Journal of Periodontology; Copyright 2014
DOI: 10.1902/jop.2014.140266
Effects of Scaling and Root Planing on Clinical Response and Serum Levels of Adipocytokines in Obese Patients With Chronic Periodontitis Tiago Eduardo Dias Gonçalves, DDS, MS*, Magda Feres, DDS, MS, PhD Professor*, Glaucia Santos Zimmermann, DDS, MS, PhD program*†, Marcelo Faveri, DDS, MS, PhD Assistant Professor*, Luciene Cristina Figueiredo, DDS, MS, PhD Assistant Professor*, Paloma Gralha Braga, DDS*, Poliana Mendes Duarte, DDS, MS, PhD Assistant Professor* *
Department of Periodontology, Dental Research Division, Guarulhos University, Guarulhos, São Paulo, Brazil.
†
Department of Dentistry, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Background: Despite several investigations suggesting that obesity is a risk indicator for periodontitis, little is known about the impact of obesity on periodontal treatment response. The aim of this study was to evaluate the effects of scaling and root planing (SRP) on clinical parameters and circulating levels of leptin and adiponectin in obese patients with chronic periodontitis (CP). Methods: Twenty-four obese and 24 non-obese patients with CP were submitted to SRP. Clinical parameters were assessed at baseline, 3 and 6 months post-therapy. Serum levels of leptin and adiponectin were evaluated at all time points, using enzyme linked immunosorbent assay. Results: SRP improved the clinical parameters of both groups at 3 and 6 months (p 30 % of the sites with concomitant probing depth (PD) and clinical attachment level (CA) ≥ 4 mm and a minimum of six teeth distributed in the different quadrants presenting at least one site with PD and CA ≥ 5 mm and bleeding on probing (BoP) at baseline. To be included, patients also needed to present glycated hemoglobin (HbA1c) < 6.5 %, fasting plasma glucose (FPG)
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70-99 mg/dl, C-reactive protein (CRP) < 6 mg/l. The levels of HbA1c (High-performance Liquid Chromatography method), FPG (Glucose Oxidase method) and CRP (LatexAgglutination-Test) were all assessed by the Guarulhos University Clinical Analysis Laboratory. Exclusion criteria were pregnancy, lactation, current smoking and smoking within the past 10 years, any medical condition requiring prophylactic antibiotic coverage before dental treatment, subgingival periodontal therapy in the previous 12 months, antimicrobial, antiinflammatory, immunosuppressive and lipid-lowering (e.g. statins) therapies during the previous 6 months, regular use of mouthrinses containing antimicrobials and use of orthodontic appliances. Patients reporting the presence of systemic conditions that could affect the progression of periodontitis and/or gain/loss of weight (e.g. diabetes mellitus, immunological disorders, osteoporosis, hypothyroidism, hyperthyroidism) were also excluded. Anthropometric Measurements and Experimental Groups One trained examiner (P.G.B) performed all anthropometric measurements, including weight (kg), height (m), waist (cm) and hip circumferences (cm). Body mass index (BMI) was calculated as the weight divided by the square of height (kg/m2). The waist-hip ratio (WHR) was calculated as the ratio of waist to hip circumference. Obese patients were defined as having BMI ≥ 30 and < 40 kg/m2 and concomitant WHR ≥ 0.85 for women and WHR ≥ 0.90 for men. Non-obese patients were defined as having BMI ranging from 20 to 29.9 kg/m2 and WHR below that determined for obesity (i.e. WHR < 0.85 for women and WHR < 0.90 for men) 22. The anthropometric measurements were re-assessed at all follow-up visits to verify if the patients did not change their obese or non-obese status during the study. Non-surgical Periodontal Therapy Initially, all patients received supragingival plaque and calculus removal, exodontia, provisional restoration and filling overhang removal, as necessary. They were also instructed regarding brushing technique and use of dental floss. A trained periodontist (G.S.Z.) performed SRP in four to six appointments lasting approximately 1 hour each, using manual curettes‡ and ultrasonic device§ under local anesthesia. Periodontal therapy was completed in 14 days. The endpoint for each SRP appointment was “smoothness of the scaled roots”. Local and systemic antimicrobials were not used. All patients received periodontal maintenance at 3 and 6 months post-therapies, including professional plaque control with abrasive sodium carbonate airpowder system, re-instruction of oral hygiene and subgingival debridement of deep sites presenting BoP. Clinical Monitoring Patients received clinical monitoring at baseline, 3 and 6 months post-therapy. One calibrated examiner (T.E.D.G.) performed all the clinical examinations. After a calibration exercise, the standard error of measurement was calculated. Intra-examiner variability was 0.22 mm for PD and 0.24 mm for CA. The agreement for categorical variables (e.g. BoP) was >85%, as calculated by the Kappa-Light test. The following parameters were assessed at six sites (mesiobuccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, disto-lingual) per tooth, excluding third molars, using a manual periodontal probe⏐⏐: visible plaque accumulation (presence or absence), marginal bleeding (MB; presence or absence), BoP (presence or
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absence), suppuration (SUP; presence or absence), PD (distance between gingival margin and the bottom of the sulcus/pocket [mm]) and CA (distance between cement-enamel junction and the bottom of the sulcus/pocket [mm]). Leptin and Adiponectin Monitoring Fasting peripheral blood was sampled on the day of the clinical examination into an appropriate tube¶. Immediately after blood collection, the serum was separated from blood by centrifugation (10 min at 1,300 rpm) and stored in aliquots at -80 °C. Aliquots of serum were analyzed by enzyme linked immunosorbent assay (ELISA) using commercially available kits for detecting adiponectin# and leptin** at baseline, 3 and 6 months. ELISA procedures were carried out according to the manufacturer’s recommendations using human recombinant standards. According to the manufacturer, the minimum detectable dose (MDD) for adiponectin is 0.246 ng/ml and the MDD for leptin is less than 7.8 pg/ml. The optical density was measured at 450 nm. The adipocytokine results were reported in concentration per milliliter of serum (pg or ng/ml). A blinded operator (P.M.D.) performed all assays. Statistical Analysis The primary outcome variable was the difference between groups for full-mouth PD change between baseline and 6 months. Non-parametric statistical tests were used to evaluate the data that did not achieve normal distribution by Shapiro-Wilk test. The percentages of sites with plaque accumulation, MB, BoP, SUP, PD ≥ 5 mm and PD ≥ 7 mm, the mean number of sites with PD ≥ 5 mm and PD ≥ 7 mm, the full-mouth mean PD and CA, the BMI, the WHR, the glycemic parameters and the adipocytokines were computed for each patient. Subsequently, all data were averaged between groups. Changes in PD and CA in the full-mouth and at initially moderate and deep sites were averaged per patient and then across patients within each group. The significance of differences between groups for age, anthropometric, and glycemic parameters at baseline were compared by unpaired Student’s t-test. The significance of differences between groups for clinical parameters and adipocytokine levels at each time-point were compared using the Mann-Whitney test. The Friedman test was used to detect differences within each group over time for the clinical parameters and the serum levels of adipocytokines. Analysis of covariance (ANCOVA) with adjustments for baseline values was used to detect differences between groups in the mean changes of PD and CA. The Chi-square test was used to compare the frequency of gender and the number of patients with (≤ 4 sites with PD ≥ 5 mm), moderate (5-8 sites with PD ≥ 5 mm) or high (≥ 9 sites with PD ≥ 5 mm) risk for disease progression 23. The level of significance was set at 5%.
RESULTS Retention This study was conducted between March 2012 and July 2013. Figure 1 presents the flow chart of the study. Of the 480 individuals screened, 432 were excluded for not meeting the inclusion criteria and 48 entered the study (24 obese and 24 non-obese). Three patients from the non-obese group and six from the obese group did not return for the 3-month follow-up visit and were excluded from the statistical analysis.
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Demographic, Anthropometric and Glycemic Results There were no significant differences between groups for gender, age and glycemic parameters at baseline (p>0.05; Table 1). As expected, BMI and WHR were higher in the obese than in the non-obese group (p 4 mm and approximately 0.14 mm greater mean PD at 2 months posttherapy than patients with normal BMI. Unfortunately, significant methodological differences among the studies hamper a more direct comparison with the results of the present study. Firstly, all the above-mentioned investigations16-20 have limited their clinical monitoring period to a maximum of 3 months. In addition, studies differed considerably regarding the nonsurgical periodontal therapy protocol employed (i.e. quadrant-wise SRP or SRP within 24 hours with or without local antimicrobials), the parameter used to define obesity (i.e. BMI and/or waist circumference), the inclusion/exclusion criteria used (e.g. smokers and diabetic patients) and the severity of obesity (e.g. inclusion or exclusion of class III obesity). One could argue that the worse mean PD observed in the obese patients after therapy could be attributed to their poorer compliance with oral hygiene and/or greater degree of gingival inflammation. However, it is important to observe that obese and non-obese patients did not differ in terms of plaque accumulation, MB and BoP at any time point (Table 2). Studies in the medical field have proposed that obese patients present altered immuneinflammatory responses and adipocytokine signaling, which may increase the susceptibility to infection and impair would healing24-25. However, at this stage, the mechanisms that could explain the worse clinical response of obese patients to periodontal therapy are still unknown. Further evaluations performing local immunological and microbiological analyses, as well as longer follow up periods are still needed in order to clarify this issue. Adipose tissue acts as an endocrine organ by secreting several pro-inflammatory and antiinflammatory factors, named adipocytokines, which are able to stimulate molecular events in inflammatory and/or autoimmune conditions6-7. Leptin is an adipocytokine with proinflammatory properties that has a fundamental role in regulating appetite and energy expenditure, but also in controlling immunity and inflammation8-9,11. Recent evidence has suggested that leptin may play a role in the metabolism, defense and regeneration of the dental and periodontal tissues26. In addition, dental and periodontal tissues seem to be important sources of leptin not only locally but also systemically26. Adiponectin is an adipocytokine with anti-inflammatory properties that is related to the improvement of insulin sensitivity, antiatherogenic actions and regulation of metabolic homeostasis10-11. In the present study, serum levels of leptin were increased in obese patients at all time-points, corroborating previous evidence that leptin concentrations are mostly higher in obese patients, compared to normalweight patients, with or without periodontitis12,27-29. As previous studies have linked periodontitis with high serum levels of leptin and lower levels of adiponectin12-15, changes in the circulating levels of these adipocytokines would be expected after periodontal treatment. 6
Journal of Periodontology; Copyright 2014
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However, in this study, although SRP have yielded clinical improvements, it did not affect the circulatory levels of leptin and adiponectin in either group. Some studies have demonstrated a decrease in serum levels of leptin in obese19 or non-obese patients 14 after SRP. On the other hand, in agreement with the present results, other investigations29-30 have also failed to show changes in the serum levels of leptin and adiponectin in patients with periodontitis after nonsurgical periodontal therapy. Although the patients of the present study were engaged in a maintenance therapy at 3month intervals, the majority of them still retained several residual pockets (PD ≥ 5mm) after the proposed non-surgical periodontal therapy (Tables 2 and 4), which reflects the failure of this therapy in promoting a periodontal condition comparable to that of the periodontally healthy patients. Possibly these residual infected/inflamed sites could be enough to maintain a systemic load able to sustain the leptin and adiponectin levels unchanged despite periodontal treatment. Furthermore, a pre-existing susceptibility for systemic inflammation, possibly unrelated to periodontal infection, may be another explanation for the unchanged levels of adipocytokines post-therapy, as previous evidence has identified groups of patients that are resistant to systemic anti-inflammatory effects after periodontal therapy30. The main strength of this study is to be the first investigation to follow the response of obese patients to SRP up to 6 months post-therapy. In addition, some aspects related to participant selection could also be considered strengths of the present investigation. Obese and non-obese patients were classified considering both WHR and BMI, which take into consideration body fat distribution and abdominal obesity. To avoid interference of other risk factors for periodontitis, patients with diabetes and smokers were not included in this study, and all patients presented low levels of CRP (< 6mg/L). Previous evidence has demonstrated that CRP directly binds leptin in extra-cellular settings, impairing its biological actions31. Therefore, the chronic elevation of CRP, commonly observed in obese patients, may worsen leptin resistance31. Finally, anthropometric measurements were revised at all follow up visits to assure that patients maintained their obese or non-obese status over the course of the study. On the other hand, this study has some limitations that should be taken into consideration. Firstly, four patients fell into the BMI classification of overweight (BMI ranged from 26.7 to 27.8 Kg/m2), although their WHRs were below those determined for obesity. Secondly, there was a pronounced drop out in the obese group that ended up with 18 patients. Fortunately, the sample size calculation estimated that 18 patients per group would be enough to provide an 85% statistical power. Furthermore, based on the periodontitis and obesity profiles of the present study population, these results cannot be extrapolated to patients with milder periodontitis and Class III obesity (i.e. BMI ≥40 Kg/m2). Finally, this study is not considered blinded. As obesity is an obvious characteristic, both operator and examiner could certainly deduce the patient group, which may be a possible bias.
CONCLUSIONS Obese patients presented a worse response to SRP than non-obese patients at 6 months posttherapy. In addition, this treatment did not affect the circulating levels of leptin and adiponectin in obese and non-obese patients with CP. CONFLICT OF INTEREST: There is no conflict of interest
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FINANCIAL SUPPORT: This study was supported by São Paulo State Research Foundation (FAPESP), São Paulo, São Paulo, Brazil (# 2011/14875-3).
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Chaffee BW, Weston SJ. Association between chronic periodontal disease and obesity: a systematic review and meta-analysis. J Periodontol 2010; 81:1708-1724.
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Suvan J, D'Aiuto F, Moles DR, Petrie A, Donos N. Association between overweight/obesity and periodontitis in adults. A systematic review. Obes Rev 2011; 12: 381-404.
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Gorman A, Kaye EK, Nunn M, Garcia RI. Changes in body weight and adiposity predict periodontitis progression in men. J Dent Res 2012; 91:921-926.
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Pataro AL, Costa FO, Cortelli SC, Cortelli JR, Abreu MH, Costa JE. Association between severity of body mass index and periodontal condition in women. Clin Oral Investig 2012; 16:727-734.
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Palle AR, Reddy CM, Shankar BS, Gelli V, Sudhakar J, Reddy KK. Association between obesity and chronic periodontitis: a cross-sectional study. J Contemp Dent Pract 2013; 14:168-173.
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Wozniak SE, Gee LL, Wachtel MS, Frezza EE. Adipose tissue: the new endocrine organ? A review article. Dig Dis Sci 2009; 54:1847-1856.
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Ouch N, Parker JL, Lugus JJ, Walsh, K. Adipokines in inflammation and metabolic disease. Nat Rev Immunol 2011; 11:85-97.
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Procaccini C, Jirillo E, Matarese G. Leptin as an immunomodulator. Mol Aspects Med 2012; 33:35-45.
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Zhou Y, Rui L. Leptin signaling and leptin resistance. Front Med 2013; 7:207-222.
10. Fantuzzi G. Adiponectin in inflammatory and immune-mediated diseases. Cytokine 2013;64:1-10. 11. Conde J, Scotece M, Gómez R et al. Adipokines: biofactors from white adipose tissue. A complex hub among inflammation, metabolism, and immunity. Biofactors 2011; 37:413-420. 12. Zimmermann GS, Bastos MF, Dias Gonçalves TE, Chambrone L, Duarte PM. Local and circulating levels of adipocytokines in obese and normal weight individuals with chronic periodontitis. J Periodontol 2013; 84:624-633. 13. Karthikeyan BV, Pradeep AR. Gingival crevicular fluid and serum leptin: their relationship to periodontal health and disease. J Clin Periodontol 2007; 34:467-472. 14. Shimada Y, Komatsu Y, Ikezawa-Suzuki I, Tai H, Sugita N, Yoshie H. The effect of periodontal treatment on serum leptin, interleukin-6, and C-reactive protein. J Periodontol 2010; 81:1118-1123. 15. Nagano Y, Arishiro K, Uene M, et al. A low ratio of high molecular weight adiponectin to total adiponectin associates with periodontal status in middle-aged men. Biomarkers 2011; 16:106-111. 16. Zuza EP, Barroso EM, Carrareto AL, et al. The role of obesity as a modifying factor in patients undergoing non-surgical periodontal therapy. J Periodontol 2011; 82:676-682. 17. Al-Zahrani MS, Alghamdi HS. Effect of periodontal treatment on serum C-reactive protein level in obese and normal-weight women affected with chronic periodontitis. Saudi Med J 2012; 33:309-314. 18. Lakkis D, Bissada NF, Saber A et al. Response to periodontal therapy in patients who had weight loss after bariatric surgery and obese counterparts: a pilot study. J Periodontol 2012; 83:684-689. 19. Altay U, Gürgan CA, Ağbaht K. Changes in inflammatory and metabolic parameters after periodontal treatment in patients with and without obesity. J Periodontol 2013; 84:13-23. 20. Suvan J, Petrie A, Moles DR et al. Body mass index as a predictive factor of periodontal therapy outcomes. J Dent Res 2014; 93:49-54.
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21. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999; 4:1-6. 22. World Health Organization. Waist Circumference and Waist–Hip Ratio: Report of a WHO Expert Consultation: Geneva, 2008, December: 1-47. 23. Lang NP, Tonetti MS. Periodontal risk assessment (PRA) for patients in supportive periodontal therapy (SPT). Oral Health Prev Dent 2003; 1:7-16. 24. Wilson JA, Clark JJ. Obesity: impediment to wound healing. Crit Care Nurs Q 2003; 26:119-132. 25. Karlsson EA, Beck MA. The burden of obesity on infectious disease. Exp Biol Med 2010; 235:1412-1424. 26. Li W, Zhu W, Hou J, Huang B, Liu K, Meng H. Leptin and its receptor expression in dental and periodontal tissues of primates. Cell Tissue Res 2014; 355:181-188. 27. El-Haschimi K, Lehnert H. Leptin resistance - or why leptin fails to work in obesity. Exp Clin Endocrinol Diabetes 2003; 111:2-7. 28. Considine RV. Human leptin: an adipocyte hormone with weight-regulatory and endocrine functions. Semin Vasc Med 2005; 5:15-24. 29. Teles FR, Teles RP, Martin L, Socransky SS, Haffajee AD. Relationships among interleukin-6, tumor necrosis factor-α, adipokines, vitamin D, and chronic periodontitis. J Periodontol 2012; 83:1183-1191. 30. Behle JH, Sedaghatfar MH, Demmer RT et al. Heterogeneity of systemic inflammatory responses to periodontal therapy. J Clin Periodontol 2009; 36:287-94. 31. Hribal ML, Fiorentino TV, Sesti G. Role of C Reactive Protein (CRP) in Leptin Resistance. Curr Pharm Des 2014; 20:609-615.
Corresponding author: Poliana Mendes Duarte, Universidade Guarulhos - Centro de PósGraduação e Pesquisa, Praça Teresa Cristina, 229 - Centro – Guarulhos, CEP: 07.023-070 – SP/ Phone: ++ 55 11 24641758 - Fax: ++ 55 11 24641758, e-mail: [email protected] (email to be published) Submitted May 5, 2014; accepted for publication July 31, 2014. Figure 1 – Flow chart of the study. Table 1 – Demographic characteristics and anthropometric and glycemic parameters of the study population at baseline (mean ± SD). SD: standard deviation; BMI: body mass index; WHR: Waist-hip ratio; HbA1c: glycated hemoglobin; FPG: fasting plasma glucose. A p-value 0.05). Groups Variable Non-obese (n = 21) Obese (n = 18) p-value 61.9 72.2 0.89 Gender (% male) 0.86 Age (years) 48.4 ± 9.5 48.8 ± 5.9
DOI: 10.1902/jop.2014.140266
Effects of Scaling and Root Planing on Clinical Response and Serum Levels of Adipocytokines in Obese Patients With Chronic Periodontitis Tiago Eduardo Dias Gonçalves, DDS, MS*, Magda Feres, DDS, MS, PhD Professor*, Glaucia Santos Zimmermann, DDS, MS, PhD program*†, Marcelo Faveri, DDS, MS, PhD Assistant Professor*, Luciene Cristina Figueiredo, DDS, MS, PhD Assistant Professor*, Paloma Gralha Braga, DDS*, Poliana Mendes Duarte, DDS, MS, PhD Assistant Professor* *
Department of Periodontology, Dental Research Division, Guarulhos University, Guarulhos, São Paulo, Brazil.
†
Department of Dentistry, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Background: Despite several investigations suggesting that obesity is a risk indicator for periodontitis, little is known about the impact of obesity on periodontal treatment response. The aim of this study was to evaluate the effects of scaling and root planing (SRP) on clinical parameters and circulating levels of leptin and adiponectin in obese patients with chronic periodontitis (CP). Methods: Twenty-four obese and 24 non-obese patients with CP were submitted to SRP. Clinical parameters were assessed at baseline, 3 and 6 months post-therapy. Serum levels of leptin and adiponectin were evaluated at all time points, using enzyme linked immunosorbent assay. Results: SRP improved the clinical parameters of both groups at 3 and 6 months (p 30 % of the sites with concomitant probing depth (PD) and clinical attachment level (CA) ≥ 4 mm and a minimum of six teeth distributed in the different quadrants presenting at least one site with PD and CA ≥ 5 mm and bleeding on probing (BoP) at baseline. To be included, patients also needed to present glycated hemoglobin (HbA1c) < 6.5 %, fasting plasma glucose (FPG)
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70-99 mg/dl, C-reactive protein (CRP) < 6 mg/l. The levels of HbA1c (High-performance Liquid Chromatography method), FPG (Glucose Oxidase method) and CRP (LatexAgglutination-Test) were all assessed by the Guarulhos University Clinical Analysis Laboratory. Exclusion criteria were pregnancy, lactation, current smoking and smoking within the past 10 years, any medical condition requiring prophylactic antibiotic coverage before dental treatment, subgingival periodontal therapy in the previous 12 months, antimicrobial, antiinflammatory, immunosuppressive and lipid-lowering (e.g. statins) therapies during the previous 6 months, regular use of mouthrinses containing antimicrobials and use of orthodontic appliances. Patients reporting the presence of systemic conditions that could affect the progression of periodontitis and/or gain/loss of weight (e.g. diabetes mellitus, immunological disorders, osteoporosis, hypothyroidism, hyperthyroidism) were also excluded. Anthropometric Measurements and Experimental Groups One trained examiner (P.G.B) performed all anthropometric measurements, including weight (kg), height (m), waist (cm) and hip circumferences (cm). Body mass index (BMI) was calculated as the weight divided by the square of height (kg/m2). The waist-hip ratio (WHR) was calculated as the ratio of waist to hip circumference. Obese patients were defined as having BMI ≥ 30 and < 40 kg/m2 and concomitant WHR ≥ 0.85 for women and WHR ≥ 0.90 for men. Non-obese patients were defined as having BMI ranging from 20 to 29.9 kg/m2 and WHR below that determined for obesity (i.e. WHR < 0.85 for women and WHR < 0.90 for men) 22. The anthropometric measurements were re-assessed at all follow-up visits to verify if the patients did not change their obese or non-obese status during the study. Non-surgical Periodontal Therapy Initially, all patients received supragingival plaque and calculus removal, exodontia, provisional restoration and filling overhang removal, as necessary. They were also instructed regarding brushing technique and use of dental floss. A trained periodontist (G.S.Z.) performed SRP in four to six appointments lasting approximately 1 hour each, using manual curettes‡ and ultrasonic device§ under local anesthesia. Periodontal therapy was completed in 14 days. The endpoint for each SRP appointment was “smoothness of the scaled roots”. Local and systemic antimicrobials were not used. All patients received periodontal maintenance at 3 and 6 months post-therapies, including professional plaque control with abrasive sodium carbonate airpowder system, re-instruction of oral hygiene and subgingival debridement of deep sites presenting BoP. Clinical Monitoring Patients received clinical monitoring at baseline, 3 and 6 months post-therapy. One calibrated examiner (T.E.D.G.) performed all the clinical examinations. After a calibration exercise, the standard error of measurement was calculated. Intra-examiner variability was 0.22 mm for PD and 0.24 mm for CA. The agreement for categorical variables (e.g. BoP) was >85%, as calculated by the Kappa-Light test. The following parameters were assessed at six sites (mesiobuccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, disto-lingual) per tooth, excluding third molars, using a manual periodontal probe⏐⏐: visible plaque accumulation (presence or absence), marginal bleeding (MB; presence or absence), BoP (presence or
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absence), suppuration (SUP; presence or absence), PD (distance between gingival margin and the bottom of the sulcus/pocket [mm]) and CA (distance between cement-enamel junction and the bottom of the sulcus/pocket [mm]). Leptin and Adiponectin Monitoring Fasting peripheral blood was sampled on the day of the clinical examination into an appropriate tube¶. Immediately after blood collection, the serum was separated from blood by centrifugation (10 min at 1,300 rpm) and stored in aliquots at -80 °C. Aliquots of serum were analyzed by enzyme linked immunosorbent assay (ELISA) using commercially available kits for detecting adiponectin# and leptin** at baseline, 3 and 6 months. ELISA procedures were carried out according to the manufacturer’s recommendations using human recombinant standards. According to the manufacturer, the minimum detectable dose (MDD) for adiponectin is 0.246 ng/ml and the MDD for leptin is less than 7.8 pg/ml. The optical density was measured at 450 nm. The adipocytokine results were reported in concentration per milliliter of serum (pg or ng/ml). A blinded operator (P.M.D.) performed all assays. Statistical Analysis The primary outcome variable was the difference between groups for full-mouth PD change between baseline and 6 months. Non-parametric statistical tests were used to evaluate the data that did not achieve normal distribution by Shapiro-Wilk test. The percentages of sites with plaque accumulation, MB, BoP, SUP, PD ≥ 5 mm and PD ≥ 7 mm, the mean number of sites with PD ≥ 5 mm and PD ≥ 7 mm, the full-mouth mean PD and CA, the BMI, the WHR, the glycemic parameters and the adipocytokines were computed for each patient. Subsequently, all data were averaged between groups. Changes in PD and CA in the full-mouth and at initially moderate and deep sites were averaged per patient and then across patients within each group. The significance of differences between groups for age, anthropometric, and glycemic parameters at baseline were compared by unpaired Student’s t-test. The significance of differences between groups for clinical parameters and adipocytokine levels at each time-point were compared using the Mann-Whitney test. The Friedman test was used to detect differences within each group over time for the clinical parameters and the serum levels of adipocytokines. Analysis of covariance (ANCOVA) with adjustments for baseline values was used to detect differences between groups in the mean changes of PD and CA. The Chi-square test was used to compare the frequency of gender and the number of patients with (≤ 4 sites with PD ≥ 5 mm), moderate (5-8 sites with PD ≥ 5 mm) or high (≥ 9 sites with PD ≥ 5 mm) risk for disease progression 23. The level of significance was set at 5%.
RESULTS Retention This study was conducted between March 2012 and July 2013. Figure 1 presents the flow chart of the study. Of the 480 individuals screened, 432 were excluded for not meeting the inclusion criteria and 48 entered the study (24 obese and 24 non-obese). Three patients from the non-obese group and six from the obese group did not return for the 3-month follow-up visit and were excluded from the statistical analysis.
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Demographic, Anthropometric and Glycemic Results There were no significant differences between groups for gender, age and glycemic parameters at baseline (p>0.05; Table 1). As expected, BMI and WHR were higher in the obese than in the non-obese group (p 4 mm and approximately 0.14 mm greater mean PD at 2 months posttherapy than patients with normal BMI. Unfortunately, significant methodological differences among the studies hamper a more direct comparison with the results of the present study. Firstly, all the above-mentioned investigations16-20 have limited their clinical monitoring period to a maximum of 3 months. In addition, studies differed considerably regarding the nonsurgical periodontal therapy protocol employed (i.e. quadrant-wise SRP or SRP within 24 hours with or without local antimicrobials), the parameter used to define obesity (i.e. BMI and/or waist circumference), the inclusion/exclusion criteria used (e.g. smokers and diabetic patients) and the severity of obesity (e.g. inclusion or exclusion of class III obesity). One could argue that the worse mean PD observed in the obese patients after therapy could be attributed to their poorer compliance with oral hygiene and/or greater degree of gingival inflammation. However, it is important to observe that obese and non-obese patients did not differ in terms of plaque accumulation, MB and BoP at any time point (Table 2). Studies in the medical field have proposed that obese patients present altered immuneinflammatory responses and adipocytokine signaling, which may increase the susceptibility to infection and impair would healing24-25. However, at this stage, the mechanisms that could explain the worse clinical response of obese patients to periodontal therapy are still unknown. Further evaluations performing local immunological and microbiological analyses, as well as longer follow up periods are still needed in order to clarify this issue. Adipose tissue acts as an endocrine organ by secreting several pro-inflammatory and antiinflammatory factors, named adipocytokines, which are able to stimulate molecular events in inflammatory and/or autoimmune conditions6-7. Leptin is an adipocytokine with proinflammatory properties that has a fundamental role in regulating appetite and energy expenditure, but also in controlling immunity and inflammation8-9,11. Recent evidence has suggested that leptin may play a role in the metabolism, defense and regeneration of the dental and periodontal tissues26. In addition, dental and periodontal tissues seem to be important sources of leptin not only locally but also systemically26. Adiponectin is an adipocytokine with anti-inflammatory properties that is related to the improvement of insulin sensitivity, antiatherogenic actions and regulation of metabolic homeostasis10-11. In the present study, serum levels of leptin were increased in obese patients at all time-points, corroborating previous evidence that leptin concentrations are mostly higher in obese patients, compared to normalweight patients, with or without periodontitis12,27-29. As previous studies have linked periodontitis with high serum levels of leptin and lower levels of adiponectin12-15, changes in the circulating levels of these adipocytokines would be expected after periodontal treatment. 6
Journal of Periodontology; Copyright 2014
DOI: 10.1902/jop.2014.140266
However, in this study, although SRP have yielded clinical improvements, it did not affect the circulatory levels of leptin and adiponectin in either group. Some studies have demonstrated a decrease in serum levels of leptin in obese19 or non-obese patients 14 after SRP. On the other hand, in agreement with the present results, other investigations29-30 have also failed to show changes in the serum levels of leptin and adiponectin in patients with periodontitis after nonsurgical periodontal therapy. Although the patients of the present study were engaged in a maintenance therapy at 3month intervals, the majority of them still retained several residual pockets (PD ≥ 5mm) after the proposed non-surgical periodontal therapy (Tables 2 and 4), which reflects the failure of this therapy in promoting a periodontal condition comparable to that of the periodontally healthy patients. Possibly these residual infected/inflamed sites could be enough to maintain a systemic load able to sustain the leptin and adiponectin levels unchanged despite periodontal treatment. Furthermore, a pre-existing susceptibility for systemic inflammation, possibly unrelated to periodontal infection, may be another explanation for the unchanged levels of adipocytokines post-therapy, as previous evidence has identified groups of patients that are resistant to systemic anti-inflammatory effects after periodontal therapy30. The main strength of this study is to be the first investigation to follow the response of obese patients to SRP up to 6 months post-therapy. In addition, some aspects related to participant selection could also be considered strengths of the present investigation. Obese and non-obese patients were classified considering both WHR and BMI, which take into consideration body fat distribution and abdominal obesity. To avoid interference of other risk factors for periodontitis, patients with diabetes and smokers were not included in this study, and all patients presented low levels of CRP (< 6mg/L). Previous evidence has demonstrated that CRP directly binds leptin in extra-cellular settings, impairing its biological actions31. Therefore, the chronic elevation of CRP, commonly observed in obese patients, may worsen leptin resistance31. Finally, anthropometric measurements were revised at all follow up visits to assure that patients maintained their obese or non-obese status over the course of the study. On the other hand, this study has some limitations that should be taken into consideration. Firstly, four patients fell into the BMI classification of overweight (BMI ranged from 26.7 to 27.8 Kg/m2), although their WHRs were below those determined for obesity. Secondly, there was a pronounced drop out in the obese group that ended up with 18 patients. Fortunately, the sample size calculation estimated that 18 patients per group would be enough to provide an 85% statistical power. Furthermore, based on the periodontitis and obesity profiles of the present study population, these results cannot be extrapolated to patients with milder periodontitis and Class III obesity (i.e. BMI ≥40 Kg/m2). Finally, this study is not considered blinded. As obesity is an obvious characteristic, both operator and examiner could certainly deduce the patient group, which may be a possible bias.
CONCLUSIONS Obese patients presented a worse response to SRP than non-obese patients at 6 months posttherapy. In addition, this treatment did not affect the circulating levels of leptin and adiponectin in obese and non-obese patients with CP. CONFLICT OF INTEREST: There is no conflict of interest
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Journal of Periodontology; Copyright 2014
DOI: 10.1902/jop.2014.140266
FINANCIAL SUPPORT: This study was supported by São Paulo State Research Foundation (FAPESP), São Paulo, São Paulo, Brazil (# 2011/14875-3).
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Corresponding author: Poliana Mendes Duarte, Universidade Guarulhos - Centro de PósGraduação e Pesquisa, Praça Teresa Cristina, 229 - Centro – Guarulhos, CEP: 07.023-070 – SP/ Phone: ++ 55 11 24641758 - Fax: ++ 55 11 24641758, e-mail: [email protected] (email to be published) Submitted May 5, 2014; accepted for publication July 31, 2014. Figure 1 – Flow chart of the study. Table 1 – Demographic characteristics and anthropometric and glycemic parameters of the study population at baseline (mean ± SD). SD: standard deviation; BMI: body mass index; WHR: Waist-hip ratio; HbA1c: glycated hemoglobin; FPG: fasting plasma glucose. A p-value 0.05). Groups Variable Non-obese (n = 21) Obese (n = 18) p-value 61.9 72.2 0.89 Gender (% male) 0.86 Age (years) 48.4 ± 9.5 48.8 ± 5.9
