Reazione di fase acuta dopo terapia parodontale

UNIVERSITÀ DI PISA

FACOLTÀ DI MEDICINA E CHIRURGIA

CORSO DI DOTTORATO IN FISIOPATOLOGIA CLINICA

Coordinatore: Prof. Fulvio Basolo

Tesi di dottorato

Reazione di fase acuta dopo trattamento parodontale

RELATORE

CANDIDATO

Dott. Stefano Gennai

ANNO ACCADEMICO 2016/2017

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Acute Phase Response following Periodontal Treatment. A Randomized Clinical

Trial.

PhD candidate: Dr. Stefano Gennai

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Abstract

Aim. Aim of this study was to compare quadrant scaling (Q-SRP) versus FM-SRP and

conservative surgical periodontal therapy (C-SPT) versus resective SPT (R-SPT) in terms of systemic acute (24 hours) and medium-term (3 months) inflammation.

Methods. 38 patients suffering from periodontitis after a baseline visit were randomly

allocated to either FM-SRP or Q-SRP and 28 to either C-SPT and R-SPT. Periodontal and anthropometric parameters were collected at baseline and after 3 months. Serum samples were drawn at baseline and 1 and 7 and 90 days after treatment. High sensitivity assays for a broad array of inflammatory and endothelial assays were performed on all samples.

Results. FM-SRP produced a greater acute phase response after 24 hours when compared

to Q-SRP (3-fold increase in C-Reactive Protein (CRP), p<0.001 and 2-fold increase in Interleukin (IL-6). All periodontal treatments produced a comparable improvement in standard clinical periodontal parameters with no between-group differences. For SRP groups treatment time was positively associated with the relative 24 hrs increase in CRP (R=0.5, p<0.001) and IL-6 (R=0.5, p=0.002), whilst the number of deeper (greater than 6mm) pockets predicted only the relative increase in IL-6 (R=0.4, p<0.05). No statistical significant differences were observed in terms of systemic biomarkers at any time points for C-SPT and R-SPT groups.

Conclusions. FM-SRP triggers a moderate acute phase response of one-week duration

compared to Q-SRP. C-SPT and R-SPT seems to not produce a significant acute phase response at 24 hours. Clinicians may choose conventional (quadrant scaling) treatment in patients with complicated medical history and/or uncontrolled co-morbidities, while it doesn’t appear important perform a conservative surgical approach to prevent a systemic inflammatory perturbation if the candidate area of intervention is restricted to less of 4

4 teeth.

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Index

6 5.3 Periodontal parameters ... 39 5.4 Systemic Biomarkers ... 41 6 Results for the SPT trial ... 50 6.1 Subjects accountability and baseline characteristics ... 50 6.2 Vital signs ... 51 6.3 Periodontal parameters ... 53 6.4 Systemic biomarkers ... 57 6.5 Patient related outcome ... 59 7 Discussion ... 60 8 Conclusions ... 64 9 References ... 65 10 Abbreviation index ... 72 11 Figures index ... 73 12 Tables Index ... 75 13 Acknowledgements ... 76

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1 Introduction

1.1 Periodontal disease

Periodontal disease is a destructive inflammatory disease of the tooth supporting tissues and is the leading cause of teeth loss (Brookes et al. 1995).

The aetiology of this disease is related to chronic infection of mixed Gram-negative bacteria.

As demonstrated by epidemiological studies about 46% of the adult population over 50 years old, and 62% over 65 years old, suffers from this disease (Eke et al. 2012, 2016). The periodontium comprises the following tissue: the alveolar bone, the cement, the periodontal ligament, and the gum. Its main function is to support the tooth in the act of chewing and its articulation with the jaw bones.

The main function of the periodontium is to attach the tooth to the bone tissue of the jaws and to maintain the integrity of the surface of the masticatory mucosa of the oral cavity. In the context of the oral cavity, the bacterial deposits have been termed dental plaque which forms a biofilm, consisting of bacteria in a matrix composed mainly of extracellular bacterial polymers and salivary and/or gingival exudate products.

Biofilms effectively protect bacteria from antimicrobial agents. Treatment with antimicrobial sub-stances is often unsuccessful unless the deposits are mechanically removed. The intercellular matrix, which constitutes approximately 20-30% of dental plaque, is represented by organic and inorganic materials from the saliva, crevicular fluid and by bacterial products.

The first phase of plaque formation includes the deposition of salivary glycoproteins on hard and soft tissues in the oral cavity. Then bacteria begin to colonize the teeth. In the early stages, gram-positive forms, such as Streptococcus sanguis and Actynomices viscosus

8 are found.

The secondary colonizers are microorganisms that initially do not adhere to teeth surfaces and include Provetella Intermedia, Porphyromonas gingivalis, Capnocytophaga species, Fusobacterium nucleatum and Prevotella Loesch. From the early to the later stages we assist to a change from the first aerobic environment, characterized by Gram-positive facultative species, to a highly oxygen-free environment in which anaerobic Gram-negative organisms predominate. These bacteria and their toxins, in susceptible individuals, start a destructive process that compromises the integrity of tissues that constitute the periodontium. Inflammatory and immune processes operate in the gingival tissues to protect against local microbial attack and prevent microorganisms from spreading or invading into the tissues. In some cases, these host defense reactions may be harmful to the host in that inflammation can damage surrounding cells and connective tissue structures. Furthermore, inflammatory and immune reactions extending deeper into the connective tissue beyond the base of the pocket may also include alveolar bone loss in this destructive process. Thus, these "defensive" processes may paradoxically account for much of the tissue injury observed in gingivitis and periodontitis.

Infection and the host response, specific or nonspecific, generate a multitude of biochemical processes that result in the production of substances (free radicals, lymphotoxin, growth factors, proteases, lysosomal enzymes) that damage cells and periodontal tissues.

1.2 Periodontal disease and systemic inflammation

Epidemiological evidence produced in the last decade largely corroborates the idea that there is a plausible relationship between oral health, especially periodontal health, and

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systemic diseases (Davé & Van Dyke 2008, Hingorani & D’Aiuto 2008). Although, the periodontal disease is a chronic pathology, acute-phase elements are part of the innate immune response. The systemic inflammatory response to acute phase results in the activation of complement factors, neutralizing the pathogenic bacteria and stimulating tissue regeneration. The acute phase proteins involved are: interleukin 6 (IL-6), plasminogen activator, fibrinogen and C reactive protein (CRP). The latter is considered a factor of considerable importance in the pathogenesis of atherosclerotic disease and high levels (eg> 2.1 mg / l) constitute a risk factor for cardiovascular events(Paraskevas et al. 2008). After non-surgical periodontal therapy these markers showed significant reductions(Ridker 2007).

The CRP is produced by the liver as acute phase reactant during a process of systemic inflammation. Elevated CRP levels have been demonstrated in patients with ischemia and myocardial infarction (Widener 2007). CRP levels above 10 mg/L are indicative of a significant inflammatory condition of the subject (Ioannidou et al. 2006). The concentration of the protein in 98% of the population is generally less than 10 mg/dl. It is unclear whether the predictive ability of this protein is linked to a possible etiologic role in the pathogenesis of atherosclerosis or whether it is merely a risk indicator of myocardial and vascular damage. CRP concentration has continuous associations with the risk of coronary heart disease, ischaemic stroke, vascular mortality and 3-fold increase of CRP determines a relative risk of fatal myocardial infarction of 1.8 (1.59-2.14) (Kaptoge et al. 2010).

Several studies have shown that there are high concentrations of CRP in patients with severe periodontal disease (D’Aiuto & Donos 2007, Paraskevas et al. 2008) (Table 1).

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Table 1 Meta-analysis form(Paraskevas et al. 2008) showing levels of CRP in patient with periodontal disease.

Many authors observed a higher inflammatory response in periodontal patients which were overweight. A possible explanation for this relationship is that adipocytes produce high amounts of TNF alpha which induce the synthesis in the liver of CRP. Similarly, the severe periodontal disease induces the release of tumor necrosis factor alpha (TNF-α) with consequent increase of CRP (Fontana). CRP can contribute to atherosclerotic processes through the following mechanisms: • Release of reactive oxygen species: CRP, activates complement, induces the release of cytokines and increases the release of reactive oxygen species by monocytes and neutrophils. All this results in changes at the level of mediators such as nitric oxide (NO) that contribute to the maintenance of endothelial vasodilation.

• Increased expression of adhesion molecules: CRP induces the expression of molecules such as ICAM-1 and VCAM-1.

• Formation of foam cells: CRP induces the uptake of LDL by macrophages cells. • Inhibition of plasminogen.

• Nitric oxide (NO) regulates the pro-atherogenic effects of CRP.

The NO and vasoconstrictor substances act on platelet aggregation, proliferation and migration of vascular smooth cells. Therefore an alteration of endothelial function may not only change the vascular tone but also play an important role in the genesis of vascular

11 damage (Ferroni et al. 2003).

Previous studies have shown that markers of systemic inflammation such as CRP, D-dimer, protein and serum amyloid leucocyte are significantly different at 24 hours a week and 3 months after periodontal surgery (Ghiadoni et al. 2008).

Patient affected by periodontal disease were found to have an impaired vascular-endothelial function. In a cohort of 5.452 patients, periodontal affected showed a greter carodtid intima media thickness (c-IMT) of 0.08 mm, this is considerable since per every 0.1mm of c-IMT difference the RR of myocardial infarction increases of 1.15 times and for stroke increases of 1.18 times (Orlandi et al. 2014) (Figure 1).

Figure 1 c-IMT histological section of periodontal patient (Orlandi et al. 2014).

Also surrogate clinical parameters for periodontal disease were found to be correleate with ECG abnormalities with a OR=1.6 (Shimazaki et al. 2004, Franek et al. 2009)(Figure 2); furthermore periodontal disease is associated with atherosclerotic vascular disease independent of known confounders.

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Figure 2 Results from a case-control study comparing heart function in periodontal affected patients.

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These evidences suggest that blood sampling at 24 hours, at 1 week and 3 months after periodontal surgery could be used as a model for assessing the state of systemic inflammation in association with periodontal surgical therapy.

1.3 Non-surgical periodontal treatment and acute phase response

Non-surgical periodontal treatment (NPT) represents the base of any therapeutic approaches to periodontitis. A broad range of benefits following PT have been shown including improvements in periodontal, systemic and patient-reported outcomes. NPT successfully decreases gingival inflammation and obtain pocket reduction/closure (Van der Weijden & Timmerman 2002) (Figure 3, Figure 4, Figure 5). It is also noteworthy to mention that NPT produces significant improvements in individuals’ overall quality of life (Shanbhag et al. 2012).

Similarly, systemic effects of PT have been extensively studied in the last decade. NPT is associated with improvement of the endothelial function and reduces biomarkers of atherosclerotic disease, especially in those already suffering from cardio vascular disease (CVD) and/or diabetes (Teeuw et al. 2014, Orlandi et al. 2014). NPT is also associated with significant improvements of glucose control (Engebretson & Kocher 2013). A variety of NPT protocols (full versus quadrant scaling, local versus systemically delivered antimicrobials) however have been adopted in intervention trials with CVD and/or metabolic outcomes. The general assumption is that each treatment modality is equally effective with regards to the improvement in periodontal outcomes, whilst less evidence is available on the potential impact of each NPT protocol on a variety of systemic outcomes.

The term intensive NPT has recently been linked with a full mouth instrumentation of the diseased dentition (FM-SRP). This approach has been consistently associated, in the first 24 hours after treatment, with one-week duration systemic acute perturbations. A sharp

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increase in a large number of inflammatory biomarkers including, C-reactive protein (CRP), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α), D-Dimer has consistently been noted (D’Aiuto et al. 2004a, Graziani et al. 2010a, b). Alteration of the haemostatic system including both endothelial cell activation and endothelial dysfunction as assessed by flow-mediated dilatation of the brachial artery are also associated with FM-SRP (D’Aiuto et al. 2013a). The reasons for such a response may be related to both the transient post-operative bacteraemia and the extension of the operative trauma (D’Aiuto et al. 2004c). There is a wealth of evidence from population studies linking inflammatory biomarkers with increased future vascular risk and mortality (Kaptoge et al. 2010). Therefore, the chosen treatment delivery in the intervention trial may confound the effect of NPT on biomarkers related to CVD. Moreover, the systemic implications of the acute effects reported following FM-SRP are not fully elucidated. A common-sense approach would suggest that perturbation of the inflammatory and haemostatic systems should be avoided in high-risk patients (i.e. with uncontrolled co-morbidities like CVD). Further research on prevention of acute perturbations after NPT has been advocated (D’Aiuto et al. 2013b).

15 Figure 3 Frontal clinical picture at baseline Figure 4 Frontal clinical picture of the case depicted in figure 3 at 3 months post-NSPT

1.4 Surgical periodontal treatment and acute phase response

Several techniques have been described for periodontal surgery. However, in all the techniques there is the need to elevate a muco-periosteal flap to expose and clean the root surfaces associated with pathological pocket. Surgical cleansing of the defect and the root surface can re-establish periodontal health when proper plaque control is established (Rosling et al. 1976) (Figure 8, Figure 9, Figure 10). However, different surgical techniques have been advocated.

Periodontal osseous lesions associated with deep pockets are frequent anatomical sequelae to periodontitis (Papapanou & Tonetti 2000). After successful cause-related therapy, the surgical treatment of persisting pockets is suggested to re-establish a periodontal anatomy able to sustain periodontal health after treatment (Lang 2000). Conservative surgical therapy consist in a surgical debridement of the pathologic periodontal sites with the tentative to conserve as well as possible soft and hard periodontal tissues (Graziani et al. 2012).

Osseous resective surgery is a well-documented treatment approach that aims at the elimination of residual periodontal pockets associated with shallow intrabony defects prevalently located at the posterior teeth (‘Consensus Report Surgical Pocket Therapy’ 1996, Carnevale & Kaldahl 2000). This treatment modality combines the use of both osteoplasty and ostectomy to re-establish the physiological bone tissue architecture around teeth in a more apical position (Ochsenbein 1958). This implies the reshaping of positive bone morphology by changing the previous bottom of the defect into the most coronal part of the new interproximal bone surface (Schluger 1949).

Osteoplasty is used to treat buccal and lingual bony ledges or tori, shallow lingual or buccal intrabony defects, thick interproximal areas and incipient furcation

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involvements that do not necessitate removing supporting bone (28, 63, 64, 70). Ostectomy is utilized to treat shallow (1–2 mm deep) to medium (3–4 mm deep) intrabony and hemiseptal osseous defects and correct reversals in the osseous topography . The endpoint of osteoplasty, used in conjunction with a modified Widman flap or an apically positioned flap, is the enhancement of tissue placement and adaptation at the time of suturing. The endpoint of ostectomy, used in conjunction with an apically positioned flap or a thinned palatal flap, is the elimination of an intrabony pocket. Osseous resective surgery is the combined use of both osteoplasty and ostectomy to re-establish the marginal bone morphology around the teeth to resemble ‘‘normal bone with a positive architecture’’, albeit at a more apical position. By definition ‘‘normal bone with a positive architecture’’ means that the surface of interdental bone is

coronal to that of the facial and lingual radicular bone. The endpoints of osseous resective surgery are minimal probing depths and a gingival tissue morphology that enhances good self-performed oral hygiene and periodontal health.

Since most clinical reports and experimental trials on regenerative procedures involve intrabony or hemiseptal defects of >3 mm depths, we believe that, as a general rule, only <4 mm intrabony or hemiseptal defects are suitable for osseous resective surgery procedures. This creates a clear-cut difference in the indications for the two surgical procedures. It is recognized that many variables with osseous resective surgery alter clinical judgment and will be assessed later. Besides the treatment of intrabony and hemiseptal defects, osseous resective surgery is also utilized in preprosthetic, restorative and cosmetic surgery to increase the clinical crown length and/or to re-establish an ‘‘adequate’’ zone of natural root surface for the gingival attachment (Carnevale & Kaldahl 2000).

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Despite the fact that the acute phase response after surgical periodontal therapy was evaluated in a single clinical study (Graziani et al. 2010b), no data are currently available for the resective surgical periodontal therapy. In the aforementioned study by Graziani et al. (2010b) a slight but not significant increase of the inflammatory markers profile was reported 24-hours after the surgical intervention (Figure 6, Figure 7). Therefore, it may be expected an acute systemic response in a more invasive or traumatic periodontal surgery techniques as the resective one.

20 Figure 6. Results of the study by Graziani et al. 2010b: Mean ± SE serum-level changes, after periodontal non-surgical (D1) and surgical therap (D201)y, of leucocyte counts (a), C-reactive protein (b). Bonferroni adjustment: *compared with BL (p<0.05) and §compared with D180 (p<0.05).

21 Figure 7 Results of the study by Graziani et al. 2010b: Mean ± SE serum-level changes, after periodontal non-surgical (D1) and surgical therap (D201)y, of serum Amyloid-A (c), D-Dimers (d). Bonferroni adjustment: *compared with BL (p<0.05) and §compa

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Figure 8 Clinical and radiological aspect prior surgical intervention

23 Figure 9 Phases of the periodontal surgical intervention: (a) flap design, (b) flap elevation, (c) defect assessment, (d) suture

24 Figure 10 Clinica and radiological aspect at 3 months form the surgical intervention

25 2 Study aim The aim of this study was to compare quadrant scaling and root planing (Q-SRP) with FM-SRP and conservative surgery with resective surgery in terms of acute phase responses following treatment. The primary aim was to compare the differences in CRP acute increase following FM-SRP versus Q-SRP therapy and conservative surgery versus resective surgery (24 hours after therapy). Secondary outcomes included changes in a broad array of inflammatory and endothelial injury markers in both groups. For these purposes two single-centred randomized clinical trials were designed, one for the non-surgical periodontal therapy (NSPT) and one for the surgical periodontal therapy (SPT).

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3 Material and methods for the NSPT trial

3.1 Experimental design and patient’s selection

This was a single-centre randomized controlled clinical trial with a 3-months follow-up (Figure 11). The protocol of the study received approval from the University Hospital of Pisa ethical committee (Pisa, Italy), it was registered within a clinical trials database (www.clinicaltrial.gov NCT02460926) and it was conducted according to the principles outlined in the Declaration of Helsinki on experimentation involving human subjects. Eligible study participants were identified among referrals for NSPT and SPT to the Unit of Dentistry and Oral Surgery of the University Hospital of Pisa (Italy). All participants gave written informed consent, full medical and dental histories were recorded and oral examination completed.

Patients presenting with proximal attachment loss of ≥3mm in ≥2 non-adjacent teeth (Tonetti & Claffey 2005), bleeding on probing on at least 25% of their total sites and documented radiographic bone loss were included in this NPT arms. Exclusion criteria were (i) age earlier than 18 and older than 70 years; (ii) pregnant or lactating females; (iii) females using contraceptive methods; (iv) reported diagnosis of any systemic illnesses including cardiovascular, renal and liver diseases; (v) any pharmacological treatment within the 3 months before the beginning of the study; (vi) NPT in the previous 6 months only for the NSPT arms.

3.2 Clinical parameters

At the baseline visit, blood samples were collected and vital signs including systolic (SBP) and diastolic blood pressure (DBP) were measured in triplicates by using an automatic oscillometric device (OMRON-705IT, Omron, Kyoto, Japan). Average BP was then calculated from the last two measurements. Weight and height were measured and body mass index (BMI) was calculated. Body temperature was measured with tympanic reading by using an ear canal thermometer (Genius TM 2, Covidien llc, USA). Fever was diagnosed when body temperature was 37.5 Celsius degrees (°C) (Sund-Levander et al. 2002). Smoking history was registered dichotomously as current or never/former and no stratification on cigarettes/day and years of smoking was made.

Standard periodontal clinical parameters were assessed using a UNC 15mm periodontal probe by a masked calibrated examiner at six sites/tooth excluding third molars. Calibration was made on a total of 10 non-study subjects affected by periodontitis. The examiner recorded full-mouth PPD and recessions at six sites per tooth (excluding third molars) on two different occasions using a manual, UNC-15 periodontal probe. Upon completion of all measurements, the intra-examiner repeatability for CAL measurement was assessed. The examiner was judged to be reproducible after meeting a percentage of agreement within±2 mm between repeated measurements of at least 98% (Graziani et al. 2010b).

Full-mouth pocket probing depth (PPD) and recession of the gingival margin (REC) were recorded with measurements rounded to the nearest millimetre. Clinical attachment level was calculated as the sum of PPD and REC. The full-mouth plaque score (FMPS) was measured as the percentage of the total surfaces showing plaque assessed dichotomously

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on six surfaces per tooth (O’Leary et al. 1972). Similarly, a full-mouth percentage bleeding score (FMBS) was calculated after assessing dichotomously the presence of bleeding on probing (Ainamo & Bay 1975).

3.3 Blood collection and analysis of the serum markers

Serum samples were collected from a venepuncture in the antecubital fossa before 8.15 AM and after an overnight fast for all patients. Blood samples were immediately processed and serum aliquots were stored at -80°C. Lipid fractions including total cholesterol, high density lipoprotein (HDL), lower density lipoprotein (LDL) and triglycerides were measured using standard laboratory chemistry procedures. Serum C-reactive protein (CRP) was measured by immunoturbidometry (Cobas, Roche Diagnostic, Mannheim, Germany). A broad panel of inflammatory biomarkers including interleukin(IL)-6, IL-8, IL-10, IL-12, interferon-γ (IFN-γ) and Tumor Necrosis Factor (TNF)-α and endothelial injury markers including. E-selectin, P-selectin, intercellular adhesion molecule-3 (ICAM-3) and thrombomodulin were assayed with a Multiplex array according to manufacturer instructions (Meso Scale Discovery, Maryland, USA). All samples were analysed at the end of the study in duplicates by staff masked to the group allocation. Intra and inter-assays coefficient of variation were less than 7%.

3.4 Patient related outcome

Pain was collected by self-reported questionnaire. After the surgical intervention a questionnaire was given to each patient. The pain/discomfort items were rated based on a visual-analog scale (VAS) of 10 cm with the following end-points: no pain/discomfort at one end and worst imaginable pain/discomfort at the other. The subject was also requested to indicate whether medications had been taken for discomfort/pain.

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Study participant numbers were assigned in ascending order at the enrolment visit. Patients were randomly assigned in a 1:1 ratio to either FM-SRP or Q-SRP therapy using a computer-generated table. Non-clinical staff masked to treatment allocation had access to the randomization list. Allocation to treatment was concealed to the clinical examiner and statistician with opaque envelopes which were opened by the clinician on the day of treatment.

3.6 Non surgical periodontal treatment

Between baseline and the day of the treatment, all patients received oral hygiene motivation sessions including oral hygiene instructions. During these sessions manual tooth brushing and usage of interdental brushing was carefully explained and showed to the included subjects. Oral hygiene instructions were then re-enforced in each clinical sessions and during follow-ups.

NSPT, consisting in both supra- and sub-gingival mechanical instrumentation of the root surface (scaling and root planing), was performed by a single periodontist. Treatment was provided using both hand and ultrasonic instrumentation with fine tips (EMS, Nyon, Switzerland). Local anaesthesia was used when needed and no time constraints were enforced. A research nurse recorded treatment time, defined as the period comprised by the injection of anaesthesia and the last moment of instrumentation, in minutes with a chronometer. FM-SRP patients received treatment within 24 hrs in two separate sessions, one side of the mouth for each session: two quadrants were instrumented in an afternoon session, whereas the other two were instrumented the following morning. Q-SRP patients received four quadrants sessions of NSPT with an interval of 1 week between sessions. First session was performed always on the upper right maxillary quadrant. Alternatively, the left maxillary quadrant was chosen if less than 6 teeth were available.

31 3.7 Re-assessment Examinations All patients were re-examined at day 1, day 7 and day 90 following the treatment. Whilst day 1 in the FM-SRP group was 24 hours after the completion of the treatment, day 1 in the Q-SRP group was defined as 24 hours after the first quadrant/session of treatment (Figure 11). Day 90 was 90 days after FM-SRP and 90 days after the last quadrant/session in the Q-SRP. Blood collection, medical history and vital signs were measured at each time point. Clinical parameters were collected at day 90. 3.8 Statistical analysis

All data is presented as mean and standard deviation unless otherwise specified. The sample size calculation was based on data of the UCL Eastman Dental Institute group on acute increase in CRP following PT. A minimum of 13 subjects were needed to demonstrate a 3.5 mg/l difference in serum CRP levels between groups after 24 hrs (90% power, a 0.05, standard deviation of 3) (D’Aiuto et al. 2004a).

A final sample of 14 participants per group was planned including a 10% drop-out rate. Changes in all biomarkers were analysed with ANOVA for repeated measures between groups at different time-points. Covariates included in the model were, age, gender, smoking, temperature and body composition differences. Periodontal clinical parameters changes between groups were considered control outcomes and analysed with analyses of covariance. Bonferroni post-hoc corrections were adopted. For those biomarkers with a statistical significant difference between groups at day 1, a relative increase was calculated as follows: day 1 serum concentrations minus baseline, divided by baseline and multiplied by 100. Comparison of relative increases at day 1 between groups was performed by t-test or equivalent non-parametric method. Non-parametric correlation analyses were performed with Spearman rank analyses. Data was graphically tested for normality and

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logarithmic or square root transformations were made as needed before applying the adequate non-parametric tests. All analyses were performed with SPSS version 23 (SPSS Inc. Chicago, IL, USA).

4 Material and methods for the SPT trial

4.1 Experimental design and patient selection

It was design another randomized clinical trial to investigate the acute phase response after SPT. The protocol of the study received approval from the University Hospital of Pisa ethical committee (Pisa, Italy. It was conducted according to the principles outlined in the Declaration of Helsinki on experimentation involving human subjects.

Time points were baseline, day of surgical intervention, 24 hours, 1 week and 3 months from the SPT.

For the SPT arms patients with indication for SPT, full mouth bleeding and plaque scores <25% and pocket probing depth ≥5mm for the candidate surgical site were included. Exclusion criteria were the same that fro NSPT trial (Paragraph 3.1), exception for the presence of NSPT 3 months prior surgery, that instead was needed.

4.2 Clinical parameters Periodontal clinical parameters were recorded at baseline and 3 months after the surgical intervention. Vital signs were collected also at 24 hours and 1 week form the intervention. FMBS, FMPS; PPD, REC as vital signs were collected and assessed as reported for the NSPT material and methods (Paragraph 3.2).

35 4.3 Blood collection analysis of the serum markers

Serum samples were collected as reported for NSPT (Paragraph 3.3). Time points were blood samples were collected were baseline, 24 hours, 1 week and 3 months after the surgical intervention.

4.4 Surgical periodontal treatment

One week prior surgery all patients were examined, only those presenting with a <25% FMPS & FMBS were confirmed for SPT and underwent a careful debridement of the surgical site. The SPT was performed by a single expert periodontal surgeon. All surgical interventions were performed between 8.00 and 11.00 AM using local anaesthesia. Conservative SPT consisted in flap elevation, resection of degranulation tissues, carefully debridement of root surfaces with hand and ultrasonic instruments (EMS, Nyon, Switzerland); soft tissues were conserved as well as possible. 4.0 silk sutures were positioned to assure wound stability. For the resective SPT the same protocol was applied with the exception that after the debridement, osteoctomy or osteotomy were performed until positive osseous architecture was achieved (Carnevale & Kaldahl 2000). A calibrated nurse registered the duration of every single phase of the intervention; notably i) incision and flap elevation time; ii) degranulation time; iii) manual debridement time; iv) ultrasonic debridement time; v) osseous resection time; and vi) suture time were recorded. 4.5 Randomization and statistical analysis Randomization and statistical analysis were performed exactly the same way for NSPT trial (Paragraphs 3.5,3.8). Randomization respected ratio 1:1 for resective and conservative surgical groups. ANOVA or t-test were performed as needed.

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5 Results for the NSPT trial

5.1 Subjects accountability and baseline characteristics

A total of 90 subjects were screened. Thirty-eight subjects were finally included in the non-surgical arms and 28 in the surgical arms. All patients recruited were Caucasians and they all completed the study providing data for the final database analyses. All groups were comparable for age, gender distribution (50% females) and at least one third of them reported to be current smokers (Table 2). Traditional and novel cardiovascular risk factors were comparable between study groups and considered average for the age-range recruited (i.e. average cardiovascular risk).

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Table 2 Demographic characteristics of the Study Sample of non-surgical arms Variable Quadrant SRP _(N=19) Full Mouth-SRP _(N=19)

Age, years 48±9 46±12 Gender, Female (%) 10(50) 9(50) Smoking, current (%) 6(30) 7(44) BMI 24.8±4.1 25.3±4.4 Systolic BP 123±19 119±23 Diastolic BP 79±12 76±13 Total Cholesterol, mmol/L 5.4±0.9 5.4±1.0 HDL, mmol/L 1.4±0.3 1.1±0.3 LDL, mmol/l 3.4±0.9 3.6±0.9 Triglycerides, mmol/l 1.2±0.7 1.4±0.8 CRP, mg/l 1.3(8.60) 1.4(12.3) IL-6, pg/ml 1.1±0.6 1.0±0.7 TNF-α, pg/ml 1.2±0.5 1.7±1.1 Number of teeth 27±4 26±3 BP, blood pressure; BMI, body mass index; CRP, C reactive protein; IL, interleukin; TNF-α, Tumor necrosis factor

38 5.2 Vital signs

No changes between groups were observed with regards to BMI, temperature, SBP and DBP from baseline to 3 months (data not shown). A statistically significant increase in body temperature was noted in the FM-SRP group compared to control. Indeed five patients in the FM-SRP group had fever at day 1 (including 3 patients who showed temperature higher than 38 C°) compared to 2 patients in the Q-SRP group (p<0.05).

39 5.3 Periodontal parameters

All patients recruited presented with similar periodontal clinical parameters (moderate periodontitis). Mean treatment time in the Q-SRP group was 35±7 min for the first quadrant (133±22 minutes for the overall treatment) whilst patients in the FM-SRP group underwent a mean 123±18 minutes of treatment. Both treatment modalities produced

significant clinical benefits in terms of all standard periodontal parameters (Table 3). Comparable reductions in whole mouth dental plaque scores (average reduction of 48%, 37-60 95%CI in the FM-SRP group and average reduction of 55%, 43-60 95%CI in the Q-SRP group, p>0.05 for comparison) and bleeding scores (average reduction of 29%, 17-41 95%CI in the FM-SRP group and average reduction of 31%, 18-43 95%CI in the Q-SRP group, p>0.05 for comparison) were achieved in both groups after PT. Similarly both groups achieved equal reduction in number and percentage of periodontal pockets after 3 months of PT (Table 3).

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Table 3 Clinical Periodontal Parameters between groups at various time points for non-surgical arms

Variable Time Quadrant-SRP _(N=19) Full Mouth SRP (N=19) p value

FMPS (%) BL 66±26 70±26 3M 17±6 15±7 0.308 FMBS (%) BL 53±13 56±25 3M 24±12 26±19 0.761 Mean PPD (mm) BL 3.24±0.50 3.63±0.64 3M 2.65±0.41 2.94±0.56 0.906 Mean PPD (mm) BL 5.83±0.65 6.35±0.72 in sites > 4mm 3M 4.23±0.73 4.54±0.90 0.902 Mean CAL BL 3.91±0.79 4.43±0.84 3M 3.45±0.74 3.76±1.07 0.577 N Pockets > 4mm BL 30±16 38±21 3M 14±13 22±18 0.121 N Pockets > 5mm BL 16±11 24±18 3M 7±5 10±9 0.285

41 5.4 Systemic Biomarkers

No substantial changes were noted in traditional risk factors including total, HDL and LDL cholesterol and triglycerides between groups at any time points (Table 3). A time-treatment statistically significant effect was observed for CRP, IL-6 and TNF- α. At day 1 a marked increase in the serum levels of CRP, IL-6 and TNF-α (p<0.01 for all) in the FM-SRP group compared to the Q-SRP group (Figure 13). No additional biomarkers were substantially different between treatment groups at all time-points (Table 4). FM-SRP patients exhibited a marked relative increase of CRP (relative difference at day 1 of 320%, 67-574 95%CI, p=0.01) and IL-6 (relative difference at day 1 of 245%, 71-418 95%CI, p=0.007) whilst only a modest increase in TNF-α (relative difference at day 1 of 20%, 2-70 95%CI, p=0.04) (Figure 17, Figure 18, Figure 19). Inflammatory biomarkers returned to levels close to baseline after 3 months (Figure 13, Figure 14, Figure 15).

At day 1 all three inflammatory markers were strongly correlated among themselves (R>0.5 for CRP, IL-6 and TNF-α, p<0.001 for all correlation analyses). Further, treatment time (recorded in minutes) and the number of deeper periodontal pockets (greater than 6mm) were positively related to the serum levels of CRP and IL-6 at day 1. In particular, serum CRP levels at day 1 were highly correlated with treatment time irrespective of group allocation (Figure 16).

42

Table 4 Changes in Systemic Biomarkers between study groups at each time point

Group Baseline Day 1 Day 7 Day 90

P value Between groups Cholesterol, mmol/l _{Q-SRP 5.4±0.9 5.3±0.9 5.5±0.7 5.3±0.9} FM-SRP 5.4±1.0 5.4±0.7 5.1±0.6 5.3±1.0 0.435 HDL, mmol/l Q-SRP 1.4±0.3 1.3±0.3 1.3±0.3 1.5±0.4 FM-SRP 1.1±0.3 1.2±0.4 1.2±0.4 1.2±0.4 0.336 LDL, mmol/l Q-SRP 3.4±0.9 3.5±0.8 3.6±0.7 3.4±1.1 FM-SRP 3.6±0.9 3.6±0.7 3.3±0.7 3.4±0.9 0.426 Triglycerides, mmol/l _{Q-SRP 1.2±0.7 1.2±0.6 1.2±0.7 0.8±0.2} FM-SRP 1.4±0.8 1.3±0.4 1.3±0.8 1.3±0.6 0.582 IFN-γ, pg/ml Q-SRP 0.5(0.4-15) 4.3(1.3-13.2) 1.4(0.9-2) 2.3(0.7-4.1) FM-SRP 0.6(0.4-12.3) 5.9(3.2-8) 0.7(0.6-1.7) 2.9(2.2-3.8) 0.183 IL-10, pg/ml Q-SRP 3.2±1.2 4.7±2.1 4.3±0.8 5.1±1.6 FM-SRP 4.4±3.2 4.5±1.7 3.2±1.4 4.9±1.4 0.489 IL-12, pg/ml Q-SRP 0.5±0.4 10.4±1.2 0.6±0.4 0.4±0.1 FM-SRP 0.6±0.2 0.8±0.9 0.4±0.4 0.4±0.3 0.830 IL-8, pg/ml Q-SRP 11.7±4.4 15.9±11.9 14.0±3.3 22.1±28.7 FM-SRP 29.0±46.5 20.6±14.0 14.7±8.0 15.3±9.2 0.705 E-selectin, pg/ml _{Q-SRP 22.3±10.3 22.9±10.1 18.7±9.2 20.9±5.4} FM-SRP 17.4±10.2 24.1±12.7 16.9±10.1 19.3±6.8 0.146 P-selectin, pg/ml Q-SRP 119.2±44.4 142.2±51.1 109.8±39.3 128.8±49.1 FM-SRP 108.2±53.7 103.9±59.3 99.7±50.9 93.0±32.7 0.165 s-ICAM3, pg/ml Q-SRP 0.6±0.2 0.7±0.2 0.6±0.2 0.6±0.2 FM-SRP 0.6±0.2 0.7±0.3 0.5±0.2 0.6±0.2 0.922 Thrombomodulin, pg/ml Q-SRP 4.6±1.2 4.6±0.8 4.0±0.8 4.5±0.7 FM-SRP 4.0±0.6 4.0±1.2 3.7±0.8 3.8±1.0 0.795

43 Figure 13 Mean values (SE) of CRP biomarkers between study non-surgical arms at various time points. Values represent means±standard errors derived from the multivariate model analysis adjusted for age, gender, smoking and body temperature

44

Figure 14 Mean values (SE) of IL-6 between study non-surgical arms at various time points for IL-6. Values represent means±standard errors derived from the multivariate model analysis adjusted for age, gender, smoking and

45 Figure 15 Mean values (SE) of TNF-α between study non-surgical arms at various time points. Values represent means±standard errors derived from the multivariate model analysis adjusted for age, gender, smoking and

46 Figure 16 Scatter plot of serum CRP levels 24 hours after treatment against Treatment Time in minutes by non-surgical arms. Please note that some values are almost identical and overlap in the figure.

47

Figure 17 Relative acute increase in serum CRP levels 24 hours after treatment between non-surgical arms

48 Figure 18 Relative acute increase in serum IL-6 levels 24 hours after treatment between non-surgical arms

49 Figure 19 Relative acute increase in serum TNF-a levels 24 hours after treatment between non-surgical arms

50

6 Results for the SPT trial

6.1 Subjects accountability and baseline characteristics

A total of 120 subjects were screened and 24 subjects were finally included. All patients recruited were Caucasians and they all completed the study providing data for the final database analyses. All groups were comparable for smoking status, clinical parameters and systemic biomarkers levels (Table 5).

Table 5 Demographic characteristics of the study sample of surgical arms Variable _{surgery (N=19)} Conservative _{surgery (N=19)} Resective

Age, years 52.64±10.01 49.50±10.30 Gender, Female (%) 10(71.4) 6(42.9) Smoking, current (%) 2(14,3) 2(14,3) BMI 24.41±2.56 23.47±2.98 Systolic BP, mmHg 120.35±11.17 115.71±12.06 Diastolic BP, mmHg 76.50±8.54 73.92±9.02 Body temperature, °C 36.57±0.43 36.48±0.47 HDL, mmol/L 60.92±11.41 52.07±12.14 LDL, mmol/l 118.42±34.83 119.35±33.08 CRP, mg/l 1.12±0.85 1.49±2.22 Number of teeth 3.00±0.78 3.21±0.80 BP, blood pressure; BMI, body mass index; CRP, C reactive protein; IL, interleukin; TNF-α, Tumor necrosis factor

51 6.2 Vital signs

A significant difference of temperature at 24 hours in the conservative group vs all other time points intragroup was observed (p<0.01). No significant differences were noted for SBP, DBP or heart rate (Table 6, Figure 20).

Table 6 Vital signs recorded for the surgical arms of the study

Group Baseline 24h 1week 3 months

Systolic BP, mmHg Conservative surgery 120.35±11.17 116.07±11.79 118.21±10.11 110.00±8.49 Resective surgery 115.71±12.06 124.07±11.79 112.85±8.56 120.00±17.60 Diastolic BP, mmHg Conservative surgery 76.50±8.54 74.64±10.46 75.00±8.32 74.50±10.91 Resective surgery 73.92±9.02 78.92±9.64 71.93±8.78 76.36±12.86 Heart rate, bpm Conservative surgery 71.78±70.64 73.71±10.46 71.14±9.72 69.30±10.41 Resective surgery 70.64±949 72.71±9.17 69.15±8.25 64.00±9.50 Body temperature, °C Conservative surgery 36.47±0.43 36.07±0.72 36.35±0.45 36.00±0.45 Resective surgery 36.48±0.47 37.18±0.51* 36.39±0.44 36.50±0.34 *P<0.01 intra-group compared with all time point

52

Figure 20 Graph representing body temperature at various time points for SPT arms

53 6.3 Periodontal parameters

No differences between baseline and 3 months were observed for FMBS and FMPS (Table 7). Mean PPD was significantly reduced in both groups, from 3.88±0.65 to 2.66±0.32 mm for the conservative arms and from 3.82±0.58 to 2.77±0.30 mm for the resective arms. Mean REC increase was noted in both groups at 3 months post-op, in the resective arm this increase was highly significant (0.47±0.64 to 1.61±0.81 mm, p=0.001) (Figure 8). For both groups, no changes were observed between baseline and the end of the study in terms of mean CAL. The number of pockets with PPD >3 and 5 mm drastically changed between baseline and 3 months post-op (p<0.0001)(Figure 21), pathological pockets were eliminated almost completely (0.10±0.31 and 0.00±0.00 mm respectively for conservative and resective arms at 3 months post NSPT). However no differences between study groups were observed in terms of clinical parameters. Mean treatment time was higher in the resective arm compared with the conservative one (39.07±10.23 vs. 31.46±9.86), the difference was not statistical significant (Table 8).

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Table 7 Clinical Periodontal Parameters between groups at various time points for surgical phase

Variable Time _{surgery (N=14)} Conservative _{surgery (N=14)} Resective between P value groups FMPS (%) BL 20.07±8.29 21.35±8.08 0.681 3M 15.8±6.86 18.36±5.35 0.356 P between time points 0.196 0.301 FMBS (%) BL 13.92±7.03 15.78±6.58 0.477 3M 13.67±6.72 13.09±7.44 0.853 P between time points 0.929 0.348 BoP(%) BL 3.71±3.19 3.71±4.12 1.000 3M 2.70±3.19 2.27±4.14 0.796 P between time points 0.452 0.396 Mean PPD (mm) BL 3.88±0.65 3.82±0.58 0.802 3M 2.66±0.32 2.77±0.30 0.456 P between time points 0.000 0.000 Mean REC (mm) BL 0.61±0.59 0.47±0.64 0.571 3M 1.36±1.50 1.61±0.81 0.641 P between time points 0.103 0.001 Mean CAL BL 4.49±0.99 4.29±0.84 0.582 3M 4.03±1.51 4.39±0.93 0.527 P between time points 0.375 0.800 N Pockets > 3mm BL 9.14±3.69 9.28±3.60 0.918 3M 2.30±2.40 2.54±1.86 0.798 P between time points 0.000 0.000 N Pockets > 5mm BL 3.07±1.9 3.92±2.52 0.327 3M 0.10±0.31 0.00±0.00 0.343 P between time points 0.000 0.000

55

Figure 21 Graph representing the mean number of site with PPD>3mm and PPD>5mm at various time points for SPT arms

56

Table 8 Intervention times recorded for the surgical phase in minutes

Time Conservative surgery Resective surgery P value between groups Intervention duration 31.46±9.86 39.07±10.23 0.056 Incision & flap elevation 7.01±2.73 8.37±3.51 0.265 Degranulation 5.17±2.28 4.53±2.38 0.475 Ultrasonic debridement 6.68±8.45 8.45±4.19 0.299 Mechanical debridement 2.99±1.74 3.32±1.95 0.647 Osseous resection - 6.18±4.53 - Suture 7.88±3.66 6.52±2.50 0.262

57 6.4 Systemic biomarkers No changes or differences were observed between groups. CRP did not show a statistically significant increase at 24 hours compared with baseline (Figure 22, Table 9). In terms of CRP in both groups at 1 week and 3 months a slight decrease was observed. On the other hand, HDL and LDL, compared with baseline, remained stable at the end of the study. Table 9 Changes in Systemic Biomarkers between study groups at each time point for the surgical arms

Marker Group Baseline 24h 1-week 3-months

CRP Conservative surgery 1.12±0.85 1.62±1.32 0.94±0.78 1.03±1.23 Resective surgery 1.49±2.22 1.35±1.19 0.87±0.76 1.23±1.40 HDL Conservative surgery 60.92±11.41 - - 54.09±16.65 Resective surgery 52.07±12.14 - - 60.63±15.73 LDL Conservative surgery 128.42±34.83 - - 128.18±40.10 Resective surgery 119.35±33.08 - - 118.80±42.86 No significant P value were observed for this data set.

58

Figure 22 Graph representing CRP at various time points for SPT arms, bars represent 95% confidence intervals

59 6.5 Patient related outcome

Twenty-four hours after SPT higher pain scores were observed in the resective group when compared to the conservative group. Furthermore, the resective group experienced in a statistically significant way far more pain independently of pain intensity (p<0.001) (Table 10).

Table 10 mean values of self-reported pain trough VAS scale

Groups Mean worst

pain Moderete Mean pain Pain at 24 hours Total pain Conservative surgery 2.66±1.93 2.66±1.32 1.66±0.70 7.00±3.24 Resective surgery 6.10±2.28 5.20±1.98 4.70±1.66 16.00±7.00 P-value between groups 0.003 0.005 0.000 0.000

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7 Discussion

To our best knowledge, this is the first clinical trial to compare the magnitude of acute phase responses following two common techniques of non-surgical and surgical periodontal therapy. Our data demonstrated that non-surgical periodontal therapy performed within 24 hours induced greater perturbations of systemic inflammation compared to conventional (quadrant) scaling. Instead or the surgical periodontal treatment an increase of acute phase response was not observed at 1 day from surgical intervention. Our data is in agreement with previous clinical trials investigating the effect of PT on inflammatory markers and haemostatic system (D’Aiuto et al. 2013a). A moderate acute inflammatory response associated consistently with an increase of early (TNF-α and IL-6) and hepatic inflammatory proteins (i.e. CRP) has been reported following non-surgical periodontal therapy performed within 24 hours (Ide et al. 2004, D’Aiuto et al. 2004b). The reason for such a response may be found in both the bacteremia and host immune response combined with the local tissue damage following subgingival instrumentation (Lofthus et al. 1991) which may determine an increase both of pro-inflammatory mediators (Birkedal-Hansen 1993) and acute-phase proteins (Gabay & Kushner 1999). Accordingly, traditional and modern models used to study human inflammation report similar short-term acute response of variable magnitude (Suffredini et al. 1995, 1999). The intravenous injection of Escherichia Coli produces a robust acute phase response with leukocytosis, fever and a considerable increase in CRP serum concentration in the 24 hours. Interestingly in our sample, patients undergoing full mouth treatment showed an increase in body temperature 24 hours after treatment as previously noticed (Vandekerckhove et al. 1996). This was not the case in the previous report of intensive periodontal therapy (whole mouth instrumentation within 4-6 hours) when compared to supragingival scaling and polishing. A

61

possible explanation of this difference could be the two-hit bacterial dissemination performed in the FM-SRP. Indeed patients received the first half-mouth treatment one evening and the other session the following morning. It is plausible that 24 hours after the second session the second bacteremia could elicit increase in body temperature. Alternatively, the use of pain-killers following periodontal therapy could explain the lack of increase in body temperature. In all clinical trials completed at UCL with an intensive periodontal therapy approach, patients were allowed to take paracetamol following the session of cleaning.

Local tissue trauma may also elicit a systemic response. According to the extent and nature of the trauma the host response might be different. Our data confirmed that differences in time needed to instrument sub-gingivally the diseased dentition closely tracked the changes in acute systemic markers. This is consistent with the previous findings we reported when a lower increase of inflammatory markers was observed after periodontal surgery if compared to full mouth treatment (Graziani et al. 2010b). On the other hand, we are missing the data on the post-operative bacteremia comparing these two approaches. The application of full-mouth protocols of debridement determines bacteremia with a prevalence varying from 13% (Kinane et al. 2005) to 73% (Forner et al. 2006). Frequency of bacteremia following periodontal therapy is affected by many factors including the study design, the microbiological methodology, the stimulus and the periodontal health status of the patients (Reyes et al. 2013). The differences in biomarkers observed could also be due to the sampling times and the presence of residual untreated periodontitis sites in the quadrant-scaling group. Further research is needed to further elucidate the exact mechanisms underpinning this finding.

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Interestingly, quadrant treatment did not produce a long-lived acute phase response. Our study only confirmed some minimal increase in IL-6 and TNF-α in the Q-SRP group 24 hours after the treatment. It is plausible to suggest that the magnitude of bacteremia/trauma created by a single session of quadrant treatment may be insufficient to trigger a host response for more than 12 hours. Earlier sampling could have elucidated the exact kinetic of host response following this treatment modality. Although increased, the early peaks in IL-6 and/or TNF- α might have been not sufficient to trigger a long-lived hepatic response. The sharp and transitory acute response after treatment may be of particular interest as inflammation could lead to an acute state of vascular dysfunction (Seinost et al. 2005, Tonetti et al. 2007) and a possible increase in the putative risk of vascular events. Acute inflammatory episodes (as those following a simple respiratory or urinary infection) triplicate the individuals’ risk of vascular events (Smeeth et al. 2004). Interestingly, the rate of important vascular events significantly increased in the first 4 weeks after invasive dental treatment independent of traditional risk factors (Minassian et al. 2010). Obviously this study was not designed to test this hypothesis nor provide evidence in support of a negative effect of periodontal therapy on future vascular risk. Future research addressing the eventual impact of invasive dental treatment procedures (including extractions or subgingival instrumentation) on the risk of vascular events is advocated.

Our trial did not indicate differences between full mouth and conventional quadrant approach in terms of both plaque and PPD/CAL reductions as it has already been noticed (Eberhard et al. 2008, Farman & Joshi 2008).

Another important confirmation is that no statistical difference was noted 3 months after therapy in terms of systemic inflammation between study groups with no superior effect noted in the full mouth versus quadrant approach. Indeed, we observed a reduction in CRP

63

from baseline to 3 months in both groups (these differences were statistically significant, data not shown). The lack of a no/delayed treatment group in this case does not allow any appropriate comparison between study groups and a possible regression to the mean effect could be responsible for this finding.

Surgical treatment of periodontal disease produced a significant decrease of all surrogate clinical parameters and indexes of healing. Nevertheless, no significant differences were observed between groups. This is somehow in contrast with the clinical perception of periodontal surgical procedure being a ‘‘trauma’’ of a greater magnitude when compared with non-surgical therapy.

In terms of systemic biomarkers no differences between the two groups or the various time points were noted. This data is in accordance with a previous study investigating the acute phase response after surgical therapy and reporting no significant differences at 24 hours compared with baseline (Graziani et al. 2010b). It may be assumed that the absence of a consistent inflammatory response 24 hours after surgical intervention is strictly correlated to the low bacterial burden of the intervention site. The low bacterial burden may be attributable to the fact that only patients presenting with FMPS/FMBS inferior to 25% one week prior to the intervention were admitted for surgery and received a prophylaxis session consisting in a careful debridement of the surgical site. Furthermore, the number of the teeth involved in the intervention was very limited (3.00±0.78 and 3.21±0.80 mean number of teeth for conservative and resective arms respectively); the reduced area of intervention may have contained the acute phase response.

The authors are aware of some limitations of this study. Although formal sample size estimation was performed, the patient sample is relatively small and mainly composed of patients suffering from moderate periodontitis presenting no other co-morbidities. These

64

studies were not powered to show differences in multiple inflammatory biomarkers but only in CRP after 24hrs of the therapy. Further research in this area is advocated.

8 Conclusions

A single session of full mouth debridement produced a 24-hour moderate acute phase response compared to conventional (quadrant scaling) treatment. Conservative and resective surgical periodontal therapy seem not to determine a significant acute phase response. Clinicians might choose conventional (quadrant scaling) non-surgical periodontal treatment in patients with complicated medical history and/or uncontrolled co-morbidities. Conservative and resective surgical periodontal therapy seem not to determine a

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