival microbiota. J Clin Periodontol 2005; 32: 68-74.
25 Haffajee AD, Socransky SS. Relationship of cigarette smoking to the subgingival microbiota. J Clin Periodontol 2001; 28: 377-388.
26 Gomes SC, Piccinin FB, Oppermann RV, Susin C, Nonnenmacher CI, Mutters R, Marcantonio RA. Periodontal status in smokers and never-smokers: clinical findings and real-time polymerase chain reaction quantification of putative periodontal pathogens. J Periodontol 2006; 77: 1483-1490.
27 Kubota M, Tanno-Nakanishi M, Yamada S, Okuda K, Ishihara K. Effect of smoking on subgingival microflora of patients with periodontitis in Japan. BMC Oral Health 2011; 11: 1.
28 Fullmer SC, Preshaw PM, Heasman PA, Kumar PS. Smoking cessation alters subgingival microbial recolonization. J Dent Res 2009; 88: 524-528.
29 Bergstrom J. Tobacco smoking and subgingival dental calculus. J Clin Periodontol 2005; 32: 81-88.
30 Ohyama H, Kato-Kogoe N, Kuhara A, Nishimura F, Nakasho K, Yamanegi K, Yamada N, Hata M, Yamane J, Terada N. The involvement of IL-23 and the Th17 pathway in periodontitis. J Dent Res 2009; 88: 633-638.
31 Gaffen SL, Hajishengallis G. A new inflammatory cytokine on the block: re-thinking periodontal disease and the Th1/Th2 paradigm in the context of Th17 cells and IL-17. J Dent Res 2008; 87: 817-828.
32 Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, Ellen RP, Penninger JM. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 2000; 106: R59-67.
33 de Heens GL, van der Velden U, Loos BG. Cigarette smoking enhances T cell activation and a Th2 immune response; an aspect of the pathophysiology in periodontal disease. Cytokine 2009; 47: 157-161.
34 Botelho FM, Gaschler GJ, Kianpour S, Zavitz CC, Trimble NJ, Nikota JK, Bauer CM, Stampfli MR. Innate immune processes are sufficient for driving cigarette smoke-induced inflammation in mice. Am J Respir Cell Mol Biol 2010; 42: 394-403.
35 Tymkiw KD, Thunell DH, Johnson GK, Joly S, Burnell KK, Cavanaugh JE, Brogden KA, Guthmiller JM. Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis. J Clin Periodontol 2011; 38: 219-228.
36 Preshaw PM, Heasman L, Stacey F, Steen N, McCracken GI, Heasman PA. The effect of quitting smoking on chronic periodontitis. J Clin Periodontol 2005; 32: 869-879.
37 Poggi P, Rota MT, Boratto R. The volatile fraction of cigarette smoke induces alterations in the human gingival fibroblast cytoskeleton. J Periodontal Res 2002; 37: 230-235.
38 Tomasi C, Leyland AH, Wennstrom JL. Factors influencing the outcome of non-surgical periodontal treatment: a multilevel approach. J Clin Periodontol 2007; 34: 682-690.
39 Roos-Jansaker AM. Long time follow up of implant therapy and treatment of peri-implantitis. Swed Dent J Suppl 2007: 7-66.
40 Chung DM, Oh TJ, Lee J, Misch CE, Wang HL. Factors affecting late implant bone loss: a retrospective analysis. Int J Oral Maxillofac Implants 2007; 22: 117-126.
41 Mascarenhas P, Gapski R, Al-Shammari K, Hill R, Soehren S, Fenno JC,Giannobile WV, Wang HL. Clinical response of azithromycin as an adjunct to non-surgical periodontal therapy in smokers. J Periodontol 2005; 76: 426-436.
42 Filoche SK, Cornford E, Gaudie W, Wong M, Heasman P, Thomson WM. Smoking, chronic periodontitis and smoking cessation support: reviewing the role of dental professionals. N Z Dent J 2010; 106: 74-77.
43 Hodge P, Binnie V. Smoking cessation and periodontal health--a missed opportunity? Evid Based Dent 2009; 10: 18-19.
44 Yanagisawa T, Ueno M, Shinada K, Ohara S, Wright FA, Kawaguchi Y. Relationship of smoking and smoking cessation with oral health status in Japanese men. J Periodontal Res 2010; 45: 277-283.
45 Arora M, Schwarz E, Sivaneswaran S, Banks E. Cigarette smoking and tooth loss in a cohort of older Australians: the 45 and up study. J Am Dent Assoc 2010; 141: 1242-1249.
46 Hanes PJ, Schuster GS, Lubas S. Binding, uptake, and release of nicotine by human gingival fibroblasts. J Periodontol 1991; 62: 147-152.
47 Tipton DA, Dabbous MK. Effects of nicotine on proliferation and extracellular matrix production of human gingival fibroblasts in vitro. J Periodontol 1995; 66: 1056-1064.
48 Tanur E, McQuade MJ, McPherson JC, Al-Hashimi IH, Rivera-Hidalgo F. Effects of nicotine on the strength of attachment of gingival fibroblasts to glass and non-diseased human root surfaces. J Periodontol 2000; 71: 717-722.
49 Benatti BB, Nogueira-Filho GR, Diniz MC, Sallum EA, Sallum AW, Nociti FH, Jr. Stress may enhance nicotine effects on periodontal tissues. An in vivo study in rats. J Periodontal Res 2003; 38: 351-353.
50 Benatti BB, Cesar-Neto JB, Goncalves PF, Sallum EA, Nociti FH, Jr. Smoking affects the self-healing capacity of periodontal tissues. A histological study in the rat. Eur J Oral Sci 2005; 113: 400-403.
51 Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans. Br J Pharmacol 2008; 154: 1558-1571.
52 Grando SA, Kawashima K, Wessler I. Introduction: the non-neuronal cholinergic system in humans. Life Sci 2003; 72: 2009-2012.
53 Yoshida M, Masunaga K, Satoji Y, Maeda Y, Nagata T, Inadome A. Basic and clinical aspects of non-neuronal acetylcholine: expression of non-neuronal acetylcholine in urothelium and its clinical significance. J Pharmacol Sci 2008; 106: 193-198.
54 Song P, Spindel ER. Basic and clinical aspects of non-neuronal acetylcholine: expression of non-neuronal acetylcholine in lung cancer provides a new target for cancer therapy. J Pharmacol Sci 2008; 106: 180-185.
55 Kawashima K, Fujii T. Basic and clinical aspects of non-neuronal acetylcholine: overview of non-neuronal cholinergic systems and their biological significance. J Pharmacol Sci 2008; 106: 167-173.
56 Grando SA. Basic and clinical aspects of non-neuronal acetylcholine: biological and clinical significance of non-canonical ligands of epithelial nicotinic acetylcholine receptors. J Pharmacol Sci 2008; 106: 174-179.
57 Fujii T, Takada-Takatori Y, Kawashima K. Basic and clinical aspects of non-neuronal acetylcholine: expression of an independent, non-neuronal cholinergic system in lymphocytes and its clinical significance in immunotherapy. J Pharmacol Sci 2008; 106: 186-192.
58 Nguyen VT, Hall LL, Gallacher G, Ndoye A, Jolkovsky DL, Webber RJ, Buchli R, Grando SA. Choline acetyltransferase, acetylcholinesterase, andnicotinic acetylcholine receptors of human gingival and esophageal epithelia. J Dent Res 2000; 79: 939-949.
59 Arredondo J, Hall LL, Ndoye A, Chernyavsky AI, Jolkovsky DL, Grando SA. Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes. J Periodontal Res 2003; 38: 79-89.
60 Dussor GO, Leong AS, Gracia NB, Kilo S, Price TJ, Hargreaves KM, Flores CM. Potentiation of evoked calcitonin gene-related peptide release from oral mucosa: a potential basis for the pro-inflammatory effects of nicotine. Eur J Neurosci 2003; 18: 2515-2526.
61 Yamane A, Ohnuki Y, Saeki Y. Developmental changes in the nicotinic acetylcholine receptor in mouse tongue striated muscle. J Dent Res 2001; 80: 1840-1844.
62 Saito T, Ohnuki Y, Saeki Y, Nakagawa Y, Ishibashi K, Yanagisawa K, Yamane A. Postnatal changes in the nicotinic acetylcholine receptor subunits in rat masseter muscle. Arch Oral Biol 2002; 47: 417-421.
63 Simons CT, Sudo S, Sudo M, Carstens E. Mecamylamine reduces nicotine cross-desensitization of trigeminal caudalis neuronal responses to oral chemical irritation. Brain Res 2003; 991: 249-253.
64 Arredondo J, Chernyavsky AI, Marubio LM, Beaudet AL, Jolkovsky DL, Pinkerton KE, Grando SA. Receptor-mediated tobacco toxicity: regulation of gene expression through alpha3beta2 nicotinic receptor in oral epithelial cells. Am J Pathol 2005; 166: 597-613.
65 Garcia de Aquino S, Manzolli Leite FR, Stach-Machado DR, Francisco da Silva JA, Spolidorio LC, Rossa C, Jr. Signaling pathways associated with the expression of inflammatory mediators activated during the course of twomodels of experimental periodontitis. Life Sci 2009; 84: 745-754.
66 Guan SM, Zhang M, He JJ, Wu JZ. Mitogen-activated protein kinases and phosphatidylinositol 3-kinase are involved in Prevotella intermedia-induced proinflammatory cytokines expression in human periodontal ligament cells. Biochem Biophys Res Commun 2009; 386: 471-476.
67 Oikawa A, Kobayashi M, Okamatsu Y, Shinki T, Kamijo R, Yamamoto M, Hasegawa K. Mitogen-activated protein kinases mediate interleukin- 1beta-induced receptor activator of nuclear factor-kappaB ligand expression in human periodontal ligament cells. J Periodontal Res 2007; 42: 367-376.
68 Lee HJ, Cho JW, Kim SC, Kang KH, Lee SK, Pi SH, Kim EC. Roles of p38 and ERK MAP kinases in IL-8 expression in TNF-alpha- and dexamethasone-stimulated human periodontal ligament cells. Cytokine 2006; 35: 67-76.
69 El Kouhen R, Hu M, Anderson DJ, Li J, Gopalakrishnan M. Pharmacology of alpha7 nicotinic acetylcholine receptor mediated extracellular signal-regulated kinase signalling in PC12 cells. Br J Pharmacol 2009; 156: 638-648.
70 Paleari L, Catassi A, Ciarlo M, Cavalieri Z, Bruzzo C, Servent D, Cesario A, Chessa L, Cilli M, Piccardi F, Granone P, Russo P. Role of alpha7-nicotinic acetylcholine receptor in human non-small cell lung cancer proliferation. Cell Prolif 2008; 41: 936-959.
71 Wada T, Naito M, Kenmochi H, Tsuneki H, Sasaoka T. Chronic nicotine exposure enhances insulin-induced mitogenic signaling via up-regulation of alpha7 nicotinic receptors in isolated rat aortic smooth muscle cells. Endocrinology 2007; 148: 790-799.
72 Darby IB, Hodge PJ, Riggio MP, Kinane DF. Clinical and microbiologicaleffect of scaling and root planing in smoker and non-smoker chronic and aggressive periodontitis patients. J Clin Periodontol 2005; 32: 200-206.
73 Heasman L, Stacey F, Preshaw PM, McCracken GI, Hepburn S, Heasman PA. The effect of smoking on periodontal treatment response: a review of clinical evidence. J Clin Periodontol 2006; 33: 241-253.
74 Nociti FH, Jr., Nogueira-Filho GR, Tramontina VA, Machado MA, Barros SP, Sallum EA, Sallum AW. Histometric evaluation of the effect of nicotine administration on periodontal breakdown: an in vivo study. J Periodontal Res 2001; 36: 361-366.
75 Liu YF, Wu LA, Wang J, Wen LY, Wang XJ. Micro-computerized tomography analysis of alveolar bone loss in ligature- and nicotine-induced experimental periodontitis in rats. J Periodontal Res 2010.
76 Gamal AY, Bayomy MM. Effect of cigarette smoking on human PDL fibroblasts attachment to periodontally involved root surfaces in vitro. J Clin Periodontol 2002; 29: 763-770.
77 Giannopoulou C, Roehrich N, Mombelli A. Effect of nicotine-treated epithelial cells on the proliferation and collagen production of gingival fibroblasts. J Clin Periodontol 2001; 28: 769-775.
78 Wang XJ, Liu YF, Wang QY, Tsuruoka M, Ohta K, Wu SX, Yakushiji M, Inoue T. Functional expression of alpha 7 nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and rat periodontal tissues. Cell Tissue Res 2010; 340: 347-355.
79 Yanagita M, Kojima Y, Kawahara T, Kajikawa T, Oohara H, Takedachi M, Yamada S, Murakami S. Suppressive effects of nicotine on the cytodifferentiation of murine periodontal ligament cells. Oral Dis 2010.
80 Cannon GB, McCoy JL, Jerome LJ, Reddick R, Alford C, Tinley V,Herberman RB. Immunologic relationship between breast carcinoma and benign breast disease as detected by the leukocyte migration inhibition assay. J Natl Cancer Inst 1978; 61: 1181-1186.
81 Hatakeyama J, Hatakeyama Y, Takahashi I, Suzuki O, Sasano Y. Proliferation and adhesion of periodontal ligament cells on synthetic biominerals. Oral Dis 2007; 13: 500-506.
82 Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329-333.
83 Chang YC, Lii CK, Tai KW, Chou MY. Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitro. J Clin Periodontol 2001; 28: 277-282.
84 Chang YC, Huang FM, Tai KW, Yang LC, Chou MY. Mechanisms of cytotoxicity of nicotine in human periodontal ligament fibroblast cultures in vitro. J Periodontal Res 2002; 37: 279-285.
85 Ghannad F, Nica D, Fulle MI, Grenier D, Putnins EE, Johnston S, Eslami A, Koivisto L, Jiang G, McKee MD, Hakkinen L, Larjava H. Absence of alphavbeta6 integrin is linked to initiation and progression of periodontal disease. Am J Pathol 2008; 172: 1271-1286.
86 Crawford JM, Watanabe K. Cell adhesion molecules in inflammation and immunity: relevance to periodontal diseases. Crit Rev Oral Biol Med 1994; 5: 91-123.
87 Liu YC, Lerner UH, Teng YT. Cytokine responses against periodontal infection: protective and destructive roles. Periodontol 2000 2010; 52: 163-206.
88 Teng YT. Protective and destructive immunity in the periodontium: Part1--innate and humoral immunity and the periodontium. J Dent Res 2006; 85: 198-208.
89 Teng YT. Protective and destructive immunity in the periodontium: Part 2--T-cell-mediated immunity in the periodontium. J Dent Res 2006; 85: 209-219.
90 Liu YF, Ge X, Wen LY, Wang XJ. Targeting nicotinic acetylcholine receptor to treat smoking-related periodontitis. Med Hypotheses 2011; 76: 244-245.
91 Park CH, Abramson ZR, Taba M, Jr., Jin Q, Chang J, Kreider JM, Goldstein SA, Giannobile WV. Three-dimensional micro-computed tomographic imaging of alveolar bone in experimental bone loss or repair. J Periodontol 2007; 78: 273-281.
92 Cantley MD, Bartold PM, Marino V, Reid RC, Fairlie DP, Wyszynski RN, Zilm PS, Haynes DR. The use of live-animal micro-computed tomography to determine the effect of a novel phospholipase A2 inhibitor on alveolar bone loss in an in vivo mouse model of periodontitis. J Periodontal Res 2009; 44: 317-322.
2 Bosco AF, Bonfante S, de Almeida JM, Luize DS, Nagata MJ, Garcia VG. A histologic and histometric assessment of the influence of nicotine on alveolar bone loss in rats. J Periodontol 2007; 78: 527-532.
3 Nogueira-Filho GR, Froes Neto EB, Casati MZ, Reis SR, Tunes RS, Tunes UR, Sallum EA, Nociti FH, Jr., Sallum AW. Nicotine effects on alveolar bone changes induced by occlusal trauma: a histometric study in rats. J Periodontol 2004; 75: 348-352.
4 Torrungruang K, Nisapakultorn K, Sutdhibhisal S, Tamsailom S, Rojanasomsith K, Vanichjakvong O, Prapakamol S, Premsirinirund T, Pusiri T, Jaratkulangkoon O, Kusump S, Rajatanavin R. The effect of cigarette smoking on the severity of periodontal disease among older Thai adults. J Periodontol 2005; 76: 566-572.
5 Saldanha JB, Pimentel SP, Casati MZ, Sallum EA, Barbieri D, Moreno HJ, Nociti FH. Guided bone regeneration may be negatively influenced by nicotine administration: a histologic study in dogs. J Periodontol 2004; 75: 565-571.
6 Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. Receptor- mediated tobacco toxicity: cooperation of the Ras/Raf- 1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of alpha7 nicotinic receptor in oral keratinocytes. FASEB J 2006; 20: 2093-2101.
7 Chernyavsky AI, Arredondo J, Karlsson E, Wessler I, Grando SA. TheRas/Raf-1/MEK1/ERK signaling pathway coupled to integrin expression mediates cholinergic regulation of keratinocyte directional migration. J Biol Chem 2005; 280: 39220-39228.
8 Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. Receptor-mediated tobacco toxicity: alterations of the NF-kappaB expression and activity downstream of alpha7 nicotinic receptor in oral keratinocytes. Life Sci 2007; 80: 2191-2194.
9全国牙病防治指导组.第3次全国口腔健康流行病学抽样调查北京:人民卫生出版社,2005
10 Vered Y, Livny A, Zini A, Sgan-Cohen HD. Periodontal health status and smoking among young adults. J Clin Periodontol 2008; 35: 768-772.
11 Bergstrom J. Periodontitis and smoking: an evidence-based appraisal. J Evid Based Dent Pract 2006; 6: 33-41.
12 Johnson GK, Hill M. Cigarette smoking and the periodontal patient. J Periodontol 2004; 75: 196-209.
13 Shimazaki Y, Saito T, Kiyohara Y, Kato I, Kubo M, Iida M, Yamashita Y. The influence of current and former smoking on gingival bleeding: the Hisayama study. J Periodontol 2006; 77: 1430-1435.
14 Dietrich T, Bernimoulin JP, Glynn RJ. The effect of cigarette smoking on gingival bleeding. J Periodontol 2004; 75: 16-22.
15 Morozumi T, Kubota T, Sato T, Okuda K, Yoshie H. Smoking cessation increases gingival blood flow and gingival crevicular fluid. J Clin Periodontol 2004; 31: 267-272.
16 Nair P, Sutherland G, Palmer RM, Wilson RF, Scott DA. Gingival bleeding on probing increases after quitting smoking. J Clin Periodontol 2003; 30: 435-437.
17 Kurtis B, Tuter G, Serdar M, Akdemir P, Uygur C, Firatli E, Bal B. Gingival crevicular fluid levels of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in patients with chronic and aggressive periodontitis. J Periodontol 2005; 76: 1849-1855.
18 Hou LT, Liu CM, Liu BY, Lin SJ, Liao CS, Rossomando EF. Interleukin-1beta, clinical parameters and matched cellular-histopathologic changes of biopsied gingival tissue from periodontitis patients. J Periodontal Res 2003; 38: 247-254.
19 Rawlinson A, Grummitt JM, Walsh TF, Ian Douglas CW. Interleukin 1 and receptor antagonist levels in gingival crevicular fluid in heavy smokers versus non-smokers. J Clin Periodontol 2003; 30: 42-48.
20 Tomar SL, Asma S. Smoking-attributable periodontitis in the United States: findings from NHANES III. National Health and Nutrition Examination Survey. J Periodontol 2000; 71: 743-751.
21 Susin C, Dalla Vecchia CF, Oppermann RV, Haugejorden O, Albandar JM. Periodontal attachment loss in an urban population of Brazilian adults: effect of demographic, behavioral, and environmental risk indicators. J Periodontol 2004; 75: 1033-1041.
22 Lappin DF, Sherrabeh S, Jenkins WM, Macpherson LM. Effect of smoking on serum RANKL and OPG in sex, age and clinically matched supportive-therapy periodontitis patients. J Clin Periodontol 2007; 34: 271-277.
23 Tang TH, Fitzsimmons TR, Bartold PM. Effect of smoking on concentrations of receptor activator of nuclear factor kappa B ligand and osteoprotegerin in human gingival crevicular fluid. J Clin Periodontol 2009; 36: 713-718.
24 Buduneli N, Baylas H, Buduneli E, Turkoglu O, Dahlen G. Evaluation of therelationship between smoking during pregnancy and subgingival microbiota. J Clin Periodontol 2005; 32: 68-74.
25 Haffajee AD, Socransky SS. Relationship of cigarette smoking to the subgingival microbiota. J Clin Periodontol 2001; 28: 377-388.
26 Gomes SC, Piccinin FB, Oppermann RV, Susin C, Nonnenmacher CI, Mutters R, Marcantonio RA. Periodontal status in smokers and never-smokers: clinical findings and real-time polymerase chain reaction quantification of putative periodontal pathogens. J Periodontol 2006; 77: 1483-1490.
27 Kubota M, Tanno-Nakanishi M, Yamada S, Okuda K, Ishihara K. Effect of smoking on subgingival microflora of patients with periodontitis in Japan. BMC Oral Health 2011; 11: 1.
28 Fullmer SC, Preshaw PM, Heasman PA, Kumar PS. Smoking cessation alters subgingival microbial recolonization. J Dent Res 2009; 88: 524-528.
29 Bergstrom J. Tobacco smoking and subgingival dental calculus. J Clin Periodontol 2005; 32: 81-88.
30 Ohyama H, Kato-Kogoe N, Kuhara A, Nishimura F, Nakasho K, Yamanegi K, Yamada N, Hata M, Yamane J, Terada N. The involvement of IL-23 and the Th17 pathway in periodontitis. J Dent Res 2009; 88: 633-638.
31 Gaffen SL, Hajishengallis G. A new inflammatory cytokine on the block: re-thinking periodontal disease and the Th1/Th2 paradigm in the context of Th17 cells and IL-17. J Dent Res 2008; 87: 817-828.
32 Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, Ellen RP, Penninger JM. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 2000; 106: R59-67.
33 de Heens GL, van der Velden U, Loos BG. Cigarette smoking enhances T cell activation and a Th2 immune response; an aspect of the pathophysiology in periodontal disease. Cytokine 2009; 47: 157-161.
34 Botelho FM, Gaschler GJ, Kianpour S, Zavitz CC, Trimble NJ, Nikota JK, Bauer CM, Stampfli MR. Innate immune processes are sufficient for driving cigarette smoke-induced inflammation in mice. Am J Respir Cell Mol Biol 2010; 42: 394-403.
35 Tymkiw KD, Thunell DH, Johnson GK, Joly S, Burnell KK, Cavanaugh JE, Brogden KA, Guthmiller JM. Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis. J Clin Periodontol 2011; 38: 219-228.
36 Preshaw PM, Heasman L, Stacey F, Steen N, McCracken GI, Heasman PA. The effect of quitting smoking on chronic periodontitis. J Clin Periodontol 2005; 32: 869-879.
37 Poggi P, Rota MT, Boratto R. The volatile fraction of cigarette smoke induces alterations in the human gingival fibroblast cytoskeleton. J Periodontal Res 2002; 37: 230-235.
38 Tomasi C, Leyland AH, Wennstrom JL. Factors influencing the outcome of non-surgical periodontal treatment: a multilevel approach. J Clin Periodontol 2007; 34: 682-690.
39 Roos-Jansaker AM. Long time follow up of implant therapy and treatment of peri-implantitis. Swed Dent J Suppl 2007: 7-66.
40 Chung DM, Oh TJ, Lee J, Misch CE, Wang HL. Factors affecting late implant bone loss: a retrospective analysis. Int J Oral Maxillofac Implants 2007; 22: 117-126.
41 Mascarenhas P, Gapski R, Al-Shammari K, Hill R, Soehren S, Fenno JC,Giannobile WV, Wang HL. Clinical response of azithromycin as an adjunct to non-surgical periodontal therapy in smokers. J Periodontol 2005; 76: 426-436.
42 Filoche SK, Cornford E, Gaudie W, Wong M, Heasman P, Thomson WM. Smoking, chronic periodontitis and smoking cessation support: reviewing the role of dental professionals. N Z Dent J 2010; 106: 74-77.
43 Hodge P, Binnie V. Smoking cessation and periodontal health--a missed opportunity? Evid Based Dent 2009; 10: 18-19.
44 Yanagisawa T, Ueno M, Shinada K, Ohara S, Wright FA, Kawaguchi Y. Relationship of smoking and smoking cessation with oral health status in Japanese men. J Periodontal Res 2010; 45: 277-283.
45 Arora M, Schwarz E, Sivaneswaran S, Banks E. Cigarette smoking and tooth loss in a cohort of older Australians: the 45 and up study. J Am Dent Assoc 2010; 141: 1242-1249.
46 Hanes PJ, Schuster GS, Lubas S. Binding, uptake, and release of nicotine by human gingival fibroblasts. J Periodontol 1991; 62: 147-152.
47 Tipton DA, Dabbous MK. Effects of nicotine on proliferation and extracellular matrix production of human gingival fibroblasts in vitro. J Periodontol 1995; 66: 1056-1064.
48 Tanur E, McQuade MJ, McPherson JC, Al-Hashimi IH, Rivera-Hidalgo F. Effects of nicotine on the strength of attachment of gingival fibroblasts to glass and non-diseased human root surfaces. J Periodontol 2000; 71: 717-722.
49 Benatti BB, Nogueira-Filho GR, Diniz MC, Sallum EA, Sallum AW, Nociti FH, Jr. Stress may enhance nicotine effects on periodontal tissues. An in vivo study in rats. J Periodontal Res 2003; 38: 351-353.
50 Benatti BB, Cesar-Neto JB, Goncalves PF, Sallum EA, Nociti FH, Jr. Smoking affects the self-healing capacity of periodontal tissues. A histological study in the rat. Eur J Oral Sci 2005; 113: 400-403.
51 Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans. Br J Pharmacol 2008; 154: 1558-1571.
52 Grando SA, Kawashima K, Wessler I. Introduction: the non-neuronal cholinergic system in humans. Life Sci 2003; 72: 2009-2012.
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