J Clin Periodontol 2013; doi: 10.1111/jcpe.

12142

The effects of stress on periodontal treatment: a longitudinal investigation using clinical and biological markers
Bakri I, Douglas CWI, Rawlinson A. The effects of stress on periodontal treatment: a longitudinal investigation using clinical and biological markers. J Clin Periodontol 2013; doi: 10.1111/jcpe.12142.

Issam Bakri1, Charles W. Ian Douglas2 and Andrew Rawlinson3

1

Oral and Maxillofacial Medicine and Surgery, Charles Clifford Dental Hospital, Shefeld, UK; 2Academic Unit of Oral and Maxillofacial Pathology, University of Shefeld, Shefeld, UK; 3Academic Unit of Restorative Dentistry, University of Shefeld, Shefeld, UK

Abstract Aim: To investigate the effects of psychosocial stress on the outcome of nonsurgical periodontal treatment (NPT). Methods: Patients were categorized as stressed or unstressed, and the degree of stress was measured. One deep bleeding and one deep non-bleeding site 6 mm were selected in each patient for detailed investigation, and the clinical parameters were recorded before and at 6 months after NPT. Elastase and C-terminal teleopeptide of type I collagen (ICTP) were measured in gingival crevicular uid (GCF) samples at both intervals. Results: The baseline, clinical parameters and biological markers were similar in both stressed and unstressed groups, other than for GCF elastase levels, which were signicantly higher in the stressed group of patients (p < 0.05). The effect of stress on the changes for clinical measurements and elastase levels in GCF was statistically signicant for deep bleeding sites, with the response to treatment being poorer in the stressed group. The effects of smoking and the degree of stress were not statistically signicant for any of the clinical or biological parameters (p > 0.05). Conclusions: Patients under psychosocial stress had a poorer outcome following NPT. The assessment of psychosocial stress may be valuable in the holistic management of periodontal disease.

Key words: markers; periodontal disease; stress; treatment Accepted for publication 6 July 2013

Psychosocial stress is thought to play an important role in the aetiology and progression of periodontal diseases (Giddon & Goldhaber 1963, Eickholz et al. 2005), and the outcome
Conict of interest and source of funding statement The authors declare that they have no conict of interests. This study was partly self-funded with some funding from the Dental School, University of Shefeld.

of treatment (Vettore et al. 2005, Kloostra et al. 2006). Individuals under high work load, those with poor marital status (Marcenes & Sheiham 1992), occupational dissatisfaction (Linden et al. 1996) and high psychological strain (Croucher et al. 1997) have more periodontal destruction than those not under such stresses. Also, several psychological disorders and determinants have been shown to be associated with chronic and aggressive periodontitis (Genco et al. 1999, Vettore

et al. 2003, 2005, Dumitrescu & Kawamura 2010, Goyal et al. 2011). The outcome measures used in most previous studies have relied on the traditional clinical measurements of periodontal clinical attachment level (CAL), pocket probing depth (PPD) and bleeding on probing (BoP), and only a few have investigated the inuence of psychosocial factors on periodontal healing after treatment (Axtelius et al. 1998, Wimmer et al. 2002). To date, there have been no studies on the effect of

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Bakri et al.
to join the study. Potentially suitable patients were invited in succession over a number of clinics until the required number were recruited. Patients were given an information sheet and invited to join the study if they wished to do so, and given at least 24 h to decide. Written consent was obtained from patients who wished to join the study. A clinical diagnosis of chronic periodontitis was made based on the criteria described by Armitage (1999). The inclusion criteria were: (i) a diagnosis of chronic periodontitis; (ii) aged >20 years; (iii) no systemic disease that may affect periodontal disease progression or the outcome of treatment. The age, gender and smoking status (smoker or non-smoker) were recorded and patients recruited to the study were assigned a study number. The study codes and the patient details were kept separately with only the investigators having access to this information, and data were stored on a passwordprotected computer, kept in a locked ofce.
Categorization of stress

stress on periodontal treatment outcomes that have included biological measures of inammation and bone turn over. Several markers can be measured in gingival crevicular uid (GCF). These include neutrophil elastase which can be used as a measure of inammation (Lamster & Ahlo 2007, Ujiie et al. 2007), and the C-terminal teleopeptide of type I collagen (ICTP) (Giannobile et al. 2002) which has been suggested to reect pathological type I collagen degradation in chronic diseases (Jakob et al. 2002). Indeed, detection of ICTP in GCF has been reported to indicate bone loss in periodontal disease (Al-Shammari et al. 2001, Taba et al. 2003, Kinney et al. 2007). The categorization of patients as being stressed or non-stressed may be undertaken according to the level of cortisol in saliva as this is considered a reliable biological marker of stress, and to be more indicative of stress than the level in plasma (Vining et al. 1983). In addition, the Perceived Stress Scale (PSS) questionnaire may be used to assess the degree of psychosocial stress (Cohen et al. 1983). The aim of this study was to investigate the effects of psychosocial stress on the outcome of non-surgical periodontal treatment (NPT) using clinical and biological markers in patients with chronic periodontitis.
Materials and Methods
Study design

unstressed. Patients with values above these levels were categorized as being stressed. This process was not blinded; however, all other parameters (clinical and biological markers) were processed and analysed blindly.
Clinical data

The exposure of interest in this study was the stress level of participants. A quasi-experimental design was used rather than an experimental design because it would have been impossible and also unethical to expose the study participants to stress randomly.
Patients

Ethical approval was obtained from the South Shefeld Research Ethics Committee, and the research was registered with Shefeld Teaching Hospitals NHS Foundation Trust for Research Governance purposes. Patients were recruited from among those attending a Periodontology Clinic by inviting those with chronic periodontitis and requiring treatment

The categorization of patient status was carried out at baseline using an assay of the salivary cortisol level, and a version of the PSS questionnaire with 10 questions was used to measure of the degree of stress as this was previously validated by Cohen & Williamson (1988). The PSS is widely used and its reliability and validity was originally determined in a group of college students, and participants in a community smoking cessation programme (Cohen et al. 1983). Further evidence for its construct validity comes from higher PSS scores being associated with for example failure to quit smoking (Cohen & Lichtenstein 1990) and failure among diabetics to control blood sugar levels (Kramer et al. 2000). A sample of saliva was obtained from each patient at about the same time of day on each occasion to remove the effects of the circadian rhythm in salivary cortisol levels. Values were regarded as normal when ranging from 3 to 10 ng/ml (AM) and 0.52.5 ng/ml (PM) (see below), and patients with these values or less were categorized as

The periodontal condition of each patient was assessed by the measurement of probing depths, attachment loss, bleeding upon probing, gingival recession and increased mobility scores. Patients with chronic periodontitis were considered for entry into the study. Two representative sites were selected for detailed study in each patient: A deep non-bleeding (DNB) site with probing depths of 6 mm (non-inamed) and a deep bleeding (DB) site with probing depths of 6 mm (inamed). Clinical measurements of probing depth (mm) and bleeding on probing (present or absent) were made using a Williams periodontal probe. The selection of these categories of sites was based on the rationale that deep bleeding sites are inamed and may be actively undergoing periodontal disease progression, and that nonbleeding sites are not clinically inamed and are more likely to be stable. We wished to investigate the effect of stress on the inammatory markers in each type of site. The assessment of examiner reliability was undertaken before the study by repeat clinical measurements to determine the percentage that were within 1 mm of the original.
Saliva

Saliva was obtained by asking patients to spit into an Eppendorf tube. The samples were transferred to the laboratory in numbered tubes and stored at 20C until ready for assay.
Gingival crevicular uid

Each site was isolated from saliva using a cotton-wool roll and dried before sampling GCF using Periopaper strips (Proow Inc., Amityville, NY, USA). The rst strip was introduced to the entrance of the gingival sulcus for 5 s to empty the crevicular pool. A second strip was left in the

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Stress and periodontal treatment

pocket or sulcus for 1 min. to obtain the sample used for investigation. The volume of GCF was measured using a Periotron calibrated using human serum and used to calculate the concentration of each enzyme in samples. The strips containing GCF were stored at 20C in sealed plastic Eppendorf tubes containing 1.5 ml of transfer buffer (PBS supplemented with 0.5% BSA) until further use.
Treatment

Patients then received full-mouth standard NPT comprising oral health education and advice on plaque control, including brushing and inter-dental cleaning. Scaling and root surface debridement of all sites 4 mm under local anaesthesia was undertaken using ultrasonic and hand instrumentation. Patients were reviewed 3 months following initial treatment at which time further oral health education and advice on plaque control was given as necessary, and root surface debridement was provided for residual sites 4 mm. Maintenance care was provided for sites demonstrating satisfactory clinical improvements. The clinical measurements and samples were recorded before treatment and at 6 months post treatment by one investigator (IB).
Biological markers

A competitive ELISA method was developed to measure salivary cortisol. Briey, the principle of the assay was to add labelled competitor cortisol at a standard concentration to the patients sample of saliva and this was then added to wells coated with anti-cortisol antibody. Under these circumstances, both the standard and the patients cortisol compete for binding to immobilized antibody with the outcome that the more cortisol in the patients sample the less labelled competitor cortisol will bind. The assay parameters were optimized and then it was tested on samples of saliva from 10 healthy volunteers. From this, the normal cortisol levels were determined to be 310 ng/ml in the morning and 0.52.5 ng/ml in the afternoon, both of which are in line with values reported in other studies (Aardal & Holm1995).

For the assay, wells were coated with monoclonal murine anti-cortisol antibody (East Coast Biologics, Inc., North Berwick, ME, USA) diluted (1:5000) in coating buffer (0.1M sodium carbonate buffer, pH 9.6) overnight at 4C. After washing with PBS-Tween and blocking of non-specic binding sites (2% (w/v) BSA in PBS, 2 h at 37C), 50 ll of patients saliva was added along with 50 ll of competitive cortisol-HRP conjugate (25 lg/ml in PBS-BSA) and incubated for 1 h at 37C. Wells were then washed and substrate added (tetramethylbenzidine 1 lg/ml; Sigma, St. Louis, MO, USA). The subsequent colour change was measured at 450 nm in a plate reader (FluoStar, BMG Labtechnologies, Allmendgruen, Germany). A cortisol standard curve (200.05 ng/ml) was run in each assay, which also served as the positive control. Negative controls included wells with no anti-cortisol capture antibody and wells with no cortisol-HRP conjugate. Each sample was assayed in triplicate. Elastase activity was measured by a modication in the method of Giannopoulou et al. (1992). Human neutrophil elastase (Sigma) was used to produce a standard curve of activity (0100 ng/ml in 50 mM PBS, pH 7.4 containing 0.05% Triton), using the uorogenic substrate Meo- Suc-Ala-Ala-Pro-Val/7-amino4-methylcoumarin (100 lg/ml). Incubation was at 37C for 6 h with the resultant uorescence measured hourly in a plate reader (350nmex, 450nmemm; FluoStar, BMG lab technologies, Germany). To measure elastase in the GCF samples, 50 ll aliquots of the eluted material were added to an equal volume of uorogenic substrate and incubated as above. Samples, including negative and positive controls, were assayed in duplicate. A commercial ELISA kit (Nordic Bioscience, Herlev, Denmark) was used for detection of ICTP. The assay was performed according to the manufacturers instructions, and the absorbance of the nal colour generated was measured in a plate reader at 450 nm.
Analysis of data

The study was designed to recruit all patients whether stressed or

unstressed and the ratio of stressed to unstressed patients was unknown at the design stage. At this early stage, a power calculation was carried out assuming a ratio of one stressed to four unstressed patients. The power calculation, based on a two-sample t-test, showed that a total sample size of 55 patients (11 stressed and 44 unstressed) would have 80% power to detect a difference of 1.5 mm assuming a standard deviation of 1.5 mm based on the changes in probing depth following non-surgical treatment previously reported by Cobb (2002). After the rst 20 patients had been recruited, it became clear that the actual ratio of stressed to unstressed patients was 1:2, this more balanced ratio would provide the same power with fewer patients and therefore the sample size was recalculated for this ratio a sample size of 39 (13 stressed and 26 unstressed) patients would also provide 80% power to detect a difference of 1.5 mm assuming a standard deviation of 1.5 mm. Therefore, a nal sample size of 40 patients was selected. A further ve patients were recruited to take into account an anticipated fall out rate in the study. The clinical measurements and the biological data were subject to tests for normality, and comparisons between baseline clinical measurements and elastase levels in GCF were performed using the independent samples t-test. Comparisons between ICTP levels were performed using the MannWhitney U-test. Linear regression models were used to study the change in values of clinical and biological data over 6 months (6 months baseline) comparing the patients who were stressed at baseline with those who were unstressed. The model tted the change from baseline as the dependent variable, the baseline value as a covariate and factors for smoking and stress group at baseline. The analysis used multiple imputation (MI) and last observation carried forward (LOCF) for missing data. A similar linear regression model was tted with the PSS total score at baseline as a continuous covariate, together with a factor for smoking and the baseline value as a covariate.

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Bakri et al.
repeat measurements were within 1 mm of the original, indicating good intra-examiner reliability and agreement. The descriptive statistics for periodontal probing depths, clinical attachment levels, elastase and ICTP for the sites investigated are shown at baseline in Table 2. The periodontal pocket probing depths, clinical attachment levels and ICTP levels for stressed and non-stressed patients at baseline were generally similar. However, the levels of elastase in GCF from DB and DNB sites in stressed patients were both signicantly higher than in non-stressed patients (p < 0.05). Table 2 also shows the changes from baseline to 6 months for all eight parameters investigated. The effect of stress on these changes is shown in Table 3 using both the MI and LOCF methods to input missing values. The effect of stress on changes in probing depths for DNB sites was statistically signicant using the MI method only, but just failed to reach signicance for the LOCF method for inputting missing values. The effect of stress on all the other parameters for DNB sites was not signicant using both methods. In contrast, the effect of stress on all the changes for probing depths, clinical attachment levels and elastase levels in GCF was statistically signicant for deep bleeding sites using both methods. The effect of stress on changes in ICTP levels at deep bleeding sites was not statistically signicant. Overall, therefore, both methods of analysis for inputting missing data gave similar results for all measurements, other than for changes in probing depths at DNB sites. In no case was there a correlation between the levels of biological or clinical measures and the degree of stress as judged by the PSS score or for smoking (p > 0.05).
Discussion

Littles test was used to demonstrate that the missing data were missed completely at random (MCAR) prior to MI, and the mean value for each group was used to substitute for missing data. This method was used because we considered that treatment was likely to have produced some effect. The baseline data were used for the method of LOCF, and as this assumes no change in the data this would tend to under estimate a treatment effect. Where both methods give the same outcome, the credibility of the results may be supported. SPSS version 20 (Armonk, NY, USA) was used for analyses. All statistical tests employed a level of signicance of p < 0.05 at a 95% condence level.
Results
Patients

Fifty patients were eligible for recruitment to the study of which 45 were recruited. The characteristics of the patients included in the study are shown in Table 1. An exploratory analysis of missing data was undertaken. Five patients failed to complete the study; however, 40 patients remained which was the number calculated to provide 80% power. All the data were recorded at baseline, but the 6 months data for the ve patients who left the study were not available. Two stressed and three unstressed patients failed to complete the study, accounting for 12.5% and 10.3% of the patients in each group respectively. Littles Test demonstrated the data were missing completely at random.
Clinical measurements

This is the rst report to our knowledge to investigate the effect of stress on treatment, using salivary cortisol and a self reported stress scale questionnaire in a cohort of patients with periodontal disease. The ndings of a better response to NPT in unstressed compared with stressed patients suggests that avenues for the exploration of adjunctive treatment, such as stress management, may be helpful as part of the holistic management of patients with periodontal diseases. A key nding from the biological data is that elastase levels were

Table 2. Mean and standard deviation of the raw data (no imputation) at baseline and change from baseline for the sites investigated in unstressed and stressed patients showing the periodontal pocket probing depths (mm), clinical attachment levels (mm), elastase (ng/ ml) and ICTP (ng/ml) for deep non-bleeding (DNB) and deep bleeding sites (DB) Baseline Change from baseline to 6 months Stressed (n = 26) Mean 1.4 1.7 7.13 1.26 1.4 1.1 9.56 1.48 SD 1.08 1.27 5.569 1.199 0.93 0.95 6.717 1.017 Unstressed (n = 14) Mean 1.8 1.9 6.26 1.13 2.4 2.3 7.89 1.74 SD 0.71 1.09 4.554 0.935 0.76 1.06 5.855 0.945

Stressed (n = 29) Mean Deep non-bleeding sites Periodontal probing 6.3 depth (mm) Clinical attachment 8.1 level (mm) Elastase (ng/ml) 26.61* ICTP (ng/ml) 3.39 Deep bleeding sites Periodontal probing 6.8 depth (mm) Clinical attachment 8.1 level (mm) Elastase (ng/ml) 30.03* ICTP (ng/ml) 3.76 SD 0.79 1.34 6.223 1.875 0.68 1.15 6.160 1.200

Unstressed (n = 16) Mean 6.1 7.5 20.22 2.88 6.8 8.6 23.04 3.61 SD 0.46 1.106 4.588 0.857 0.71 0.99 5.275 1.170

The assessment of examiner reliability showed that more than 90% of

Table 1. Characteristics of patients according to gender, smoking habits and age Stressed Gender (male/female) Smokers Mean age (years) (SD) *p > 0.05. 7/9 5 49.8 (9.7) Unstressed 9/20 10 44.6* (10.3)

*p < 0.05 stressed versus unstressed. ICTP, C-terminal telopeptide of type 1 collagen. 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Stress and periodontal treatment

Table 3. Estimated effect of stress (stressedunstressed) on the change from baseline to 6 months calculated from the linear regression models. Each regression model included factors for stress and smoking and a covariate for the baseline measure. Smoking was non-signicant (p > 0.05) in all models, the baseline covariate was signicant in all models (p < 0.05) Showing effect size for stress and p-value Multiple imputation Effect size for stress Changes in deep non-bleeding sites Periodontal probing depth Clinical attachment level Elastase level ICTP level Changes in deep bleeding sites Periodontal probing depth Clinical attachment level Elastase level ICTP level *p < 0.05. ICTP, C-terminal telopeptide of type 1 collagen. 0.63 0.55 1.708 0.090 0.93 0.88 3.611 0.184 95% CI 0.08 .10 1.776 0.415 0.50 0.23 0.411 0.124 1.18 1.20 5.193 0.236 1.36 1.54 6.812 0.493 p-value 0.025* 0.096 0.336 0.589 <0.001* 0.008* 0.027* 0.238 Effect size for stress 0.54 0.54 2.171 0.135 0.94 0.84 4.418 0.369 LOCF 95% CI 0.05 0.16 1.324 .431 0.36 0.22 1.094 .073 1.13 1.23 5.665 0.702 1.51 1.46 7.742 0.810 p-value 0.070 0.125 0.217 0.632 0.002* 0.009* 0.010* 0.099

statistically higher within the stressed group at baseline and remained higher throughout the study for all sites. In addition, we found the effect of stress on the changes in elastase levels at deep bleeding sites was statistically signicant. In previous studies, it has been demonstrated that GCF elastase levels were positively correlated with PPD and CAL in systemically healthy patients with periodontitis (Alpagot et al. 1996). Chen et al. (2000) and Figueredo et al. (2004) have also shown that a general improvement in periodontal healing occurred after NPT and that this corresponded to a signicant decrease in total elastase activity. The ndings of our investigation are in agreement with the general reduction in elastase activity following treatment, but additionally report the effect of stress on GCF elastase levels both before and in response to treatment. To our knowledge, no previous studies have reported on the effect of stress on this inammatory marker in GCF. The level of cortisol in saliva is a better measure of adrenal cortical function than the level of serum cortisol (Vining, et al. 1983), and so was considered a valid measure to categorize patients. The PSS questionnaire on the other hand provided a means to measure the degree of stress being experienced by the participants (Cohen et al. 1983). However, the results failed to show a statistically signicant effect of the degree of stress on the changes seen in the clinical or biological data. It was the categorization as stressed or

unstressed that was the important factor in this study. Our ndings showed that there was a decrease in GCF ICTP levels in all sites following treatment, although the effect of stress on the changes in this marker of bone turnover was not statistically signicant. Reductions in GCF ICTP levels were slightly greater in unstressed patients at deep bleeding sites, suggesting possibly greater reductions in bone turnover at 6 months. In general, these ndings are in agreement with those of previous studies in terms of decreases in GCF ICTP levels in response to interventional therapies (Perez et al. 2002, Wilson et al. 2003, Kinney et al. 2007), but additionally we have investigated the effect of stress on this marker. It is possible that stress might simply be a surrogate for other risk factors that may also impact on periodontal health such as smoking. However, smoking was found to be non-signicant for changes in any of the clinical measurements or biological markers. Other confounders such as plaque and age may also affect periodontal health. Plaque scores were not recorded specically for this study and the presence of plaque may have an effect on the inammatory status of the site being investigated. However, the inammatory status of the sites investigated was taken into account in their selection as being either inamed or not, and the mean age of both groups was not statistically different (p > 0.05).

The mechanisms whereby stress may affect the outcome of periodontal treatment is unclear. The immune response may be affected and slow healing, and some studies have reported the association between anxiety and periodontal health (Castro et al. 2006). This may be due to a disruption in the balance between oral bacteria and the hosts immune system, through alteration in the cytokine response (Genco 1992, Seymour et al. 1993, Ainamo & Ainamo 1996, Genco et al. 1998). Psychological stress signicantly increases IL-1b, IL-6 and IL-10, and decreased IFN-c production (Paik et al. 2000). Interestingly, most of the known periodontal disease risk factors are known to induce HypothalamicPituitaryAdrenal axis hypersensitivity (Genco et al. 1998, Breivik et al. 2000). Alterations in the production of cytokines seems to inuence the TH1/TH2 balance (Breivik et al. 1996), resulting in a shift towards a pattern of cytokine production by TH2 lymphocytes (Ramirez et al. 1996), which may, at least in part, explain the association. The clinical responses to treatment are similar to those published by Cobb (1996, 2002) and suggest the variations in the biological markers observed, are unlikely to be a consequence of an atypical response to treatment. Finally, the limitations of a quasi-experimental design in comparison with an experimental design should be considered. These include the lack of controls, the groups may not be comparable at baseline and

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the lack of random assignment to study groups, which in this study was determined by the presence or absence of stress. The uneven loss of two stressed and three unstressed patients from the study amounting to an attrition rate of 11% may also have affected our ndings, by introducing a selection bias due to the slightly uneven attrition rate between the two groups of patients. However, the numbers of patients remaining in each group were the same as the number determined in the power calculation. In future research, the investigation of individual factors such as personality and coping should also be explored as inuencing factors.
Conclusions

Our ndings support the hypothesis that psychosocial stress is associated with a poorer outcome of NPT on the basis of clinical changes and biological markers. This knowledge may be helpful in the holistic management of patients with chronic periodontitis.
Acknowledgement

The authors acknowledge the statistical assistance of Rosie Taylor, Statistical Services Unit, University of Shefeld, in this study.
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Stress and periodontal treatment

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Address: Andrew Rawlinson Academic Unit of Restorative Dentistry, University of Shefeld, Claremont Crescent, Shefeld S10 2TA UK E-mail: a.rawlinson@shefeld.ac.uk

Clinical Relevance

Scientic rationale for the study: To investigate the effects of psychosocial stress on the outcome of nonsurgical periodontal treatment using clinical and biological markers.

Principle ndings: Patients under psychosocial stress have a poorer response to non-surgical periodontal treatment. Practical implications: Stress may affect the outcome of periodontal treatment according to clinical and

biological markers. This information may be useful in the holistic management of patients undergoing periodontal treatment.

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd