Periodontology 2000, Vol. 67, 2015, 187–210 © 2014 John Wiley & Sons A/S.

& Sons A/S. Published by John Wiley & Sons Ltd

Printed in Singapore. All rights reserved PERIODONTOLOGY 2000

Current perspective of the impact

of smoking on the progression
and treatment of periodontitis
F R A N C I S C O H. N O C I T I J R , M A R C I O Z. C A S A T I & P O L I A N A M E N D E S D U A R T E

Several developed and developing countries are expe- previous worldwide estimates (11%). The highest
riencing an epidemic in diseases caused by cigarette smoking prevalence was among the youngest age
smoking and tobacco use. Tobacco consumption pro- groups (25–34 and 35–44 years) and smoking was
motes increased risk for the development of cardio- found to start mostly during adolescence (41).
vascular disease (e.g. ischemic and peripheral heart The dangers of passive smoking have also been
disease and stroke), lung cancer and chronic obstruc- emphasized as some evidence has linked passive
tive pulmonary disease. Despite numerous smoke- smoking to death and diseases in nonsmokers (57,
free initiatives, governmental tobacco-control policies 60). In Latin America, considerable exposure to sec-
and the well-recognized risks associated with smok- ond-hand tobacco smoke has been reported to occur
ing, tobacco consumption still remains excessively both in the home and in the workplace. A previous
prevalent. The World Health Organization estimates study assessed the concentrations of second-hand
that the number of smokers worldwide is more than smoke in public places in the capital cities of Argen-
1 billion and is expected to increase to 1.7 billion by tina, Brazil, Chile, Costa Rica, Paraguay, Peru and
2025 (153). Based on these trends, it is predicted that, Uruguay. High levels of airborne nicotine were found
by 2030, approximately 10 million people will die to be extensively distributed in public places in
annually as a result of tobacco use, with 70% of such several Latin-American cities, particularly in Argen-
deaths occurring in low- and middle-income tina and Uruguay (117), for review, see Mu € ller &
countries (81). Wehbe (114).
Data on the incidence and prevalence of cigarette/ As a result of the excessive consumption of ciga-
tobacco consumption in Latin America are insuffi- rette/tobacco, the harm caused by this habit and the
cient and controversial. It is estimated that 8–10% of consequences of environmental tobacco smoke have
smokers live in Latin America (114). The frequency of spread significantly in Latin America and countries
smoking is particularly elevated in developing coun- worldwide. Therefore, in addition to the damaging
tries because of the low socio-economic conditions impact of smoking on general health, there is interest
and low level of education in these countries. Cham- in studying the effect of cigarette/tobacco consump-
pagne et al. (41) evaluated the prevalence of tobacco tion on oral health, including dental implant failures,
smoking in seven major cities of Latin America (Bar- oral cancers and periodontal diseases. Overall, smok-
quisimeto/Venezuela, Bogota/Colombia, Buenos ing has been considered as the most important pre-
Aires/Argentina, Lima/Peru, Mexico City/Mexico, ventable risk factor for periodontitis among all
Quito/Ecuador and Santiago/Chile). The authors lifestyle factors. Experimental and clinical studies in
showed that although the prevalence of smoking was the periodontal field are unanimous in demonstrating
high in all cities studied, the patterns of smoking var- the detrimental influence of smoking on periodontal
ied among the cities. The prevalence of male smokers cells and tissues and consequently on periodontal
(31.3–49.4%) ranged between that of other developed disease progression and treatment response. The
and developing regions (47%), whereas the preva- present review will provide an overview of the current
lence of female smokers (10.5–43.3%) was higher than scenario regarding the impact of smoking on the

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progression and treatment of periodontitis; data from never smokers and there was a dose–response rela-
studies from our and other Latin American research tionship between the number of cigarettes smoked
groups will be presented. per day and the odds of periodontitis. In 2005, Heitz-
Mayfield (77), reviewing studies on the individual pre-
dictive factors associated with the susceptibility to
Clinical findings progression of periodontitis, reported that cigarette
smoking is a strong dose-related predictor of peri-
This section presents classical data and recent find- odontitis progression.
ings on the clinical relationship between smoking Ojima et al. (124) demonstrated a higher preva-
and periodontal diseases. Table 1 summarizes some lence and higher probability of periodontal disease in
clinical findings, published between 2007 and 2011, current smokers than in non- and former smokers.
on the relationship between smoking and periodontal Kibayashi et al. (86) evaluated the association
disease in different populations. These studies are between smoking and periodontal disease progres-
unanimous in demonstrating a positive relationship sion (i.e. three or more sites presenting a probing
between smoking habit and periodontitis severity, depth increase of at least 2 mm over 4 years). The
thereby corroborating the findings of classical results demonstrated that 38.5% of disease progres-
studies. sion was related to current smoking. In addition,
The first observations on the relationship between pack-years of smoking demonstrated a positive dose–
smoking and periodontal tissues occurred in the response correlation with periodontitis progression.
1940s, when Pindborg demonstrated that necrotizing Hanioka et al. (75) evaluated the association between
ulcerative gingivitis was associated with tobacco con- smoking and tooth loss in 3,999 Japanese subjects.
sumption (134, 135). Remarkably, until the 1980s Based on logistic regression models, a positive associ-
there were no longitudinal studies, controlled for ation was demonstrated between tooth loss and cur-
confounding factors, which provided strong evidence rent, but not former, smokers after controlling for
of smoking as an actual risk factor for periodontitis. confounding factors (e.g. age, frequency of brushing,
Subsequently, several epidemiological studies, pre- body mass index and alcohol consumption). Compa-
senting adequate methodologies and statistical analy- rable findings were demonstrated in another study in
ses (i.e. adjustments for confounding variables), have which current pipe/cigar smokers had a 20%
clearly demonstrated a strong association between increased risk of tooth loss compared with never and
tobacco use/smoking habit and periodontal diseases former smokers (51). Similarly, Ojima et al. (123),
in diverse populations (20, 42, 52, 72, 177, 181). In evaluating the association between cigarette smoking
general, evidence indicates that smokers have more and tooth-loss experience among Japanese adults,
severe periodontal disease, with increased bone, confirmed that tooth loss was more frequent among
attachment and tooth loss, and gingival recession, as current smokers (40.6%) than among former smokers
well as pocket formation, than do nonsmokers (16, (23.1%) and nonsmokers (27.9%). Tobacco use was
29, 87, 95). also associated with periodontitis prevalence in a
Risk assessment, based on several previous investi- population composed of 161 Mexican men (112). In
gations, indicates that the tobacco attributable risk New Zealand, long-term smokers (15–32 years of age)
odds ratio of periodontal disease progression is high, had a very high odds ratio for having one or more
especially in heavy smokers (e.g. those smoking ≥20 sites with attachment loss of ≥5 mm. In addition,
cigarettes/day) (19, 62, 172). Linden & Mullally (98) there were no significant differences in periodontal
found that the odds ratio for the presence of peri- health between never smokers and those who had
odontitis and smoking was 14.1 (95% confidence stopped smoking after 26 years of age (171).
interval: 1.5–132.9) in a young heavy-smoking popula- Smokers presented a higher mean tooth loss than
tion who had smoked for an average of did nonsmokers in a population from Sana’a, Repub-
11.8  7 years. Tomar & Asma (172), based on the lic of Yemen (3). In addition, Brazilian smokers
National Health and Nutrition Examination Survey III showed significantly higher alveolar bone loss com-
study, provided one of the major pieces of evidence pared with never smokers (3.3 mm vs. 2.2 mm,
of smoking as a risk for periodontitis. Among 12,329 respectively) in the anterior teeth (96). Likewise, a
subjects evaluated, approximately half of all peri- previous study in an untreated isolated Brazilian pop-
odontitis cases could be related to smoking. In addi- ulation demonstrated, using multivariate analysis,
tion, current smokers were about four times more that smoking was a risk indicator for clinical attach-
likely to present with periodontitis compared with ment level ≥5 mm (odds ratio = 2.4) and for clinical

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Table 1. Summary of clinical findings on the relationship between smoking and periodontal disease in different popu-
lations over the past 5 years

Reference Population Sample size Principal parameters Principal findings

evaluated

Oceania

Thomson et al. (171) New Zealand 810 Gingival recession, probing Smokers had a very high odds ratio
depth, clinical for having one or more sites with
attachment level attachment loss of ≥5 mm
Do et al. (52) Australia 3,161 Gingival recession, probing Smoking was a risk for moderate-to-
depth, clinical severe periodontitis. The
attachment level prevalence of severe periodontitis
was positively correlated with the
severity of smoking status
Arora et al. (11) Australia 99,663 Questionnaire about tooth Smoking was a significant predictor
loss of tooth loss, with current and
former smokers having a greater
risk compared with never smokers
North America

Dietrich et al. (51) USA 43,112 health Tooth missing The risk of tooth loss increased
professionals progressively with increasing
smoking intensity. Current
smokers had twice the risk of tooth
loss compared with never smokers
Phipps et al. (132) USA 1,347 older men General periodontal Heavy smoking was associated with
parameters* tooth retention, clinical
attachment level, probing depth
and severity of periodontitis
Iida et al. (80) USA 5,110 women Presence of gingivitis or Subjects with detectable cotinine
periodontitis levels were more likely to have
gingivitis than were those with
undetectable cotinine. Current
smokers were more likely to have
periodontitis than were never- or
former smokers
Asia

Ojima et al. (123) Japan 1,314 Tooth missing Current smokers presented more
tooth loss than did former and
nonsmokers. A positive
exposure-related correlation was
observed between cigarette
smoking and tooth loss
Hanioka et al. (75) Japan 3,999 Tooth missing Positive association between tooth
loss and current smokers after
controlling for confounding factors
Kibayashi et al. (86) Japan 219 Periodontitis progression 38.5% of periodontal disease
(three or more sites with progression was attributable to
an increase of probing current smoking. Risk for disease
depth of ≥2 mm over progression presented a
4 years) dose-dependent relationship
with increasing pack-years
Al-Bayaty et al. (3) Republic 2,506 Tooth missing Smokers had higher mean tooth loss
of Yemen compared with nonsmokers
Chatrchaiwiwatana Thailand 1,843 General periodontal Smoking is a risk indicator for
et al. (42) parameters* periodontitis

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Nociti et al.

Table 1. (Continued)

Reference Population Sample size Principal parameters Principal findings

evaluated

Yanagisawa et al. (181) Japan 1,088 Community Periodontal The odds ratio of current smokers
Index and number of having more than eight missing
teeth teeth and periodontitis were 1.67
and 1.74, respectively
Zini et al. (187) Israeli 254 Community Periodontal Tobacco smoking was significantly
Index associated with severe chronic
periodontitis
Latin America

Minaya-Sa nchez Mexico 161 General periodontal The odds ratio of periodontitis
et al. (112) parameters* (defined as ≥4 mm of clinical
attachment level) was greater in
current and former tobacco users
than in never tobacco users
Corraini et al. (44) Brazil 214 General periodontal Current and former smokers were
parameters* two and eight times more likely to
have a clinical attachment level of
≥5 mm and ≥7 mm, respectively,
than were nonsmokers
Lima et al. (96) Brazil 80 Radiographic bone loss Radiographic bone loss in anterior
teeth was higher in smokers than
in never-smokers
Rosa et al. (144) Argentine 81 General periodontal Clinical attachment loss and
parameters* and recession were significantly
radiographic bone loss greater, whereas bone height and
over 545 days density were lower, in smokers
over time
Gomes et al. (66) Brazil 45 Gingival crevicular fluid Smokers had significantly smaller
volume gingival crevicular fluid volumes
than did never-smokers
Silva-Boghossian Brazil 491 General periodontal Nonsmokers presented more teeth,
et al. (160) parameters* lower mean probing depth and
plaque accumulation than did
smokers. Smokers had higher
chances of clinical attachment loss
than did nonsmokers with similar
bleeding on probing and plaque
levels
Susin et al. (167) Brazil 612 General periodontal Heavy smokers had a significantly
parameters* higher chance of having chronic
periodontitis than did nonsmokers
Europe

Adler et al. (1) Sweden 293 Probing depth Smokers presented deeper probing
depth compared with nonsmokers.
Heavy smokers have greater
percentage of palatal pockets
≥6 mm than do nonsmokers and
light smokers
Sarfati et al. (155) France 2,074 Mid-buccal gingival Smoking was a risk factor for the
recession extent of gingival recessions

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 Smoking and periodontitis: current perspective

Table 1. (Continued)

Reference Population Sample size Principal parameters Principal findings

evaluated

Vouros et al. (177) Greek 115 General periodontal Heavy smokers had worse
parameters* periodontal condition and less
bleeding than did moderate,
intermittent or never-smokers
Hugoson and Sweden 1,591 General periodontal Smokers had a higher risk of severe
Rolandsson (79) parameters* and periodontal disease and lower risk
radiography of gingivitis than did nontobacco
users
*Clinical evaluation of periodontal parameters including probing depth, gingival bleeding, clinical attachment level, tooth missing, suppuration, gingival recession
and plaque accumulation.

attachment level ≥7 mm (odds ratio = 8.2) (44). periodontitis in a large population-based sample,
Swedish smokers had significantly deeper periodontal consisting of 612 adolescents and young adults from
pockets (probing depth ≥6 mm) compared with non- South Brazil. Based on a multivariable logistic regres-
smokers (1). Young Argentine smokers showed worse sion analysis, older age, low socio-economic status,
periodontal conditions and lower alveolar bone heavy smoking and higher levels of calculus were sig-
height and density compared with nonsmokers (144). nificantly associated with periodontitis in this popu-
Furthermore, Silva-Boghossian et al. (160) showed lation. Zini et al. (187) confirmed the association of
that age, smoking and bleeding on probing were all tobacco smoking and severe chronic periodontitis in
risk indicators associated with moderate and severe an Israeli population.
attachment loss in subjects attending a public dental Although smokers may present increased plaque
school in Brazil. The negative impact of heavy smok- accumulation and exacerbated disease progression,
ing on tooth retention, probing depth, clinical attach- paradoxically, their gingival inflammatory signs and
ment level and severity of periodontitis was symptoms are suppressed. Preber & Bergstrom (138)
confirmed by Phipps et al. (132) in an older popula- demonstrated that the number of bleeding sites in
tion of American men. American women with detect- smokers (27%) was lower than that in nonsmokers
able cotinine levels were more likely to have gingivitis (40%). When examining 369 subjects with periodonti-
than were those with undetectable cotinine, and cur- tis, fewer gingival bleeding sites were reported for
rent smokers were more likely to have periodontitis smokers (25%) than for nonsmokers (51%) (137). The
than were never smokers or former smokers (80). same research group also evaluated the influence of
In 2010, a multivariate linear regression model indi- smoking on experimental gingivitis (18) in a group of
cated that age, gender, plaque index and tobacco nonperiodontitis dental students in which the smok-
consumption were associated with the extent of gin- ers had smoked for at least 4 years. The results
gival recession (155). Current and former smokers revealed that the number of sites with bleeding, the
had significantly higher odds of experiencing edentu- volume of gingival exudate and the number of gingi-
lism compared with never smokers (odds ratio = 2.51; val sites with redness were significantly lower in
95% CI = 2.31–2.73 for current and former smokers smokers with plaque-accumulation levels compara-
vs. odds ratio = 1.50; 95% CI = 1.43–1.58 for never ble with those of nonsmokers. Later, a study reported
smokers) (11). These findings were confirmed by a that, after 28 days of plaque-induced gingivitis, the
recent systematic review which showed that a causal intensity of the vascular reaction in smokers was only
association between smoking and tooth loss is highly 50% of that observed for nonsmokers (17). Rosa et al.
likely and that there is an evident decrease in the risk (145) showed lower levels of crevicular fluid and gin-
of tooth loss for former smokers (119). Recently, gival index in smokers than in nonsmokers without
Hugoson & Rolandsson (79), using multiple logistic periodontitis. In 2005, Apatzidou et al. (10) showed
regression analysis, demonstrated that cigarette that smokers presented significantly less gingival
smokers had a significantly higher frequency of prob- inflammation and a lower gingival crevicular fluid
ing depth ≥4 mm and a higher incidence of severe volume compared with nonsmokers. A more recent
periodontitis compared with nontobacco users, after study from a Brazilian research group demonstrated
adjusting for age, gender and sociodemographic vari- that smokers had a significantly lower gingival
ables. Susin et al. (167) described risk indicators for crevicular fluid volume than did never smokers (66).

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Table 2. Microbiological findings on the relationship between smoking and periodontal diseases over the past 5 years

Reference Type of Microbiological Principal findings

study technique

Kibayashi et al. (86) Clinical PCR No significant differences in the proportions of Porphyromonas gingivalis,
Tannerella forsythia, Aggregatibacter actinomycetemcomitans,
Treponema denticola, Prevotella intermedia and Prevotella nigrescens
were observed between current and noncurrent smokers
Gomes et al. (66) Clinical PCR The levels of Dialister pneumosintes and Parvimonas micra were higher in
deep periodontal sites, and the levels of D. pneumosintes, P. micra and
P. gingivalis were higher in moderate sites (probing depth ≥3 mm
but ≤5 mm) of smokers compared with nonsmokers
Bagaitkar et al. (13) In vitro RT-PCR Porphyromonas gingivalis exposed to cigarette smoke extract reduced
the proinflammatory response from monocytes and peripheral
blood mononuclear cells
Cogo et al. (43) In vitro Cell culture Cotinine increased the ability of P. gingivalis to invade epithelial cells

Teixeira et al. (170) Clinical RT-PCR Porphyromonas gingivalis genotype fimA IV presented a significant,
positive association with periodontitis severity in Brazilian smoker
subjects with chronic periodontitis
Shchipkova et al. (158) Clinical Cloning and High prevalence and abundance of disease-associated pathogens in
Sequencing smokers than in nonsmokers with periodontitis
Kumar et al. (89) Clinical Cloning and Smokers presented early acquisition and colonization of
Sequencing biofilm-forming pathogens
Kubota et al. (88) Clinical PCR Odds ratio = 1.7 for colonization with Campylobacter rectus in smokers

Meulman et al. (110) Clinical Cloning and In smokers, supragingival periodontal therapy only slightly affected the
Sequencing subgingival biofilm biodiversity, compared with nonsmokers

Corroborating the abovementioned findings, Hugo- DNA–DNA hybridization, immunoassay, etc.) and
son & Rolandsson (79) observed that cigarette smok- experimental designs among studies. Table 2 pre-
ers had significantly less gingivitis. It is presumed that sents a summary of the microbiological findings on
smoking may decrease gingival bleeding and crevicu- the relationship between smoking and periodontal
lar fluid volume as a result of changes in the propor- diseases, published between 2007 and 2011.
tion of blood vessels and vascular alterations in Darby et al. (49) evaluated the presence of
periodontal tissues. periodontal pathogens, including Aggregatibacter
actinomycetemcomitans, Porphyromonas gingivalis,
Prevotella intermedia, Tannerella forsythia and Trepo-
Microbiological findings nema denticola, by PCR, in the subgingival biofilm of
smokers and nonsmokers with chronic and aggressive
Some studies have hypothesized that microbiological periodontitis. There were no significant differences in
differences between smokers and nonsmokers could the prevalence of periodontal pathogens between
justify the detrimental influence of smoking on peri- groups. Later, Apatzidou et al. (10) also evaluated the
odontal tissue breakdown (176, 184). The highest frequency of the aforementioned periodontal patho-
growth of anaerobic bacterial species in smokers gens in the subgingival plaque of smokers and non-
could be related to the increased anaerobiosis in the smokers. Smoking status was self-reported and
smoker’s oral cavity. On the other hand, other studies confirmed by cotinine enzyme inhibition assay. In
(10, 23, 49) have shown minor or no differences in the agreement with the findings of Darby et al. (49), the
composition of the subgingival biofilm between authors did not observe any significant differences in
smokers and nonsmokers. Therefore, it remains the periodontal pathogens detected between smoker
unclear whether, and to what extent, microbiological and nonsmoker groups. Kibayashi et al. (86) also dem-
dissimilarities actually exist between smokers and onstrated no significant differences in the proportions
nonsmokers, probably because of the numerous of P. gingivalis, T. forsythia, A. actinomycetemcomitans,
differences in microbiological techniques (e.g. immu- T. denticola, P. intermedia and Prevotella nigrescens
nofluorescence microscopy, PCR, checkerboard between current and noncurrent smokers.

192
 Smoking and periodontitis: current perspective

Other investigations have suggested that smoking promotes early acquisition and colonization of bio-
is related to a peculiar pattern of periodontal patho- film-forming pathogens. Kubota et al. (88) showed
gen colonization, as well as to a higher prevalence that the prevalence of C. rectus was higher in Japa-
and/or levels of pathogenic species. In addition, nese smokers than in Japanese nonsmokers, suggest-
some cigarette compounds may be able to alter the ing a possible influence of smoking on the
behavior of pathogens (13, 43, 55, 88, 176, 184). Zam- colonization of specific periodontal pathogens.
bon et al. (184), using immunofluorescence micros- An unpublished study from the Guarulhos Univer-
copy, demonstrated that cigarette smoking increased sity research group (Sa ~o Paulo, Brazil) evaluated,
the chance of subgingival infection with critical using the checkerboard DNA–DNA hybridization
pathogens. The risk of subgingival infection with technique, the recolonization of subgingival bacteria
T. forsythia in current smokers was 2.3 times higher in current and never smokers with chronic periodon-
than that observed in former and nonsmokers. The titis, up to 6 months after scaling and root planing.
subgingival biofilm of smokers was also more likely The subgingival biofilm profile did not differ consid-
to harbor P. gingivalis than that of nonsmokers. erably between never and current smokers before
Later, van Winkelhoff et al. (176) also noticed that therapy. However, scaling and root planing was inef-
the smoking habit may favor the colonization of spe- fective at reducing the levels of some periodontal
cific clusters of known and suspected periodontal pathogens in smokers. In addition, after such treat-
pathogens (e.g. T. forsythia, Parvimonas micra, Fuso- ment, smokers were more susceptible to the re-estab-
bacterium nucleatum and Campylobacter rectus). lishment of a pathogenic subgingival biofilm than
Haffajee & Socransky (74) demonstrated, in a retro- were nonsmokers. Similarly, the modest effect of scal-
spective study, higher numbers of periodontal patho- ing and root planing alone in reducing the levels and/
gens from the red and orange microbial complexes or proportions of some periodontal pathogens in
in periodontal sites, with a probing depth of <4 mm, smokers has also been confirmed in other studies (69,
in smokers compared with such sites of nonsmokers. 70, 73, 108, 176). According to Grossi et al. (70), smok-
Eggert et al. (55) confirmed the hypothesis that ers had smaller reductions in the subgingival levels of
smoking promotes a favorable environment for colo- T. forsythia and P. gingivalis than did nonsmokers at
nization with periodontal pathogens, especially in 3 months after scaling and root planing. Likewise,
shallow sites (probing depth ≤5 mm). A Brazilian Haffajee et al. (73) demonstrated that the prevalence
research group demonstrated that smokers and of P. gingivalis, T. forsythia and T. denticola
never smokers exhibit similar and significant reduc- decreased significantly in nonsmokers, but not in
tions in total bacteria in the subgingival microbiota smokers, after scaling and root planing. In addition,
after rigorous supragingival plaque control. However, Darby et al. (48) showed that some periodontal
deep sites of smokers harbored higher numbers of pathogens, especially T. forsythia, were detected
total bacteria during the study (65). more frequently in smokers than in nonsmokers at
Porphyromonas gingivalis, when exposed to ciga- 2 months after scaling and root planing. As the clini-
rette smoke extract, induced a lower proinflammatory cal outcome after therapy depends upon a suitable
response (i.e. regarding the levels of tumor necrosis reduction in periodontal pathogens, these microbio-
factor-alpha, interleukin-6 and interleukin-12) from logical findings may explain the inefficiency of scaling
monocytes and peripheral blood mononuclear cells and root planing at maintaining long-term periodon-
compared with unexposed bacteria (13). In addition, tal health stability in smokers with periodontitis.
cotinine may increase the ability of P. gingivalis to Therefore, these data highlight the necessity for the
invade epithelial cells (43). Moreover, there was a sig- development of more efficient periodontal therapies
nificant association between the P. gingivalis geno- for smokers, as reported below under the heading
type fimA IV and disease severity in Brazilian smokers ‘Response to periodontal treatments in smokers’.
with chronic periodontitis (170).
Shchipkova et al. (158) showed that the microbial
profile of smoking-associated periodontitis is distinct Immunoinflammatory effects of
from that of nonsmokers, with a high prevalence and smoking on periodontal tissues and
abundance of disease-associated pathogens. Kumar cells
et al. (89) demonstrated that smokers had different
patterns of bacterial acquisition and colonization In recent years, several experimental and clinical
over 7 days, compared with nonsmokers, in the investigations have focused on the immunoinflam-
marginal and subgingival microbiomes, as smoking matory systems that link tobacco use, its specific

193
Nociti et al.

components and periodontal diseases. However, to during the act of smoking and quickly generates
date, the actual cellular and molecular mechanisms higher levels of tobacco products in corporeal tissues
that could precisely explain the exacerbated severity and fluids (i.e. serum, saliva and gingival crevicular
and progression of periodontitis in tobacco/cigarette fluid), whereas chronic exposure provides low, but
users have not been completely elucidated. Overall, persistent, levels of tobacco products. Therefore,
smoking may affect periodontal breakdown through inconsistent findings among studies may be
different pathways, including dysfunction in the mi- explained by differences in study designs, methodolo-
crocirculatory, inflammatory and immune systems. gies and the types of smoking components to which
Tobacco smoke is a cytotoxic and carcinogenic com- subjects were exposed.
bination of more than 5,000 constituents, among A brief explanation of the role of some mediators
which nicotine, carbon monoxide, reactive oxidant and cells involved in the innate and adaptive immune
substances and acrolein are the most important tox- responses will help to provide a better understanding
ins with immunomodulatory potential (94, 168). The of the actions of smoking in the pathogenesis of peri-
various toxic compounds of cigarette smoke may trig- odontitis. In short, when an infection, such as peri-
ger pro- and suppressive immunoinflammatory odontitis, is established, the recognition of pathogen-
effects on mucosal surfaces and periodontal tissues. associated molecular patterns by pathogen recogni-
These toxins include trace components of microbial tion receptors triggers antimicrobial innate immune
cell parts, including lipopolysaccharide, which can responses, including the induction of proteinases,
provoke host immunoinflammatory responses. It is elastases, metalloproteinases, chemokines and cyto-
suspected that cigarette smoke components may kines, such as interleukin-1beta, tumor necrosis fac-
impair innate defenses against pathogens, alter anti- tor-alpha, interleukin-8 and interleukin-6 (5). Both
gen presentation and modulate the adaptive immune neutrophils and macrophages are phagocytic cells
response, for a review, see Lee et al. (94). Our group that link the innate and adaptive immune responses
has performed a recent review of the mechanisms and effectively promote inflammatory resolution and
involved in the effects of smoking on inflammation, tissue healing (156). The innate immune response ini-
for review, see Goncßalves et al. (67). It has been tiates the development of long-lasting adaptive
reported that the effects of smoking on tissues could immunity through B- and T-lymphocytes. T-cells dif-
be attributed to oxidative stress and alterations in im- ferentiate into CD8+ cytotoxic T-cells and CD4+ helper
munoinflammatory systems, including suppression T-cells. CD4+ T-cells may differentiate into T-helper
of the immune and inflammatory systems. This 1, T-helper 2 or T-helper 17 phenotypes, with differ-
review also provides a detailed explanation about the ent cytokine profiles and functions, or into the regula-
smoking-induced functional changes in the main tory T lineage, with suppressor functions (58). T-
immune cells (e.g. neutrophils, macrophages, natural helper 1 and T-helper 17 cells mediate proinflamma-
killer cells, mast and dendritic cells, eosinophils and tory responses and functionally oppose the protective
B- and T-lymphocytes) and indicates multiple intra- T-helper 2 and regulatory T-cells, respectively. The
cellular signaling pathways as possible mechanisms proinflammatory cytokines, interferon-gamma and
to explain the effects of smoking on cytokine produc- interleukin-17, are key markers of T-helper 1 and T-
tion in periodontal tissues. helper 17 responses, respectively, while interleukin-4
Before discussing the mechanisms by which smok- and interleukin-10 are anti-inflammatory T-helper 2
ing affects the periodontal tissues, it is relevant to cytokines. Regulatory T-cells exert regulatory func-
address some general characteristics of the studies tions through the suppressive cytokines, including
available on this topic. The majority of existing stud- interleukin-10 and transforming growth factor-beta.
ies have assessed the levels and functions of immune Interleukin-23 plays a role in stabilization of the T-
and periodontal cells and their mediators of the helper 17 lineage and expansion of T-helper 17
innate and adaptive immune systems. Such investiga- responses. Transforming growth factor-beta up-regu-
tions used different types of studies (i.e. cell culture, lates the transcription factor, forkhead box P3, and
animal and human), samples (fluids and tissue), generates regulatory T-cells (58). When an infection is
tobacco exposure models and immunological/molec- recognized, interleukin-6 inhibits the generation of
ular biology techniques (e.g. quantitative PCR, immu- regulatory T-cells and induces the differentiation of
nohistochemistry, ELISA and flow cytometry). It is T-helper 17 cells in the presence of transforming
important to note that acute and chronic tobacco growth factor-beta (21). Among others, the activation
exposure may have different impacts on the immuno- of these mechanisms from the innate and adaptive
inflammatory responses. Acute exposure occurs immune responses is required for pathogen clearance

194
 Smoking and periodontitis: current perspective

and for re-establishing the integrity of the tissues. significantly lower compared with those from
Therefore, disorders and behaviors such as diabetes nonsmokers. In addition, the levels of matrix
mellitus and smoking, which can interfere in these metalloproteinase-8 mRNA had increased 8 weeks
sequences of events, may put pathogen elimination after smoking cessation. Almasri et al. (7) showed that
and tissue destruction at risk. In general, the majority nicotine significantly increased the expression of
of studies have evaluated the impact of smoking on growth-regulated oncogene-alpha, interleukin-7,
innate, T-helper 1 and T-helper 2 responses, whereas, interleukin-10 and interleukin-15 from gingival fibro-
to date, little evidence exists regarding its role in T- blasts, compared with the untreated control. In
helper 17 and regulatory T-cell responses. addition, the combination of nicotine and lipopoly-
In-vitro studies have evaluated the effects of single saccharide has an additional effect on the levels of
or multiple products of cigarette smoke, cigarette regulated on activation, normal T cell expressed and
smoke extract or cigarette smoke as a whole on differ- secreted (RANTES) and interferon-gamma. Recent
ent types of animal and human periodontal cells, investigations demonstrated that cigarette smoke
including epithelial, immune and bone cells and gin- condensate affects the proliferation of human gingi-
gival and periodontal ligament fibroblasts. Overall, val fibroblasts and increases the collagen-degrading
the literature agrees that smoking and nicotine pres- ability of these cells by changing the production and
ent deleterious stimuli in the function of all of these localization of matrix metalloproteinase and its inhib-
types of cells. Ryder et al. (149) incubated peripheral itors (tissue inhibitor of matrix metalloproteinase)
neutrophils from the blood of heavy smokers (>20 (185, 186). Katono et al. (83) showed that the supple-
cigarettes/day) and nonsmokers with cigarette smoke mentation of osteoblast culture with nicotine and/or
(0–5 min exposure) and nicotine at different concen- lipopolysaccharide increased the expression of matrix
trations. Cigarette smoke up-regulated the expression metalloproteinases 1, 2 and 3 and tissue-type plas-
of adhesion integrin (CD11/18 integrin) and down- minogen activator and decreased the expression of
regulated the surface expression of selectin (L-selec- tissue inhibitor of matrix metalloproteinases 1, 3 and
tin) on neutrophils. The same research group demon- 4. In addition, nicotine suppressed the expression of
strated later that alterations in F-actin kinetics may bone sialoprotein, a mineralized tissue-specific pro-
affect neutrophil functions, which may impact on the tein, in rat osteoblast-like cells (116). Nicotine nega-
pathogenesis of periodontal diseases in smokers tively regulated the differentiation and mineralization
(151). A culture of rat gingival fibroblasts, supple- of murine periodontal ligament cells, as the expres-
mented with high concentrations of nicotine, pre- sion of extracellular matrix and osteoblastic transcrip-
sented diminished proliferation and dilated tion factor genes were reduced in these cells treated
mitochondria and vacuolization (92). Wendell & Stein with this cigarette component (182). Epithelial cell
(179) evaluated the effects of nicotine, in combination growth was inhibited, through an apoptosis/necrosis
or not with lipopolysaccharide from pathogens, on pathway, following exposure to whole cigarette
cell cultures of gingival fibroblasts from subjects with smoke (157). Periodontal ligament cells treated with
chronic periodontitis. Nicotine per se stimulated the various concentrations of cigarette smoke extract pre-
production of interleukin-6 and interleukin-8, sented reduced survival and altered expression of
whereas the association of high doses of nicotine with molecules involved in the structural integrity of the
lipopolysaccharide synergistically up-regulated the ligament (28).
production of these mediators. Another investigation In humans, the local and circulating levels of im-
assessed, using microarray analysis, the effects, in vi- munoinflammatory cells and mediators in smoking-
tro, of acute smoke exposure on the expression of related periodontitis have been evaluated; however,
mRNA in primary peripheral mononuclear blood inconclusive results have been observed. Bostro €m
cells. Cells exposed to smoke for 5 min presented ele- et al. (24) observed higher levels of tumor necrosis
vated expression of 20 genes previously reported to factor-alpha in the gingival crevicular fluid of smokers
be associated with the pathogenesis of periodontitis and former smokers than in the gingival crevicular
(150). Morozumi et al. (113) evaluated the expres- fluid of nonsmokers matched for periodontitis sever-
sions of interleukin-1beta, tumor necrosis factor- ity. Later, the same research group demonstrated no
alpha, interleukin-8, vascular endothelial growth fac- significant effects of smoking habit on the levels of
tor and matrix metalloproteinase-8 by neutrophils interleukin-6, interleukin-1beta and interleukin-1
isolated from the peripheral blood of smokers, before receptor antagonist in gingival crevicular fluid from
and after smoking cessation. The expression of mRNA diseased sites (25, 26). Although a previous study
from all genes of neutrophils from smokers was demonstrated that smokers had a higher functional

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Nociti et al.

elastase activity (a neutrophil proteinase) in the gingi- granulocyte–macrophage colony-stimulating factor,

val crevicular fluid compared with nonsmokers (162), interleukin-1beta, interleukin-2, interleukin-8, inter-
others showed that the levels of elastase did not differ leukin-9, interleukin-12, interferon-gamma, RANTES,
between smokers and nonsmokers, both with peri- tumor necrosis factor-alpha and vascular endothelial
odontitis (130). Rawlinson et al. (142) observed that growth factor than did nonsmokers during one or
the gingival crevicular fluid levels of interleukin- more days of plaque development. These data sug-
1beta, in deep bleeding pockets, and interleukin-1 gest that smokers present a highly proinflammatory
receptor antagonist, in all categories of pockets, were response to early bacterial colonization.
lower in smokers than in nonsmokers. Giannopoulou Our laboratory has performed some investigations
et al. (63) demonstrated, using multiple linear regres- to study the effects of smoking habit on the mRNA
sion analysis, that smoking was associated with the and protein levels of some immunoinflammatory
levels of interleukin-4, interleukin-6 and interleukin-8 mediators of the innate and adaptive immune
in gingival crevicular fluid. Erdemir et al. (56) did not responses, using human gingival biopsies. The gingi-
find significant effects of smoking on the levels of val samples included oral, sulcular and junctional
tumor necrosis factor-alpha and interleukin-6 in the epithelia and connective gingival tissue from teeth
gingival crevicular fluid of patients with chronic peri- indicated for exodontia as a result of advanced peri-
odontitis. The concentration of interleukin-1alpha odontitis. Periodontally healthy gingival samples with
was reported to be reduced in the gingival crevicular no signs of clinical attachment loss and inflammation
fluid of the diseased sites of smokers, compared with were used as controls. The levels of pro- and anti-
nonsmokers (130). In addition, the salivary levels of inflammatory cytokines (interleukin-1beta, interleu-
biomarkers (e.g. prostaglandin E2, lactoferrin, albu- kin-1receptor antagonist, interleukin-6, interleukin-8,
min, aspartate aminotransferase, lactate dehydroge- interleukin-10, interferon-gamma and tumor necrosis
nase and alkaline phosphatase) were significantly factor-alpha), matrix metalloproteinase-2, matrix me-
lower in current smokers than in nonsmokers, sug- talloproteinase-8 and osteoclastogenesis-related fac-
gesting a possible role of smoking in suppressing the tors (osteoprotegerin and RANKL) were first assessed
host-defense system (86). Lappin et al. (93) observed in the gingival tissues of smokers and nonsmokers
a lower serum concentration of osteoprotegerin, an with chronic periodontitis (37, 38). The mediators
osteoclastogenesis suppressor, and a higher ratio of studied are thought to be critical in the initiation,
receptor activator of RANKL/osteoprotegerin, in progression and/or suppression of periodontitis. The
smokers than in nonsmokers. In addition, the levels subjects were divided into the following groups: non-
of osteoprotegerin in smokers had a significant, nega- smoking nonperiodontitis subjects (control); never-
tive correlation with tobacco consumption. Tang smokers with advanced periodontitis; and heavy
et al. (169) showed that increased lifetime exposure smokers (>20 cigarettes/day for at least 10 years) with
to cigarette smoking suppresses osteoprotegerin pro- advanced periodontitis. Gene expression and protein
duction and leads to an increased RANKL/osteopro- levels were analyzed using quantitative PCR and
tegerin ratio in gingival crevicular fluid. Ozcßaka et al. ELISA, respectively. In general, the results showed
(126) reported that smokers with chronic periodonti- that the levels of the majority of the proinflammatory
tis exhibited significantly lower circulating osteopro- cytokines, matrix metalloproteinase-2 and the pro-os-
tegerin concentrations and a higher RANKL/ teoclastogenesis factor, RANKL, were increased in
osteoprotegerin ratio than did healthy smoker con- sites with periodontitis compared with healthy con-
trols. It was recently reported that smoking subjects trol sites, independently of smoking condition. On
with periodontitis exhibited decreased amounts of the other hand, the levels of the anti-osteoclastogene-
proinflammatory cytokines (interleukin-1alpha, inter- sis factor, osteoprotegerin, were reduced in sites with
leukin-6 and interleukin-12), chemokines (interleu- periodontitis. The levels of interleukin-1beta, inter-
kin-8, monocyte chemoattractant protein-1, leukin-8, interleukin-10, tumor necrosis factor-alpha,
macrophage inflammatory protein-1 and RANTES) matrix metalloproteinase-2, metalloproteinase-8,
and regulators of T-cells and natural killer cells (inter- RANKL and osteoprotegerin levels were reduced,
leukin-7 and interleukin-15) than did nonsmokers. whereas those of interleukin-6, interferon-gamma
These findings suggest an immunosuppressive effect and interleukin-1 receptor antagonist were increased
of smoking on periodontal tissues, which may con- in smokers with periodontitis, compared with never-
tribute to the high susceptibility to periodontitis smokers with periodontitis. However, increased
(175). In contrast, Kumar et al. (89) observed that RANKL/osteoprotegerin and interleukin-6/interleu-
smokers presented significantly higher levels of kin-10 ratios were found at sites with periodontitis in

196
 Smoking and periodontitis: current perspective

smokers compared with nonsmokers. Therefore, a and function, alterations in the proportion of subpop-
high frequency and duration of smoking has been ulations of CD4+ and CD8+ cells and smoking-
found to modulate the levels of critical biomarkers of induced immunosuppression (94). The role of B-cells,
innate and adaptive responses in periodontitis. The one principal function of which is immunoglobulin
mechanism of action of smoking on periodontal tis- production, in smoking-related periodontitis has
sues seems to be the suppression of some essential been assessed by studying immunoglobulin levels. Al-
mediators for pathogen elimination, as well as the Ghamdi & Anil (4) compared the levels of serum im-
exacerbation of some mediators involved in tissue munoglobulins (IgG, IgA and IgM) in current, never
destruction which, together, may lead to an enhanced smokers with chronic periodontitis with those of
susceptibility to periodontitis. Interestingly, we healthy controls. A possible suppression of B-cell
recently noticed that type 2 diabetes mellitus, but not function and immunoglobulin production was sug-
smoking, up-regulated the levels of interleukin-17- gested because the serum levels of IgG and IgA were
positive, interleukin-15-positive and forkhead box P3- significantly lower in smokers than in nonsmokers
positive cells (factors related to T-helper 17 and regu- and healthy controls. Jointly, the above-described
latory T-cell responses) and increased the amounts of findings suggest that cigarette smoking may be asso-
fibrosis in sites of chronic periodontitis (53). In addi- ciated with the suppression of the number and func-
tion, unpublished data from our laboratory have also tions of several immune cells from the innate and
shown that the expression of matrix metalloprotein- adaptive responses, which may be a potential mecha-
ase genes and the specific tissue inhibitor of matrix nism by which smoking exacerbates periodontal
metalloproteinases were down-regulated in peri- breakdown.
odontitis sites of smokers compared with similar sites Oxidative stress is an important factor in the patho-
of subjects with type 2 diabetes. Collectively, these genesis of several diseases, including periodontitis.
findings suggest that, although smoking and diabetes The literature suggests a relationship among oxidative
mellitus are both recognized risk factors for periodon- status, periodontitis and smoking-related periodonti-
titis, the immunoinflammatory mechanisms by which tis, in which the progressive damage of the periodon-
they interfere in the pathogenesis of periodontitis tal tissues may partly be caused by an oxidant–
seem to be different. antioxidant imbalance. Polymorphonuclear cells in
In relation to the immune cells, some investigations infected sites are activated by inflammatory media-
have reported no major differences in the neutrophil tors, produce reactive oxygen species (e.g. hydrogen
profile between smoking and nonsmoking subjects peroxide and hydroxyl radical) and generate oxidative
with periodontitis (129, 131). On the other hand, stress (115). In addition, neutrophils exposed to
Gu€ ntsch et al. (71) showed that heavy smokers pre- tobacco smoke display elevated destructive oxidative
sented a lower number of neutrophils and that the burst products, with the release of superoxide and
viability of neutrophils and their ability to phagocy- hydrogen peroxide, which may also cause oxidative
tose were lower in light, moderate and heavy smokers damage in several tissues (148). There are some
than in nonsmokers. Souto et al. (165) demonstrated mechanisms to decompose oxidative species and
that the densities of inflammatory infiltrate and Lan- control tissue injury, including the formation of anti-
gerhans’ cells from the sulcular epithelium and lam- oxidant compounds such as superoxide dismutase,
ina propria were lower in gingival biopsies of smokers catalase, glutathione peroxidase and peroxiredoxins.
with chronic gingivitis than in nonsmokers with However, the levels of superoxide dismutase were
chronic gingivitis. With regard to T-cells, Orbak et al. decreased in the gingival crevicular fluid and saliva of
(125) reported that T-lymphocyte (CD4+ and CD8+) smokers compared with nonsmokers. In addition,
levels were lower in the gingival tissues of smokers there was a significant reduction in the levels of
than nonsmokers. Loos et al. (100) demonstrated that superoxide dismutase in the gingival crevicular fluid
heavy smokers (at least 10 cigarettes/day) presented a and saliva of heavy smokers compared with light
higher number of total leukocytes and neutrophils in smokers and nonsmokers (2).
peripheral blood compared with nonsmokers, former Another potential mechanism by which smoking
smokers and light smokers. In addition, heavy smok- may affect periodontal diseases is through up-regula-
ers had a higher number of CD3+ and CD4+ T-cell tion of the receptor of advanced glycation end-prod-
subsets, and increased T-cell proliferation, after stim- ucts (RAGE) (84, 85). RAGE is a multiligand receptor
ulation with mitogens, than did nonsmokers. Current expressed in few cell types. Its biological function is
data suggest destructive effects of cigarette smoke on dependent on the presence of its various ligands (e.g.
T-cells, including a reduction in T-cell proliferation advanced glycation end-products, S100-calcium

197
Nociti et al.

binding protein/calgranulins, high-mobility group cies of bacteria which hybridize to probes of peri-
protein 1, amyloid-beta-peptides and the family of odontal bacterial species, including Fusobacterium,
beta-sheet fibrils), known to be elevated in chronic P. nigrescens- and P. micra-like species and P. gingi-
metabolic, malignant and inflammatory diseases (6). valis- and A. actinomycetemcomitans-like species,
A previous study revealed that human gingival cells commonly observed in humans. The rats were ran-
exposed to nornicotin, a product of cigarette smoke, domly divided into one of the following groups trea-
up-regulated the expression of RAGE (84). Later, the ted with intraperitoneal injections of: saline solution
same research group (85) studied the expression of (control; group A); 2 ll/g of body weight of 0.13 ll of
RAGE in gingival tissues of smokers and the impact of nicotine/ml of saline solution (group B); 2 ll/g of
nornicotine on RAGE expression in gingival epithelial body weight of 0.19 ll of nicotine/ml of saline solu-
cells. Compared with nonsmokers, biopsies from tion (group C); and 2 ll/g of body weight of 0.26 ll of
smokers expressed a higher level of RAGE and the nicotine/ml of saline solution (group D). Thirty days
cells treated with nornicotine presented a time- later, serial histological sections were obtained and
dependent increase in RAGE expression. Together, the bone loss in the furcation area of the rat molar
these findings suggest that RAGE might be associated was determined by histometric analysis. Greater bone
with periodontal disease related to smoking. loss was found in the ligated teeth of the test groups
More excitingly, we recently reviewed the molecu- (group B, 1.01  0.61 mm2; group C, 1.14 
lar intracellular signaling of cytokine production 0.72 mm2; and group D, 1.36  0.60 mm2) compared
probably related to the modulation of inflammation with the ligated teeth of the control group (group A:
by smoking, for review see Goncßalves et al. (67). This 0.64  0.62 mm2). No differences in bone loss were
mechanism mainly involves the nuclear factor-kap- observed in the teeth without ligatures. These find-
paB family, through the activation of inhibitor of IjB ings suggest that nicotine negatively affects alveolar
kinase-dependent and -independent pathways. In bone loss resulting from biofilm accumulation. How-
addition to nuclear factor-kappaB activation, a num- ever, a dose-dependent effect of nicotine on bone loss
ber of transcription factors, including GATA, PAX5 was not observed. Therefore, it was subsequently
and Smad 3/4, have also been implicated in smoking- hypothesized that higher concentrations of nicotine
related inflammation. Therefore, smoking-induced could induce a dose-dependent effect on the ligature-
cytokine expression seems to be a complex and mul- induced bone loss (120). The same ligature model
tistep system with participation and cooperation of was used in a subsequent study; however, high nico-
multiple signaling pathways. However, to date, these tine concentrations were tested in the following
systems have only been explored in lung inflamma- experimental groups: saline solution (group A);
tion and cancer, and no evidence exists in the peri- 0.37 mg of nicotine/kg (group B); 0.57 mg of nico-
odontal field. Thus, the aforementioned review tine/kg (group C); and 0.73 mg of nicotine/kg (group
highlights the need for future research exploring the D). The results confirmed our previous findings as
molecular intracellular signals of cytokine production greater bone losses were observed around the ligated
in smoking-related periodontitis. teeth of the animals that received nicotine. In addi-
tion, the amount of bone loss indicated a dose-
dependent response to nicotine (0.90  0.64 mm2,
Animal studies 3.08  2.30 mm2, 3.88  1.89 mm2 and 6.90  1.88
mm2 for groups A, B, C and D, respectively). Interest-
Table 3 summarizes the Latin American studies ingly, a direct effect of nicotine was observed on bone
showing the effects of smoking on periodontal tissues around unligated teeth, suggesting that nicotine may
in rats. Our group performed a series of experimental cause alveolar bone loss regardless of plaque
studies in rats to assess the impact of nicotine admin- accumulation. In 2003, our group performed another
istration and cigarette smoking on peri-implant and investigation in rats to evaluate the effects of stress
periodontal tissues (32–36, 39, 121). In a first investi- associated with nicotine administration on periodon-
gation (121), the impact of different doses of nicotine tal breakdown resulting from ligature-induced bone
on ligature-induced bone loss was evaluated. A cotton loss (15). The animals received a saline solution
ligature was placed in the cervical area of the first (group A), 0.73 mg of nicotine/kg/day (group B),
mandibular molar in order to induce bone loss, while stress (immobilization for 2 h/day) plus saline solu-
the contralateral molar was left without ligature. A tion (group C) or stress (immobilization for 2 h/day)
previous study (54) demonstrated that the ligature plus 0.73 mg of nicotine/kg/day (group D). Although
model accumulates biofilm that contains various spe- stress alone did not affect ligature-induced bone loss,

198
 Smoking and periodontitis: current perspective

Table 3. Summary of the animal studies by our group on the effect of smoking on periodontal tissues in rats

Reference Type of exposure Type of evaluation Principal findings

Nociti et al. (121) Low levels of nicotine Ligature-induced bone loss Different doses of nicotine induced an
increase in bone loss
Nociti et al. (120) High levels of nicotine Ligature-induced bone loss Daily administration of nicotine
increased the bone loss in ligated
and unligatedteeth in a dose-dependent
manner
Pinto et al. (136) Nicotine Socket healing after tooth Nicotine delayed alveolar healing in
extraction terms of connective tissue and
osteoneogenesis in a dose-dependent
manner
Benatti et al. (15) Stress + nicotine Ligature-induced bone loss Stress significantly enhanced the
negative effects of nicotine on alveolar
bone loss
Nogueira-Filho Nicotine + occlusal Ligature-induced bone loss Occlusal trauma significantly enhanced
et al. (122) trauma the negative effects of nicotine on
alveolar bone loss
sar-Neto et al. (36) Cigarette smoke
Ce Ligature-induced bone loss and Cigarette smoke inhalation increased
inhalation gingival matrix matrix metalloproteinase-2 levels and
metalloproteinase-2 expression activity in gingival tissue and increased
bone loss in ligated and unligated teeth
Benatti et al. (14) Nicotine and cigarette Self-healing of periodontal tissues Cigarette smoke inhalation induced a
smoke inhalation reduction in bone repair
sar-Neto et al. (32) Cigarette smoke
Ce Ligature-induced bone loss Cigarette smoke inhalation increased
inhalation and mandibular bone density ligature-induced bone loss and
and its interruption negatively affected mandibular bone
density. Cigarette smoke inhalation
interruption reverted the negative
effects of smoking on the mandibular
bone
sar-Neto et al. (33) Cigarette smoke
Ce Bone density in the furcation Cigarette smoke inhalation decreased
inhalation alveolar bone density, regardless of
and its interruption plaque accumulation. Cigarette smoke
inhalation interruption reverted the
negative effects of smoking on bone
density
Bosco et al. (22) Nicotine Bone loss in the furcation Daily systemic administration of nicotine
resulted in significantly greater bone
loss, regardless of plaque accumulation
Giorgetti et al. (64) Cigarette smoke Gene expression in alveolar Cigarette smoke inhalation significantly
inhalation sockets healing after tooth affected the expression of key genes of
extraction bone healing
Milanezi de Almeida Nicotine Counting of fibroblast-like Nicotine induced a reduction in the
et al. (111) cells in the connective tissue number and proliferation of
of molars fibroblast-like cells

it significantly enhanced the effects of nicotine on cantly higher bone loss in the furcation of rats,
alveolar bone loss. Subsequently, our research group compared with administration of saline solution (22).
also demonstrated that nicotine enhanced alveolar Later, the same research group observed a lower
bone loss induced by occlusal trauma in rat molars number and a reduced proliferation of fibroblast-like
with or without ligature (122). In 2007, another cells in the periodontal tissues of rats treated with
Brazilian research group confirmed that daily sys- injections of nicotine, compared with control rats that
temic administration of nicotine promoted signifi- received saline solution (111). Liu et al. (99), testing

199
Nociti et al.

higher doses of nicotine (0.83 mg and 1.67 mg of nico- of carbon monoxide each) in order to simulate the
tine/kg/day) and using micro-computed tomography, cotinine serum levels observed in smokers. Rat blood
also confirmed that nicotine, in a dose-dependent samples were taken 15 min after exposure and the
manner, increased bone loss and decreased bone concentration of nicotine/cotinine was monitored by
mineral density, bone volume fraction and trabecular high-pressure liquid chromatography (36). Using the
number in ligated and unligated teeth. abovementioned protocol of cigarette exposure, the
Although our initial investigations provided impor- mean serum levels of nicotine and cotinine were
tant and original information regarding the role of 346.1  114.3 ng/ml and 265.4  109.8 ng/ml,
nicotine administration in alveolar bone loss, some respectively. These serum levels were similar to those
limitations of the aforementioned studies should be obtained with subcutaneous injections of 3 mg/kg of
highlighted. Nicotine and cotinine serum levels were nicotine (376.03  53.85 ng/ml for nicotine and
not measured and therefore whether, and to what 294.38  41.24 ng/ml for cotinine) (36). This cigarette
extent, nicotine serum levels were comparable with smoke exposure protocol resulted in an increase in
those reported for smokers could not be determined. cotinine levels that were similar to those observed in
In addition, nicotine was administered by intraperito- smokers who smoke 10–20 cigarettes a day (68).
neal injections once a day; this procedure does not Therefore, the effects of intermittent cigarette smoke
adequately simulate the daily frequency and route of inhalation on ligature-induced bone loss and gingival
nicotine intake in humans. Furthermore, nicotine is levels of matrix metalloproteinase-2 were evaluated
only one of the main components of cigarette smoke using histometry and zymography, respectively (36).
and therefore nicotine administration alone did not The rats were randomly assigned to control (nonciga-
mimic the effect of cigarette smoke as a whole. In an rette smoke inhalation) or cigarette smoke-inhalation
attempt to overcome such limitations, our research groups. The results demonstrated that the cigarette
group thereafter used a passive smoking model to smoke inhalation group presented a higher ligature-
assess the effect of cigarette smoke inhalation on induced bone loss (0.64  0.36 mm2 vs. 1.50 
periodontal tissues, with or without biofilm accumu- 0.50 mm2, for control and cigarette smoke-inhalation
lation, and on periodontal healing. This model pre- groups, respectively). In addition, matrix metallopro-
sents some advantages compared with other passive- teinase-2 levels were higher in the exposed rats com-
smoking models. It is effective in producing pulmo- pared with the nonexposed rats, suggesting that
nary emphysema during a short period of time (31) metalloproteinase-2 may be one of the molecules
and requires a simple and inexpensive device. responsible for the increased tissue degradation
Although the cigarette smoke inhalation model used observed in the periodontal tissues of smokers. The
presents advantages over nicotine administration and analysis of the teeth without ligature confirmed our
other passive smoking models, it also differs from previous results with nicotine administration (120),
active smoking by a smoker. The passive smoking showing that smoke inhalation increased bone loss in
model does not reproduce the effect of the high tem- the absence of plaque accumulation (36). In general,
perature of smoke in the airways and the release of the results from this passive smoking model corrobo-
cigarette smoke compounds during tobacco burning, rated those obtained using nicotine injections, sug-
as observed in active smokers. gesting that nicotine is certainly able to intensify the
The rats were exposed to passive smoking, using a periodontal destruction in smokers and contribute, at
cigarette smoke exposure chamber, to evaluate the least in part, to the negative impact of cigarette
effects of cigarette smoke inhalation, and its cessa- smoke on periodontal tissues. With regard to peri-
tion, on ligature-induced bone loss. Pilot studies were odontal tissue healing, we investigated the impact of
used to determine the most appropriate experimental cigarette smoke inhalation and nicotine administra-
conditions, including the characteristics of the expo- tion on the self-healing capacity of the periodontal
sure device and the appropriate timeline of exposure tissues (14). Cigarette smoke inhalation had a signifi-
to cigarette smoke to achieve serum nicotine/cotinine cant, negative effect on bone healing (i.e. of newly
levels comparable to those experienced by moderate formed bone and the extent of bone filling inside the
to heavy smokers. The exposure device was com- defect) in fenestrations created in the alveolar bone
posed of a 45 9 25 9 20-cm3 clear acrylic chamber, of rats. These results indicate that cigarette smoke
an air-pump and two inflow/outflow tubes. Five rats has a direct negative impact on the self-healing ability
per chamber were exposed to cigarette inhalation, of the alveolar bone, even in the absence of biofilm.
three times per day for 8 min (10 cigarettes each con- More recently, our group evaluated the effects of
taining 1.3 mg of nicotine, 16.5 mg of tar and 15.2 mg cigarette smoke inhalation on the expression of genes

200
 Smoking and periodontitis: current perspective

related to bone repair during 14 days of alveolar bone with previous findings (172), together, these data sug-
healing after mandibular molar extraction (64). In gest that the undesirable effects of smoking on peri-
general, the results showed that cigarette smoke inha- odontal tissues may be reversible after its
lation interfered with the expression of a number of interruption and highlight smoking cessation as a
genes that play crucial roles in bone healing, leading relevant approach to dealing with smokers in clinical
to a predominantly catabolic response, which may be practice.
detrimental to new bone formation. The deleterious In summary, this series of studies in rats from our
effects of nicotine on bone healing after tooth extrac- and other laboratories confirm clinical data demon-
tion were also demonstrated by another Brazilian strating that tobacco consumption is a risk factor for
research group and were based on the histological periodontal diseases and healing. In addition, our
analysis of osteoneogenesis and angiogenesis in rats findings provide important models for the in-vivo
(136). These findings in rats support our previous analysis of the effects of smoking on periodontal tis-
clinical study regarding the influence of smoking on sues as some aspects of the relationship between cig-
alveolar ridge remodeling after tooth extraction. arette smoking and periodontitis may not be
Radiographic evaluations demonstrated that smokers clinically evaluated as a result of ethical and method-
presented with a lower bone density in alveolar sock- ological limitations.
ets and a tendency toward lower alveolar process
height and width than that observed in nonsmokers,
6 months after extraction (152). Response of smokers to periodontal
Based on the possible benefits of smoking interrup- treatments
tion for systemic and oral health, subsequent studies
from our group evaluated the impact of the interrup- We have presented above some findings in rats on
tion of smoke exposure on ligature-induced bone the deleterious effects of smoking on periodontal
loss, periodontal healing and alveolar bone density healing. Overall, previous investigations have also
and quality (14, 32, 33). These studies provided rele- reported that smoking is a risk factor for other types
vant contributions not only to periodontology, but of wound healing, especially in clinical situations in
also to the implantology field, as poor bone quality which tissues are exposed to low oxygen tension (e.g.
and smoking are factors well recognized to be associ- lower-extremity amputations and mastectomies) (97,
ated with early/late implant failures. The rats were 164) and/or are in direct contact with cigarette
assigned to the following groups: noncigarette smoke smoke, such as in head and neck surgery (90).
inhalation (control group); 83 days of cigarette smoke Clinical studies have long compared the response
inhalation before ligature placement (cessation of smokers and nonsmokers to various types of peri-
group); and 90 days of cigarette smoke inhalation odontal treatments, including nonsurgical and surgi-
before and 60 days after ligature placement (uninter- cal therapies, for review, see Johnson & Hill (82). In
rupted exposure group). The results revealed similar general, the results have shown that smoking pro-
levels of bone loss for control and cessation groups, motes an unfavorable clinical response (i.e. worse
whilst the uninterrupted exposure group presented a reductions in probing depth and lower gains in clini-
significantly higher bone loss. In addition, continuous cal attachment) to nonsurgical and surgical periodon-
exposure to cigarette smoke promoted a significantly tal therapies, as well as to regenerative and plastic
lower bone density in the basal mandibular bone of periodontal procedures (8, 27, 47, 76, 106, 128, 137,
the rats (32). The proportion of mineralized tissue in 159, 163). Smoking is also a major risk factor for poor
the furcation area of teeth without ligature was also response to initial treatment in subjects with general-
compared among rats treated with noncigarette ized aggressive periodontitis and treated with nonsur-
smoke inhalation, 2 months of cigarette smoke inha- gical periodontal therapy (78). Previous data revealed
lation, 3 months of cigarette smoke inhalation and that approximately 90% of cases of refractory peri-
2 months without noncigarette smoke inhalation odontitis are observed in smokers (101, 105) and that
(n = 16) and 5 months of cigarette smoke inhalation. heavy smoking is a risk factor for disease progression
The results corroborated our previous findings and after active periodontal therapy (109).
revealed that cigarette smoke inhalation affects Scaling and root planing, the most usual periodon-
tooth-supporting bone as early as 2 months after the tal therapy, produces well-recognized clinical and
initial exposure. In addition, cessation of smoke expo- microbiological periodontal benefits, including
sure may revert the negative impact of cigarette improvements in clinical parameters and the re-
smoke inhalation on alveolar bone (33). In agreement establishment of a microbial community with

201
increased proportions of host-compatible microor-
ganisms (73). Studies have shown that smokers pres-
ent a worse clinical response to scaling and root
planing than do nonsmokers (69, 76, 178). As the
smoking habit reduces the effect of scaling and root
planing, several studies have proposed the use of
adjunctive therapeutic approaches (i.e. local/systemic
antimicrobials and anti-inflammatory agents) to
improve the effects of basic periodontal therapy in
smokers. With regard to antimicrobial therapies, a
systematic review has revealed that the evidence for
an additional benefit of adjunctive antimicrobial ther-
apy in smokers with chronic periodontitis is lacking
and questionable. Although some studies have dem-
onstrated advantages of the use of local and systemic
antimicrobials in smokers, the risk of bias in the
evaluated studies was high and inadequate, and
inconsistent data hampered the performance of
meta-analyses (9).
Previous studies from our research group (102–104)
evaluated the clinical and microbiological effects of
local doxycycline as an adjunctive to scaling and root
planing in smokers. In general, the results showed
that 10% doxycycline, delivered locally after scaling
and root planing, produced better clinical results
compared with mechanical therapy alone in the
treatment of smokers with periodontitis. In the first
study, 43 smokers with chronic periodontitis were
randomly assigned to receive scaling and root planing
alone (control group) or scaling and root planing plus
a local application of doxycycline (test group). The
adjunct use of doxycycline gel promoted a greater
attachment gain (1.63  0.93 mm for the test group
vs. 1.04  0.71 mm for the control group) at
6 months post-therapy. In addition, initially deep
pockets (≥7 mm) from the test group showed a higher
probing-depth reduction (3.78  1.41 mm for the test
Nociti et al.

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