Resistance to Cigarette Smoke Is Increased in Periodontal Ligament Cells by Attachment to Collagen and Fibronectin

Background: The toxic effects of cigarette smoke often presents in smokers as increased incidence and severity of periodontal disease. These patients demonstrate symptomatic inflammation, increased probing depth, and tooth loss likely attributable to the direct effects of cigarette smoke on periodontal ligament (PDL) fibroblasts. The goal of this in vitro study is to investigate the direct effects of smoking on PDL fibroblasts, focusing on cell–extracellular matrix (ECM) interactions and cell survival. Methods: PDL cells were plated for various times on tissue culture plastic, PDL‐derived ECMs, collagen Type I, or fibronectin. Cells were exposed to various concentrations of cigarette smoke extract (CSE) at different times during the cell attachment process. Subsequently, cell survival was quantified using calcein‐acetoxymethyl ester compound and a fluorescent plate reader. Results: After exposure to CSE, PDL cell survival increased with increased cell attachment time to plastic. These observations were independent of soluble factors present in PDL cell–conditioned media. PDL‐derived ECMs and collagen Type I–pretreated plates promoted increased cell survival after 1 day of cell attachment. Fibronectin‐pretreated plates demonstrated increased cell survival after 3 days of cell attachment. Conclusions: Cell–ECM interactions increase survival of PDL cells exposed to CSE. It is suggested that the increased survival is attributable to PDL cells altering their ECM, potentially by depositing collagen and fibronectin. This may imply that cells embedded in an ECM would be more resistant to the toxic effects of cigarette smoke, leading to increased cell death near the exposed edges of a wound.     

Tobacco‐product usage as a risk factor for dental implants

The oral cavities of tobacco smokers and users of smokeless tobacco products are exposed to high concentrations of nicotine. A limited number of animal studies have assessed the effect of nicotine on osseointegration. Results from experimental studies have reported a statistically significant decrease, at 4 weeks of follow‐up, in bone‐to‐implant contact among rats exposed to nicotine compared with unexposed rats. Nicotine increases the production of inflammatory cytokines (such as interleukin‐6 and tumor necrosis factor‐alpha) by osteoblasts. Waterpipe, pipe, and cigarette smokers are at increased risk of developing oral cancer, periodontal disease, and alveolar bone loss. One explanation for this is that smokers (regardless of the type of tobacco product) are exposed to similar chemicals, such as nicotine, tar, oxidants, polyaromatic hydrocarbons, and carbon monoxide. Moreover, raised levels of proinflammatory cytokines have been identified in the gingival crevicular fluid of cigarette smokers with peri‐implant diseases. Therefore, it is hypothesized that nicotine and chemicals in tobacco smoke induce a state of oxidative stress in peri‐implant tissues (gingiva and alveolar bone), thereby increasing the likelihood of peri‐implant disease development via an inflammatory response, which if left uncontrolled, will result in implant failure/loss. In this regard, tobacco smoking (including cigarettes, waterpipe, and pipe) is a significant risk factor for peri‐implant diseases. The impact of vaping electronic cigarettes using nicotine‐containing e‐juices remains unknown. Habitual use of smokeless tobacco products is associated with oral inflammatory conditions, such as oral precancer, cancer, and periodontal disease. However, the effect of habitual use of smokeless tobacco products on the success and survival of dental implants remains undocumented.     

Altered cell motility and attachment with titanium surface modifications

Background: Titanium implants are widely used in dentistry to replace lost teeth. Various surface modifications have been used to improve implant retention and osseointegration. This study is designed to compare the ability of three titanium surfaces to promote cell attachment and cell motility of cells relevant to periodontal tissues. Methods: Three clinically relevant surfaces were tested: 1) machined titanium; 2) a titanium surface roughened through acid etching (dual thermal‐etched titanium [DTET]); and 3) a titanium surface roughened with nanometer‐scale calcium phosphate deposition (nanoscale calcium phosphate–impregnated titanium [NCPIT]). Cell attachment and migration were examined for four cell types: rat osteosarcoma cells, human osteoblasts, and gingival and periodontal ligament (PDL) fibroblasts. Results: All four cell types attached to each of the three titanium surfaces equally by 2 hours, and the PDL and gingival fibroblasts generally displayed less attachment than the osteosarcoma cells and osteoblasts. The cells displayed differential motility and long‐term attachment to each of the titanium surfaces. Osteosarcoma cells displayed preferential motility on NCPIT, whereas PDL fibroblasts were more motile on machined titanium, and gingival fibroblasts moved more rapidly on both DTET and NCPIT. Osteoblasts displayed little motility on any of the titanium surfaces and lost viability on NCPIT after 24 hours. Gingival fibroblasts lost attachment to machined titanium. Conclusions: Periodontal cells displayed differential motility and long‐term attachment to titanium surfaces. Selective modification of titanium surface properties in various regions of an implant may be useful in guiding specific cell populations to specific locations where they might best aid in osseointegration and soft tissue remodeling.     

Effects of cigarette smoke and nicotine on cell viability,migration and myofibroblastic differentiation

Silva D, Cáceres M, Arancibia R, Martínez C, Martínez J, Smith P. Effects of cigarette smoke and nicotine on cell viability, migration and myofibroblastic differentiation. J Periodont Res 2012; 47: 599–607. © 2012 John Wiley & Sons A/S Background and Objective: Several studies have analysed the role of nicotine as a prominent agent affecting wound repair in smokers. However, tobacco smoke contains several components that may alter gingival wound healing. The present study aimed to analyse the roles of cigarette smoke condensate (CSC) and nicotine on cell viability, cell migration/invasion and myofibroblastic differentiation using primary cultures of human gingival fibroblasts. Material and Methods: To compare the effects of CSC and nicotine, gingival fibroblasts were stimulated with CSC (0.4–500 μg/mL) and the corresponding nicotine concentrations (0.025–32 μg/mL) present in research cigarettes (1R3F). Cell viability was evaluated through the MTS assay. Cell migration and invasion were assessed through scratch wound assays, collagen nested matrices and transwell migration. α‐Smooth muscle actin production was evaluated by western blotting. Results: Cigarette smoke condensate at 50 μg/mL induced a moderate increase in cell viability, whereas the corresponding nicotine concentration (3.2 μg/mL) did not produce this response. Cigarette smoke condensate at 250 μg/mL, but not nicotine at 16 μg/mL (the corresponding nicotine concentration), induced cell death. Both nicotine and CSC stimulated cell migration (50 μg/mL CSC; 3.2 μg/mL nicotine). At 150 μg/mL, CSC inhibited cell migration; however, the corresponding concentration of nicotine (9.6 μg/mL), did not have this effect. Although both nicotine and CSC inhibited α‐smooth muscle actin production, only the latter induced a statistically significant effect on this response. Conclusion: Cigarette smoke condensate may stimulate cell survival and migration at low concentrations and inhibit these cell responses at higher levels of exposure. Moreover, CSC may interfere in myofibroblastic differentiation. These results show that cigarette smoke, but not nicotine, may significantly alter cell viability, cell migration and myofibroblastic differentiation in gingival mesenchymal cells.     

Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts

Background and Objective: Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen‐degrading ability of human gingival fibroblasts and its mechanism. Material and Methods: Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water‐soluble tetrazolium‐1 and lactate dehydrogenase, respectively. The collagen‐degrading ability of human gingival fibroblasts was evaluated in collagen‐coated six‐well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Results: Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 μg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 μg/mL. Cigarette smoke condensate increased the collagen‐degrading ability of human gingival fibroblasts, especially at a concentration of 100 μg/mL (1.5‐fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP‐1, proMMP‐2, MMP‐14 and TIMP‐1, and decreased the production of TIMP‐2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP‐2, MMP‐14 and TIMP‐2 in membrane extracts, especially at concentrations of 50–100 μg/mL. Conclusion: Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of ≥ 400 μg/mL. Cigarette smoke condensate can increase the collagen‐degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.     

Human gingival fibroblast cytoskeleton is a target for volatile smoke components.

BACKGROUND: Several in vitro investigations have indicated that the particulate phase of cigarette smoke as nicotine affects many cell types including gingival fibroblasts, but few studies have examined the effect of volatile fraction on cellular structures involved in cell functions such as adhesion and proliferation. Since gingival fibroblast survival and reproduction are fundamental to maintaining the oral connective tissue as well as to wound healing, the effects of acrolein and acetaldehyde, volatile fractions of cigarette smoke, on cytoskeleton were examined in human gingival fibroblasts (HGFs) in vitro. METHODS: Human gingival fibroblast (HGF) strains from healthy subjects with non-inflamed gingiva were utilized. The cells were incubated in different concentrations of acrolein and acetaldehyde. Cell adhesion was evaluated after 3 hours. The influence of both substances on cytoskeletal structures, tubulin and vimentin intermediate filaments (VIF), was investigated using indirect immunofluorescence technique. RESULTS: The results show that both substances produced similar effects, resulting in a dose-dependent inhibition of HGF adhesion. Disturbance of HGF cytoskeleton consisted of a disruption of microtubules and vimentin microfilaments with alterations in cell shape. CONCLUSIONS: Our experimental findings suggest that volatile fractions of cigarette smoke such as acrolein and acetaldehyde, because their ability to bind and interact with the cytoskeleton, prevent HGF adhesion. Consequently the maintenance of the oral connective tissue and integrity and remodeling could be impaired. According to our morphological evidence, these findings confirm other clinical and epidemiological investigations reporting that volatile components of cigarette smoke could lead to the initiation and progression of periodontal disease.     

尼古丁和烟草浸提液对人牙龈成纤维细胞生长和贴附能力的影响

目的：体外观察尼古丁和烟草浸提液(ST)对人牙龈成纤维细胞的生长和贴附影响。方法：用不同浓度的尼古丁和ST作用于人牙龈成纤维细胞，用MTT法检测细胞的生长和贴附情况。结果：随尼古丁和ST浓度的增加，细胞的生长率逐渐下降，尼古丁和ST对细胞生长半抑制浓度(IC50)分别为0．095g／L和11．88g／L；与对照组相比，当尼古丁及ST浓度分别大于0．01g/L和6．2g／L时差异有显著意义。而对细胞贴附的影响表明，随着尼古丁和ST浓度的增加，细胞的贴附率逐渐下降，尼古丁和ST对细胞贴附的IC50分别是0．6g／L和10．33g/L；与对照组相比，当尼古丁及ST浓度分别大于0．1g／L和12．4g／L时差异有显著意义。结论：尼古丁和浸提取液均可抑制人牙龈成纤维细胞的生长和贴附，提示尼古丁和烟草在牙周病发病中起作用。     

烟草浸提液对牙龈成纤维细胞在钛板表面分泌胶原的影响

目的探讨烟草浸提液对钛板表面牙龈成纤维细胞分泌胶原的影响。方法取细胞悬浮液接种于24孔板内钛板表面，孵育24h后，更换含0、0．01、0．1、1、5、10、25g/L烟草浸提液和0．03、0．15、0．3g／L尼古丁（10种浓度）的培养液。继续孵育48h后，检测细胞培养上清液中羟脯氨酸含量。结果随着烟草浸提液及尼古丁浓度的增加．细胞培养上清液中羟脯氨酸的含量逐渐减少。结论烟草浸提液及尼古丁可抑制牙龈成纤维细胞胶原的产生．提示烟草在破坏牙种植体“袖口”中起重要作用。     

The up-regulation of heme oxygenase-1 expression in human gingival fibroblasts stimulated with nicotine

BACKGROUND: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Heme oxygenase-1 (HO-1) is known as a stress-inducible protein and functions as an antioxidant enzyme. There is limited information on the expression of HO-1 in smoking-associated periodontal disease. OBJECTIVES: The aim of the present study was to investigate the effects of nicotine on the expression of HO-1 protein in cultured human gingival fibroblasts in vitro and further to compare HO-1 expression in gingival tissues obtained from cigarette smokers and non-smokers in vivo. METHODS: Western blot assay was used to investigate the effects on human gingival fibroblasts exposed to nicotine. In addition, antioxidants catalase, superoxide dismutase (SOD), and N-acetyl-l-cysteine (NAC) were added to test how they modulated the effects on nicotine-induced HO-1 expression. Gingival biopsies taken from the flap surgery of 20 male patients with periodontal disease (10 cigarette smokers and 10 non-smokers) were examined by immunohistochemistry. RESULTS: The exposure of quiescent human gingival fibroblasts to 10 mm nicotine resulted in the induction of HO-1 protein expression in a time-dependent manner (p < 0.05). The addition of glutathione (GSH) precursor NAC inhibited the nicotine-induced HO-1 protein expression (p < 0.05). However, SOD and catalase did not decrease the nicotine-induced HO-1 protein expression (p > 0.05). The results from immunohistochemistry demonstrated that HO-1 expression was significantly higher in cigarette smokers (p < 0.05). HO-1 was noted in the basal layers of epithelium, inflammatory cells, and fibroblasts in specimens from cigarette smoking. CONCLUSIONS: Taken together, these results suggest that HO-1 expression is significantly up-regulated in gingival tissues from cigarette smokers, and nicotine may, among other constituents, be responsible for the enhanced HO-1 expression in vivo. The regulation of HO-1 expression induced by nicotine is critically dependent on the intracellular GSH concentration.     

The volatile fraction of cigarette smoke induces alterations in the human gingival fibroblast cytoskeleton

Several in vitro investigations have indicated that the particulate phase of cigarette smoke, such as nicotine, affects many cell types, including gingival fibroblasts. However, few studies have been performed on the effects of the volatile fraction on the cellular structures that are involved in cell functions, such as adhesion and proliferation. Since the survival and reproduction of gingival fibroblasts are fundamental in maintaining the integrity of the oral connective tissue, as well as in wound healing, the effects on the cytoskeleton of acrolein and acetaldehyde, which are the volatile fractions of cigarette smoke, were examined in vitro for human gingival fibroblasts (HGFs). HGF strains that were taken from healthy subjects with non-inflamed-gingiva were utilized in this investigation. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde. Cell adhesion and viability were evaluated after incubation for 3 h and 5 days, respectively. The influence on cytoskeletal structures (tubulin, actin and vimentin intermediate filaments) was investigated with the indirect immunofluorescence technique. The results show that both substances produced similar effects, which resulted in a dose-dependent inhibition of HGF adhesion and viability. Disturbance of the HGF cytoskeleton consisted of disruption of the microtubules, actin filaments and vimentin microfilaments, which was accompanied by alterations to cell shape. Our experimental findings suggest that the volatile fractions of cigarette smoke, such as acrolein and acetaldehyde, have a cytotoxic effect on HGFs, with the result that they lose their capacity for adhesion and proliferation. The consequences of this could be impairment of the maintenance, integrity and remodelling of the oral connective tissue. According to our morphological evidence, these findings show that cigarette smoke can lead to the development and progression of periodontal disease, and indicate the need for appropriate therapy.     

烟草烟雾提取物对人牙龈成纤维细胞在3Y-TZP表面生长状态的影响

目的：观察比较不同浓度烟草烟雾提取物（Cigarette Smoke Extract,CSE）对人牙龈成纤维细胞（HumanGingival Fibroblasts,HGFs）在3mol%氧化钇作为稳定剂的四方氧化锆多晶体陶瓷（3Y-TZP）种植材料表面粘附、增殖、形态伸展及细胞状态的影响。方法：原代培养HGFs并进行细胞来源鉴定;SRB法检测不同浓度CSE作用下HGFs在材料表面粘附、增殖情况;免疫荧光染色,病理图像分析系统计数评价HGFs细胞铺展面积及形状系数改变;激光共聚焦显微镜下观察拍照,观察凋亡形态;扫描电镜观察细胞粘附的形态结构改变。结果：细胞波形丝蛋白染色阳性,角蛋白染色阴性;随CSE浓度升高,HGFs在材料表面粘附、增殖、铺展面积、形状系数及形态伸展均呈下降趋势,各实验组与对照组间均存在显著差异（P〈0.05）,抑制作用随浓度升高更加明显,呈浓度依赖性。结论：CSE对HGFs在3Y-TZP种植材料表面的生长可产生明显抑制作用。提示吸烟对陶瓷种植体植入后的软组织界面产生不良影响,其损害程度可能与烟草有害物质的接触量有相关性。     

Cigarette smoke condensate stimulates urokinase production through the generation of reactive oxygen species and activation of the mitogen activated protein kinase pathways in human gingival fibroblasts

Background and Objective:  Tobacco smoking is a significant risk factor for periodontal disease. It has been suggested that smoking may alter connective tissue remodeling in the periodontium. In the present study, we investigated whether cigarette smoke condensate modulates the production of the serine protease urokinase in human gingival fibroblasts.
Material and Methods:  Primary cultures of human gingival fibroblasts were stimulated with cigarette smoke condensate. Urokinase production was evaluated through casein zymography and western blotting. Plasmin activation was assessed by means of a radial diffusion assay. The roles of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and reactive oxygen species in cigarette smoke condensate-stimulated urokinase production were studied using distinct selective inhibitors (SP600125, PD98059, N -acetyl cysteine). Reactive oxygen species production was determined using a fluorometric assay. Activation of ERK and JNK pathways were evaluated using western blots.
Results:  In gingival fibroblasts, cigarette smoke condensate potently stimulated urokinase production and plasmin activation. Cigarette smoke condensate-stimulated urokinase production was dependent on the activity of ERK/JNK pathways and was inhibited by the reactive oxygen species scavenger, N -acetyl cysteine. Cigarette smoke condensate strongly stimulated ERK and JNK phosphorylation and the generation of reactive oxygen species.
Conclusion:  Cigarette smoke condensate stimulates urokinase production and plasmin activation in gingival fibroblasts. Moreover, cigarette smoke condensate-stimulated urokinase production depends on both the activation of ERK/JNK pathways and on the generation of intracellular reactive oxygen species. These results show that cigarette smoke may alter connective tissue remodeling by inducing production of the urokinase-type plasminogen activator through specific signaling pathways.     

热休克蛋白27在烟草烟雾提取物介导的牙龈成纤维细胞损伤中的表达变化

目的 观察在不同质量分数烟草烟雾提取物（CSE）干预下,热休克蛋白（HSP）27在人牙龈成纤维细胞（HGFs）损伤过程中的表达。方法 取原代培养并经过鉴定的3~8代HGFs,采用细胞划痕试验检测不同刺激质量分数（0、2.5%、5.0%、12.5%、25.0%、50.0%）的CSE对HGFs体外迁移的影响,并采用Western blot方法检测HSP27在HGFs中的表达。结果 CSE质量分数越高,细胞的迁移能力越弱;HSP27在正常HGFs中呈弱阳性表达,在CSE刺激后的HGFs中呈强阳性表达,且随CSE质量分数的增高,HSP27的相对表达量有逐渐增高的趋势,与细胞迁移能力相反。结论 HSP27在CSE介导的HGFs损伤中表达升高,在CSE介导的上皮损伤中有重要作用。     

Impact of tobacco use on periodontal status

This article reviews the effects of smoked and smokeless tobacco on periodontal status, including the impact of smoking on periodontal therapy and potential mechanisms for the adverse effects of tobacco on the periodontium. Approximately half of periodontitis cases have been attributed to either current or former smoking. Both cigar and cigarette smokers have significantly greater loss of bone height than nonsmokers, and there is a trend for pipe smokers to have more bone loss than nonsmokers. Unlike smokers, who experience widespread periodontal destruction, the most prevalent effects of smokeless tobacco are localized to the site of placement, in the form of gingival recession and white mucosal lesions. Smoking has an adverse effect on all forms of periodontal therapy, and up to 90 percent of refractory periodontitis patients are smokers. The pathogenesis of smoking-related periodontal destruction has been attributed to alterations in the microflora and/or host response. Some data indicates that smoking may increase levels of certain periodontal pathogens, but there is more evidence that smoking has a negative effect on host response, such as neutrophil function and antibody production. An encouraging finding is that periodontal disease progression slows in patients who quit smoking and that these individuals have a similar response to periodontal therapy as nonsmokers. The facts presented in this paper will assist dental health professionals in treatment-planning decisions and provide them with important information to share with patients who use tobacco products.     

Nicotine-induced alterations in human primary periodontal ligament and gingiva fibroblast cultures

Various in vivo and in vitro investigations have indicated that tobacco smoking as well as the use of smokeless tobacco products may be important risk factors for the development and severity of inflammatory periodontal disease. The purpose of this study was to determine the cytotoxicity of nicotine by means of human primary oral fibroblast cultures and a permanent cell line. The cytotoxicity of nicotine was evaluated by determination of cell growth, cell membrane integrity, protein content, and alterations of the cytoskeleton. Furthermore, recovery following nicotine exposure was assessed by vital staining (trypan blue). Dose-dependent toxic effects of nicotine were measured within a range of 0.48 mM to 62 mM. Growth of fibroblasts was decreased by nicotine concentrations higher than 7.8 mM. Additionally, the protein content was significantly decreased and cell membranes were damaged. Morphological alterations of microtubules and vimentin filaments were observed at concentrations higher than 3.9 mM. Nicotine-exposed cells revealed atypical shapes and vacuoles. The toxic effects of nicotine became irreversible in the range between 10.5 and 15.5 mM, whereas at lower concentrations cells recovered after the withdrawal of nicotine. Our results confirm clinical oberservations regarding the important role of nicotine as a risk factor in the etiology and progression of periodontal disease.     

Microfilaments in human cementoblasts and periodontal fibroblasts

An electron microscopic survey was carried out on the human periodontal ligament (PDL), including a part of the gingival connective tissue attached to extracted tooth roots (11 functioning premolars and 6 nonfunctioning third molars) in order to examine the characteristics of microfilaments (6 nm) in cementoblasts and PDL fibroblasts. Microfilaments which were grouped in bundles with semiperiodic dense nodes or in meshworks just beneath the cell membrane were seen predominantly in the cells characterized by their ultrastructurally immature appearance. These microfilaments were more commonly observed in third molar PDL than in premolar PDL and, in general, more conspicuous in cementoblasts than in fibroblasts. The significance of microfilaments in human PDL is discussed, particularly in relation to cell differentiation and morphogenesis.