Effects of Smoking on Non-surgical Periodontal Therapy in

Patients with Periodontitis Stage III or IV, and Grade C

Burcu KANMAZ* DDS, PhD

David F LAPPIN† BSc PhD
Christopher J NILE† BSc PhD
Nurcan BUDUNELİ* DDS, PhD, Professor

*
Department of Periodontology, School of Dentistry, Ege University, Izmir, Turkey.
†
Oral Sciences Research Group, School of Medicine, Dentistry and Nursing, Glasgow Dental
School, University of Glasgow, UK.

Number of words: 4,171 (excluding the abstract and references)

Number of tables: 2

Number of figures: 4

Number of references: 44

Running title: Smoking and non-surgical treatment

One sentence summary: Smoker periodontitis patients exhibit higher detection rates of
Gram-negative bacteria before and after non-surgical periodontal therapy.

Corresponding author:

Dr. Nurcan Buduneli

Department of Periodontology
School of Dentistry
Ege University
35100 Bornova, İzmir / Turkey

This is the author manuscript accepted for publication and has undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1902/jper.10424.

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Tel: + 90 232 311 4556
Fax: + 90 232 388 0325
E-mail: nurcan.buduneli@ege.edu.tr

ABSTRACT

Aim: To evaluate possible effects of smoking on clinical, biochemical, and microbiological

outcomes of non-surgical periodontal treatment in patients with periodontitis stage III or IV

and Grade C.

Material and Method: Conventional quadrant-wise non-surgical periodontal treatment was

performed and whole-mouth periodontal measurements were recorded at baseline, 1-, 3-, 6-

months after completion of treatment. Saliva, gingival crevicular fluid, subgingival plaque,

and blood samples were obtained at the same time points. Inflammatory cytokine levels,

presence, and quantities of 11 different bacterial species were determined. Smoking status

was validated by cotinine assay.

Results: Fourteen smoker and 13 non-smoker patients completed the study protocol and

revealed similar clinical findings except for the higher plaque scores in the non-smokers at 6-

months (p<0.01). Significant differences were found between the study groups in biofluid

cytokine levels at 1-, 3-months (p<0.01). Gram-negative bacteria were more abundant in the

smokers at baseline and so were Gram-positive bacteria in the non-smokers (p<0.01). Gram-

negative bacteria repopulated in the smokers faster than in the non-smokers (p<0.01).

Conclusion: The present findings suggest that smoker patients with periodontitis stage III

and IV, Grade C respond well to the non-surgical periodontal treatment during the 6-month

follow-up. However, smokers exhibit faster repopulation of Gram-negative bacteria.

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MeSH Key Words: Bacteria; cytokines; gingival crevicular fluid; periodontitis; smoking

INTRODUCTION

Smoking is the major preventable risk factor in the initiation and progression of periodontal

diseases. Smokers have more severe periodontal disease; increased probing depth, significant

bone loss, clinical attachment loss, gingival recession, and considerable susceptibility for

tooth loss.1 Furthermore, smoking adversely affects the outcomes of non-surgical periodontal

treatment.2 Less reduction in probing depth and clinical attachment gain have been found in

smoker periodontitis patients compared to their non-smoker counterparts following non-

surgical periodontal treatment.3 Smoker patients with aggressive periodontitis (AgP) have

been reported not to respond well to mechanical non-surgical periodontal treatment.4

Accordingly, smoking has been associated with an increased risk for recurrence of

periodontitis in AgP patients.5

AgP is characterized by attachment loss that is usually inconsistent with the amount of

plaque and dental calculus.6 Few studies have investigated the success of non-surgical

periodontal therapy in AgP patients and it is often not possible to predict the prognosis of

various treatment options.7 Rosalem et al.8 compared the response to non-surgical periodontal

therapy in GAgP and generalized chronic periodontitis patients using clinical, immunologic

and microbiological data and suggested that the treatment response was similar in both

groups.

The interleukin-17 (IL-17) family is composed of six members (IL-17A-IL-17F). IL-

17A, IL-17F homodimers and IL-17A/F heterodimer have been demonstrated to drive

expression of antimicrobial peptides, cytokines, chemokines, and matrix metalloproteinases

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from fibroblasts, endothelial and epithelial cells and therefore, have pro-inflammatory

properties.9 In contrast, IL-17E has been shown not only to be a driver of a T helper-2

immune response but also a suppresser of the destructive pro-inflammatory responses.10

Indeed, IL-17E has been shown to inhibit IL-17A and P. gingivalis induced nuclear factor-

kappa B (NFB) activity.10 Therefore, IL-17E is likely to have opposing biological effects to

IL-17A, IL-17F and IL-17A/F. During the inflammatory response, pro-inflammatory

cytokines such as IL-1β, IL-6, IL-11, IL-17A, IL-17F and tumour necrosis factor-alpha

(TNF-α) can induce alveolar bone resorption by increasing the expression of receptor

activator of NFB ligand (RANKL) and reducing osteoprotegerin (OPG) production from

osteoblasts and stromal cells.10 In a recent study, it was stated that systemic cytokine

signaling via IL-17 is impaired in smokers and this impairment is associated with bacterial

colonization by opportunistic pathogens locally in the airways.11 Many studies suggested that

IL-17 has a significant role in the pathogenesis of periodontal diseases.12-14

Smoking affects host cytokine levels in biofluids15 and pathopionts are more abundant

in the subgingival microflora of smokers.16 However, the exact mechanisms of the

detrimental effects of smoking on periodontal tissues remain unclear.17 It was hypothesised

that non-smoker patients with GAgP will respond better in terms of the clinical, biochemical

and microbiological parameters to the conventional mechanical non-surgical periodontal

treatment compared to the smoker counterparts. Therefore, the aim of this study was to

evaluate possible effects of smoking on the outcomes of non-surgical periodontal treatment.

MATERIALS and METHODS

Study population

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A total of 48 Caucasian patients were screened and 34 patients with the clinical and the

radiological diagnosis of GAgP were recruited for this study between June 2014 and August

2015. The diagnosis of GAgP was assigned in accordance with the clinical criteria stated in

the consensus report of the World Workshop in Periodontitis.18 According to the consensus

report published by Papapanou et al.19 the patients included in the present study comply with

the definition of periodontitis Stage III or IV with regard to the extent and severity and Grade

C due to the early onset of the disease. These patients had at least one site with probing depth

(PD) and clinical attachment level (CAL) ≥5 mm in their incisors and/or first molars and at

least six other teeth with similar PD and CAL measurements, and familial aggregation (all

individuals were asked if they had any family member with current or history of severe

periodontal disease). Smokers were those who reported smoking more than 10 cigarettes/day

for more than 5 years, while non-smokers were those who reported that they had never

smoked. Smoking status was validated with salivary cotinine analysis.

The study was approved by the Ethics Committee of Ege University, İzmir, Turkey

(Protocol number; 14-9/28) and registered to the US National Institutes of Health Clinical

Trials Registry site with the ID number of NCT03512938. Full accordance with ethical

principles including the World Medical Association’s Declaration of Helsinki as revised in

2000 was provided. The study protocol was explained and written informed consent was

received from each individual before clinical periodontal examinations and sampling.

Medical and dental histories were obtained. Exclusion criteria included presence of less than

15 teeth, known medical disorders such as diabetes mellitus, rheumatoid arthritis,

immunological disorders (which could affect local and systemic inflammatory status and the

cytokine levels in oral fluids) and history of antibiotic, anti-inflammatory or periodontal

treatment in the preceding 6 months.

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Sample Size Calculation

A two-sided two-sample t-test with a significance level of 5% was used for the sample size

calculation. Based on this analysis, a sample size of 12 subjects for each group was estimated

to be necessary to achieve 80% power to detect a difference of 1 mm (SD 0.85) between the

two groups in the mean PD reduction. A total of 34 were to be selected to compensate for a

possible drop-out during the course of the study.

Non-surgical periodontal therapy

Patients were motivated and instructed to brush twice a day with the modified Bass technique

and use interdental toothbrushes and dental floss once a day regularly. Each patient

underwent quadrant-wise scaling and root planing (SRP) over a 4-week period. Root planing

(RP) was performed under local anaesthesia (2% lidocaine, epinephrine 1:100.000) and a

standard curette set‡ newly sharpened with a stone§ was used for each patient. A single

researcher (BK) performed SRP in all patients. The procedures were continued until a hard,

smooth root surface was sensed with an explorer tip.

Clinical measurements

Clinical periodontal measurements were performed by a calibrated periodontist (BK) at

baseline that is before the treatment and also 1-, 3-, and 6-months after completion of SRP.

Saliva, serum, gingival crevicular fluid (GCF), and subgingival plaque samples were

collected at the same time points. Calibration of the investigator was performed with

measurements in five patients, who were diagnosed to have chronic periodontitis and were

not included in this study. PD and CAL measurements were repeated with two days intervals.

The intraexaminer kappa scores were 0.96 for PD and 0.85 for CAL. PD, CAL, bleeding on

‡ Hu-Friedy, Chicago, IL.

§ Arkansas Stone SS4, Hu-Friedy, Chicago, IL.

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probing (BOP) (BOP deemed positive if bleeding occurred within 15 seconds after

probing)20, and plaque index (PI)21 were recorded at six sites of all teeth present using a

manual periodontal probe. In each patient, one site with the greatest PD in each quadrant was

selected for GCF and subgingival plaque sampling.

Saliva and serum sampling

In the morning, following an overnight fast, during which subjects were requested not to

drink (except water) or to chew gum, whole saliva samples were obtained simply by

expectorating into polypropylene tubes thus active sampling technique with minimal

stimulation was used. The samples were immediately frozen and stored at -40ºC until the

sample collection period was completed.

Nine milliliters of venous blood were taken from the antecubital vein by a standard

venipuncture method and centrifuged for 10 minutes at 3000 rpm, separating serum from the

cells. The serum samples were immediately frozen and stored at -40ºC.

GCF and subgingival plaque sampling

GCF samples were obtained from buccal aspects of one interproximal site in each quadrant.

Supragingival plaque was removed carefully by sterile curettes; the surfaces were dried and

isolated by cotton rolls. Filter paper strips‖ were placed in the orifice of the pocket for 30

seconds. Care was taken to avoid mechanical trauma, and strips contaminated with blood

were discarded. The absorbed GCF volume was estimated by a calibrated instrument¶. Then,

the strips were placed into polypropylene tubes and immediately frozen and stored at -40ºC.

GCF samples were converted to an actual volume (μL) by reference to a standard curve.

‖ Periopaper, ProFlow, Amytyville, NY.

¶ Periotron 8000, Oraflow, Plainview, NY.

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 Following GCF sample collection, subgingival plaque samples were collected from the

same sites by sterile paper points#. Paper points inserted into the deepest part of the pocket

and kept for 5 seconds, were then placed into sterile propylene tubes, immediately frozen and

stored at -40ºC.

Biochemical analyses

Smoking status of each patient was determined by cotinine analysis in the salivary samples

using an Enzyme Immunoassay (EIA)** . GCF samples from each patient were eluted in 1 mL

of phosphate-buffered saline containing 0.05% polysorbate surfactant†† with a 60-minute

incubation on a rotary mixer at +4ºC. Serum and saliva samples were used neat. Enzyme-

linked immunosorbent assay (ELISA) kits were purchased for IL-17A, IL-17E, IL-17A/F, IL-

6‡‡, IL-1β and TNF-᧧ analysis. The ELISA assays were carried out on 50 μL samples of

GCF in duplicate and on 50 μL of serum/saliva in triplicate according to the manufacturers’

recommendations. The minimum detection limits in the assays were 1.9 pg/mL for IL-17A

and IL-17E, 7.9 pg/mL for IL-17A/E, and 0.4 pg/mL for IL-1β and TNF-α, and 0.8 pg/ml for

IL-6. The results were expressed as picograms per 30 seconds for the total amounts in the

samples and as picograms per milliliter for concentrations.

Preparation and assessment of genomic DNA

A Gram-positive DNA isolation kit‖‖ was used to prepare genomic DNA from the pelleted

microbes present in the paperpoint sample and from known quantities of laboratory strains of

target microorganisms. The DNA in standards and plaque samples was measured by

# Diadent Group International, Seoul, Korea.

**Alere toxicology, London, UK.
††Tween 20, Sigma-Aldrich, Poole, UK.
‡‡ R&D Systems, Abingdon, UK.
§§ Thermo Fisher Renfrew, UK.
‖‖ Masterpure, Illumina, San Diego, CA.

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fluorimetric analysis¶¶. DNA content of the target species was used to fix the copy number of

its bacterial genome to use as a standard for each realtime quantitative polymerase chain

reaction (RTqPCR) assay.

RT-qPCR

Real-time reagents were purchased from##,*** and previously validated assays22 were used for

detection and quantification of bacterial cell copy numbers in 1 µg plaque DNA for the

following bacteria: P.gingivalis, A. actinomycetemcomitans, P. intermedia, P. micros, T.

forsythia, F. nucleatum T. denticola S. mutans, F. nucleatum, S. oralis, A. naeslundi, V.

dispar. The PCR reaction was performed on a thermocycler†††, under the following

conditions: 10 minutes at 95ºC and 40 cycles of 30 seconds at 95ºC and 1 minute at 60ºC.

PCR assay efficiency was determined as follows: efficiency (E) = 10(-1/slope) -1 and deemed

to be acceptable (between 91% and 104%). None of the primer amplified DNA purified from

microbial standards other than the target species.

Statistical analysis

Microsoft Excel and commercially available statistical softwares‡‡‡,§§§ were used to analyse

the data. Sample size calculation included an estimated alpha error of 1% to allow for

multiple testing of two treatment groups (smokers and non-smokers) and beta error of 20%

with an effect size =1 for differences in the levels of biomarkers or copy numbers of several

putative pathogens. For a two-sided test, a minimum of 27 subjects were required (13 in one

¶¶ CyQUANT assay system, Invitrogen, Thermo Fisher Scientific, Waltham, MA.

## Thermo Fisher Scientific, Paisley, UK.
*** Eurogentech, Liege, Belgium.
††† MRX3000 Agilent Thermocycler, Agilent, Edinburgh, UK.
‡‡‡
SPSS Inc. version 21 IBM, Chicago, IL.
§§§ Graphpad Prism version 5, La Jolla.

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group and 14 in the other group) for a longitudinal study a minimum of 34 (17 per group)

were selected to compensate for possible drop-outs during the course of the study and allow

for initial cross-sectional comparisons in an explorative study design. It was anticipated that

larger effect sizes of 1.5 would be required in analyses where multiple testing was considered

in cross-sectional analysis if there were any drop-outs from the study participants and

alpha =0.01.

Only the data of those patients who completed the entire study protocol were included

in the longitudinal analyses. Cross-sectional analyses were performed on all available

participants at each time point (Figure 1). Mean values of PD, CAL, and PI measurements

were calculated for each patient and BOP was presented as percentages. CAL, PD, BOP, and

PI data were compared between the study groups using Mann Whitney-U test. The Friedman

test and Dunn's multiple comparison test was used for post hoc comparisons of the repeat

sample tests. Although it was not relevant to the outcome, the baseline relationship between

clinical, biochemical, and microbiological data was evaluated by Spearman correlation

analysis.

RESULTS

Clinical measurements

Four smoker and three non-smoker patients had to be excluded from the study due to their

non-compliance with appointments. The study was completed with 13 non-smoker patients (9

females, 4 males) aged between 23-38 years (mean age 31.00±4.90) and 14 smoker patients

(4 females, 10 males) aged between 26-38 years (mean age 32.93±3.37). The study groups

differed in gender distribution (p<0.05), but were similar in mean ages. The patients’ medical

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and dental histories, as well as socio-economical states were similar in the smoker and non-

smoker groups. All patients had middle-income levels on the study registration forms and

reported 12-14 years of education. The mean value of salivary cotinine concentration was

37.65 ng/ml in the smoker group (min-max; 32.92-50.12 ng/ml) and 0.65 ng/ml in the non-

smoker group (min-max; 0.48-0.76 ng/ml) (p<0.001) (Table 1). Smoking habits of the

patients did not change during the study.

At baseline, the clinical periodontal measurements were similar in the smoker and non-

smoker patient groups (Table 2). At 1-month, PD, CAL, and BOP decreased significantly in

the smoker group compared to the baseline (p<0.0001). The 3- and 6-month evaluations

revealed significant reductions in all clinical periodontal parameters compared to the baseline

values in both the smoker and non-smoker patient groups (p<0.0001). At 1-month, both

groups revealed significant decreases in the number of sites with PD > 6 mm compared to the

baseline (p<0.0001). Only the non-smoker group exhibited significant decreases in the counts

and percentages of sites with PD > 6 mm at 3-month compared to the 1-month (p<0.0001)

(Table 2).

Biochemical analysis

Smoking status of each patient was determined by cotinine analysis in the salivary samples.

Patients who had cotinine levels greater than 8 ng/ml are defined to active smokers23 were

included to the smoker group.

Intergroup comparisons (i.e., between baseline and follow-up) revealed no statistically

significant differences in serum IL-17A, IL-17E, IL-1β, TNF-α, IL-6 cytokine levels between

the groups at majority of the time points (Figure 1). At 1-month, IL-17E levels were higher in

the non-smoker group (p<0.01). At 6-month, IL-17A levels were higher in the smoker group

(p<0.01). Intragroup comparisons (i.e., between smokers and non-smokers) showed that

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TNF-α and IL-6 decreased significantly at 1-month in both groups (p<0.0001). At 3-month

IL-1β decreased only in the smoker group (p<0.0001), while both TNF-α and IL-6 exhibited

significant decreases compared to the baseline values (p<0.0001). At 6-month, only IL-6

levels decreased significantly in both groups (p<0.0001).

There were no intergroup differences in salivary cytokine levels at baseline (Figure 2).

At 1-month, IL-17A and IL-1β levels were higher in the smoker group (p <0.01). At 3-month

IL-1β, and IL-6 levels were higher in the non-smoker group (p<0.01).

The GCF sample volumes were similar in the smoker and non-smoker groups at

baseline and decreased significantly in both groups at 1-, 3-, and 6-months (p<0.0001) (Table

1). At baseline, GCF cytokine levels were similar (Figure 3). At 1-month; IL-17A levels were

higher in the non-smoker group (p <0.01). There was no significant difference between the

two groups in the 3-month GCF data (p>0.05).

Microbiological analysis

At baseline, copy numbers of P.gingivalis, P.intermedia, T.denticola and T. forsythia (Figure

4) were higher in the smoker group (p<0.01). Gram-positive bacteria such as S. oralis were

higher in the non-smoker group (p<0.01). The frequency of carriage did not differ between

smokers and non-smokers (data not shown). At 1-month, and at 3-month, the copy numbers

of all bacteria were similar in both groups. At 6-month copy numbers of F. nucleatum, P.

gingivalis, P. intermedia, S. oralis, T. denticola and T. forsythia were significantly higher in

the smoker group (p<0.01). Similar results were obtained when the proportions of each

organisms to the total bacterial DNA content were compared (data not shown). Total bacterial

counts at 3- and 6-months were similar in both groups and decreased significantly from

baseline to 1-, 3-, and 6-months (p <0.0001) (Table 2).

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Spearman correlations

At baseline, there were associations between clinical data (such as, PD, CAL, the number of

sites with PD >5mm) and levels of certain cytokines (TNF-α, IL-17A, IL-6) in serum and

saliva (all Rho >0.32, p<0.05), levels of TNF-α, IL-17A, and IL-6 in GCF (all Rho>0.32,

p<0.05) and several correlations were observed between IL-1β, TNF-α, IL-6 and IL-17A and

microbiological parameters (all Rho>0.333, p<0.05). As anticipated these relationships were

not observed following treatment.

DISCUSSION

Smoking is the most common modifiable risk factor for periodontitis worldwide and it is

among the major determining factors for the grade of periodontitis in the 2017

classification.19 In the present study, the response to mechanical non-surgical periodontal

treatment was evaluated comparatively in smoker and non-smoker patients with periodontitis

stage III or IV, and grade C using clinical, biochemical and microbiological parameters.

Baseline clinical periodontal status is of upmost importance when comparing the response to

treatment among two or more study groups. The present study groups revealed similar

baseline clinical findings in terms of oral hygiene, gingival inflammation and visual signs of

tissue destruction. Moreover, the groups were comparable with regard to the socio-economic

status and educational attainment. After non-surgical periodontal treatment, the clinical

periodontal measurements decreased similarly in both smoker and non-smoker patients.

Potential biomarkers for monitoring periodontal diseases may be sought in different

biofluids; saliva, GCF, or serum. All these media have their own benefits; GCF provides site-

specific data, whereas saliva reflects the oral health as a whole and serum gives information

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also about the systemic health or environmental factors such as smoking. In the present study,

all three biofluid samples were investigated to have a comprehensive information.

Adverse effects of smoking on clinical healing have been reported in previous

studies24-29. Hughes et al.4 reported that smoker patients with GAgP did not respond well to

non-surgical periodontal treatment. Smokers have been reported to have a higher risk for

unresponsive pockets and further breakdown during supportive periodontal treatment.2 Non-

smoker patients with periodontitis exhibited better healing responses than smokers.16

However, another study reported contradicting data as both non-smokers and smokers

exhibited similarly significant reductions in PD and bleeding scores between baseline and 6

months after non-surgical periodontal treatment.30 Accordingly, the present study indicated

similar outcomes for CAL and BOP in the smoker and non-smoker groups. Hawthorne effect

may have had a role in the similar clinical outcomes in smoker and non-smoker groups.

Moreover, it is likely that the observed statistically significant differences in microbiological

and immunological parameters may precede the clinical periodontal parameters and/or may

be too small to be reflected in clinical findings. It is also possible that statistically significant

differences in clinical periodontal findings may be detected between the smoker and non-

smoker groups with longer follow-up periods or larger scale studies.

Some studies suggest that the use of antibiotics in the treatment of aggressive

periodontitis provides short-term benefits.31-33 Considering the potential side-effects34

together with limited benefits of short-term duration, no additional antibiotics were used in

the present study.

Suzuki et al.,35 found no correlation between GCF IL-1β levels and clinical parameters

in AgP and the current study partly agreed with their results. Serum IL-17 affects the

production of IL-1, IL-6 and TNF-α, and this cytokine was increased in AgP patients

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compared to healthy individuals and it was suggested to play a role in the pathogenesis of

AgP.12 GCF IL-17A levels were found to be significantly elevated in patients with AgP.13

GAgP patients exhibited higher GCF IL-11:IL-17A ratio than healthy controls and this has

been suggested to have a role in the pathogenesis of the disease.14 Teles et al.,36 also reported

increased GCF levels of IL-1β:IL-10 in patients with GAgP. On the other hand, patients with

AgP have been shown to have higher serum and salivary IL-1β and pentraxin-3

concentrations than the healthy individuals.37 Similar findings have been reported for salivary

TNF-α levels.29 In another study, serum concentrations of TNF-α, IL-4, IL-17A and IL-23

have been investigated in AgP patients at baseline and at 6-months after SRP.38 The authors

have documented higher IL-17A and TNF-α levels in AgP patients than those of

periodontally healthy subjects. Moreover, TNF-α levels remained high in GAgP patients 6-

months after completion of non-surgical periodontal therapy.

De Lima Oliveira et al.39 stated that the IL-1β:IL-10 ratio may play an important role in

the pathogenesis of AgP. Moreover, IL-17A, IL-23 and myeloperoxidase maintained high

GCF levels in GAgP after SRP.40 Serum IL-17E levels have been suggested as a good

indicator of favourable treatment outcome in periodontitis patients after scaling and root-

planing.41 Rosalem et al.8 found that GCF IL-1β levels decreased significantly 3 months after

therapy in GAgP patients. Another study reported that GCF concentrations of IL-6 and TNF-

α decreased from baseline to the 6-month in non-smokers as well as smokers.30 Although

similar trends were observed, the current study agreed only in part with the earlier

investigations presumably because of individual differences or by the complex nature of

GAgP and possible presence of disease subgroups. This might be explained by the transient

post-treatment falls and increases observed; paticularly for TNF-α. In the current study, IL-

17E levels in biofluids other than serum decreased following non-surgical periodontal

treatment. The reason for this difference remains obscure. However, other key cytokines such

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as TNF-α and IL-17A are increased in these biofluids and this may have a direct impact on

local production of IL-17E. One might also speculate that the increase in pro-inflammatory

cytokines TNF-α and IL-17A in GCF and saliva appears to coincide with a re-colonization of

the tissue by pathogenic microorganisms. Correlations between several Gram-negative

organisms and pro-inflammatory molecules were evident particularly at baseline, these

associations were weaker during the follow-up after SRP, but this was probably due to the

reduction in the number study participants as well as to the reduction in biomarker and

bacterial counts.

Significant positive correlations between PD, CAL, P. gingivalis and T. forsythia have

been reported in GAgP patients.42 However, the current study indicated few correlations

between the microorganisms and the clinical indices, and that these were only obseved at

baseline. The major finding of the present study is that potentially pathogenic Gram-negative

bacteria copy numbers were significantly higher in the smokers. In contrast to the smokers,

non-smoker patients revealed higher copy numbers of Gram-positive bacteria. The present

findings are in line with those of Darby et al.16 in terms of significantly higher detection rates

of A. actinomycetemcomitans and P. intermedia in smoker patients than the non-smoker ones.

While the results of the current study are not strictly analogous; the smoker group exhibited

significantly higher copy numbers of P. intermedia, P. gingivalis, T. denticola and T.

forsythia at baseline while the carriage frequencies were similar. The present finding of lower

baseline proportions of P. gingivalis in non-smokers is parallel to the previous studies

showing that smokers are more likely to harbor higher numbers of P. gingivalis, and to

exhibit more persistent infection.43,44 Copy numbers of A. actinomycetemcomitans were

higher in the smoker group at 1- and 6-months after treatment and the copy numbers of P.

gingivalis, P.intermedia, T. denticola and T. forsythia were higher in the smoker group at 6-

months. In the smoker patients, carriage rates decreased following non-surgical periodontal

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treatment but incresed to the baseline levels by the 6-month reassessment and the copy

numbers of Gram-negative bacteria increased to a greater extent in the smokers.

The major limitation of the present study is the rather low number of patients mainly

due to the stringent inclusion and exclusion criteria and the dropout of patients during the

follow-up period which resulted in some of the later cross-group comparisons having less

than 80% statistical power. While the longitudinal component of the study retained sufficient

statistical power (at an effect size =1) the follow-up period was shorter than desired because

of the failure of sufficient patients to return at later time points. Short follow-up period could

be considered also as a limitation in the present study. Another limitation may be the lack of

blinding to the study group during the measurement of the clinical periodontal parameters as

one researcher performed the SRP and clinical recordings in all patients. However, stainings

on the teeth, increased gingival pigmentation, and the characteristic cigarette smell make

blinding of the clinician extremely difficult if not impossible in studies that compare smokers

and non-smoker counterparts.

CONCLUSION

Within the limits of the present study, it may be concluded that smoker patients with

periodontitis stage III or IV and grade C exhibit higher detection rates and faster

recolonisation of Gram-negative bacteria in subgingival plaque samples. Adjunctive

treatment modalities such as local antimicrobials and/or laser therapy were beyond the scope

of this study, while the particular aim was to compare smoker and non-smoker periodontitis

patients in terms of their response to conventional mechanical periodontal treatment. New

larger scale studies with additional therapy modalities and longer follow-up periods may be

undertaken to provide more data for the literature.

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CONFLICTS OF INTEREST

The authors have stated explicitly that there are no conflicts of interest in connection with this

article.

ACKNOWLEDGEMENTS This study was supported by a grant from the Ege University

Research Foundation (Project No: 2014 DIS 013) and funds from the University of Glasgow.

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Figure legends

Figure 1. Cytokine levels in serum of smokers and non-smokers with aggressive periodontitis before
and after non-surgical therapy. A. Serum IL-17A Level Changes B. Serum IL-17E Level Changes C.
Serum IL-17A/E Level Changes D. Serum IL-1β Level Changes E. Serum TNF-α Level Changes F.
Serum IL-6 Level Changes
*
Significant difference between smokers and non-smokers (p<0.01) †Significant difference from
baseline data in the group (p<0.0001). ‡Significant difference from 1-month data in the group
(p<0.0001). §Significant difference from 3-month data in the group (p<0.0001).

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Figure 2. Cytokine levels in saliva of smokers and non-smokers with aggressive periodontitis before
and after non-surgical therapy. A. Saliva IL-17A Level Changes B. Saliva IL-17E Level Changes C.
Saliva IL-17A/E Level Changes D. Saliva IL-1β Level Changes E. Saliva TNF-α Level Changes F.
Saliva IL-6 Level Changes

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*
Significant difference between smokers and non-smokers (p<0.01) †Significant difference from
baseline data in the group (p<0.0001). ‡Significant difference from 1-month data in the group
(p<0.0001). §Significant difference from 3-month data in the group (p<0.0001).

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Figure 3. Cytokine levels in GCF of smokers and non-smokers with aggressive periodontitis before
and after non-surgical therapy. A. GCF IL-17A Level Changes B. GCF IL-17E Level Changes C.
GCF IL-17A/E Level Changes D. GCF IL-1β Level Changes E. GCF TNF-α Level Changes F. GCF
IL-6 Level Changes

*
Significant difference between smokers and non-smokers (p<0.01) †Significant difference from
baseline data in the group (p<0.0001). ‡Significant difference from 1-month data in the group
(p<0.0001). §Significant difference from 3-month data in the group (p<0.0001).

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Figure 4. Real-time PCR detection of Copy numbers of DNA from Gram negative bacteria. in the
dental plaque of smokers and nonsmokers with Aggressive periodontitis before and after non-surgical
therapy. A) A. actinomycetemcomitans, B) A. naeslundii, C) F. Nucleatum, D) P. gingivalis, E) P.
intermedia, F) P. oralis, G) S. mitis, H) S. Oralis, I) T. denticola, J) T. forsythia, K) V. dispar.

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*
Significant difference between smokers and non-smokers (p<0.01) †Significant difference from
baseline data in the group (p<0.0001). ‡Significant difference from 1-month data in the group
(p<0.0001). §Significant difference from 3-month data in the group (p<0.0001).

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 Table 1. Demographic findings in the study groups.

Non-smoker
Demographic Variable Smoker Group
Group

Patient count (n) 13 14

Gender (female/ male) 9/4 4 / 10

Age (year) (min - max) 23 - 38 26 - 38

Age (year)
31.00 ± 4.90 32.93 ± 3.37
(Mean ± SD)

Cigarette consumption/day
0 (0-0) 15 (10-20)
(median (Q1-Q3))

Pack-Year
0.00 ± 0.00 11.75 ± 4.76
(Mean ± SD)

Cotinine
0.65 (0.48-0.76) 37.65 (32.92-50.12)
(ng/ml) (median (Q1-Q3))

Table 2. Inter and intra group comparisons of various parameters. All data are expressed as
median (Q1-Q3) unless otherwise noted.

Variable Time Non-smokers (n=13) Smokers (n=14)

Baseline 4.19 (3.89-4.89) 4.43 (4.35-5.20)

PD (full mouth) 1. month 2.72 (2.52-3.50) 3.06 (2.87-3.48)*

(mm) 3. month 2.59 (2.40-3.26)* 2.89 (2.74-3.41)*

6. month 2.58 (2.26-3.16)* 2.98 (2.82-3.27)*

Baseline 4.85 (4.28-5.02) 5.42 (4.40-6.25)

CAL (full mouth)
1. month 4.18 (3.24-4.38) 4.57 (3.24-5.29)*
(mm)
3. month 3.82 (3.09-4.21)* 4.10 (3.11-4.87)*

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 6. month 3.70 (2.91-4.14)*† 3.81 (3.29-5.16)*

Baseline 79.76 (56.94-100.00) 66.87 (52.28-94.07)

BOP (full mouth) 1. month 19,15 (15,48-24,01) 18,83 (9,17-26,60)*

(%)
3. month 14.88 (12.90-17.76)* 15.91 (9.68-18.00)*

6. month 15.48 (8.83-17.26)* 18.10 (7.86-21.83)*

Baseline 3.28 (3.12-3.59) 3.47 (3.28-3.57)

1. month 2.43 (2.29-2.65)* 2.47 (2.23-2.63)*

PI (full mouth)
(0-5) 3. month 2.45 (2.21-2.60)* 2.31 (2.13-2.50)*

6. month 2.48 (2.37-2.58)*‡ 2.19 (2.11-2.44)*

Baseline 8.5 (7.25-9.50) 8.38 (7.44-9.69)

1. month 4 (3.30-6.50)* 4.5 (4.25-5.63)*

PD (sample sites)
(mm)
3. month 4 (3.75-5.50)* 4.63 (4.00-5.13)*

6. month 3.75 (3.25-4.50)* 3.88 (3.50-4.25)*

Baseline 9 (7.25-10.00) 9 (7.00-10.00)

1. month 5 (4.00-8.00)* 6 (4.00-8.00)*

CAL (sample sites)
(mm) 3. month 5 (3.25-7.00)* 5.50 (4.00-7.00)*

6. month 4 (3.00-6.75)* 5 (4.00-6.25)*

Baseline 21.00 (10.50-47.50) 29.00 (17.00-41.50)

1. month 0.00 (0.00-24.00)* 2.50 (0.00-17.00)*

PD (sites with PD> 6 mm)
(n) 3. month 0.00 (0.00-12.50)*† 2.00 (0.00-8.00)*

6. month 0.00 (0.00-8.00)*† 0.50 (0.00-3.00)*†§

Baseline 13.46 (6.48-26.47) 17.90 (11.22-34.29)

1. month 0.00 (0.00-14.29)* 1.93 (0.00-10.32)*

PD (sites with PD> 6 mm)
(%) 3. month 0.00 (0.00-8.01)*† 1.26 (0.00-5.55)*

6. month 0.00 (0.00-4.97)*†§ 0.33 (0.00-1.84)*†§

Baseline 1 (0.67-1)‡ 0.71 (0.48-1)

GCF volume (µl)
1. month 0.5 (0.24-0.67)* 0.42 (0.18-0.75)*

3. month 0.46 (0.29-0.66)* 0.52 (0.18-0.8)*

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 6. month 0.51 (0.28-0.66)* 0.45 (0.12-0.74)*

Baseline 11.31x108 (8.44x108 -12.69x108) ‡ 7.09x108 (4.55x108 - 9.73x108)

1. month 5.81x108 (4.50x108 -7.56x108) * ‡ 3.31x108 (1.92x108 - 4.56x108) *

Total Bacterial Count (n)
3. month 6.56x108 (3.50x108 -7.06x108) * 5.16x108 (3.70x108 - 6.34x108) *

6. month 6.13x108 (5.00x108 -8.25x108) * 5.22x108 (4.36x108 - 7.66x108) *

*
Significantly lower than the baseline value (p<0.0001).
†
Significantly lower than the 1-month value (p<0.0001).
‡
Significant difference between the study groups (p<0.05).
§
Significantly lower than the 3-month value (p<0.0001).

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