J Clin Periodontol 2015; doi: 10.1111/jcpe.12393

Antimicrobial photodynamic effect to treat residual pockets in periodontal patients: a randomized controlled clinical trial

^ nica F. Carvalho1, Priscila V. C. Vero Andrade1, Michelle F. Rodrigues1, Mario H. Hirata2, Rosario D. C. Hirata2, Claudio M. Pannuti1, Giorgio De Micheli1 and Marina C. Conde1 1

Department of Periodontology, School of Dentistry, University of Sao Paulo, Sao Paulo, Brazil; 2Department of Clinical and Toxicological Analyses; School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil

Carvalho VF, Andrade PVC, Rodrigues MF, Hirata MH, Hirata RDC, Pannuti CM, De Micheli G, Conde MC. Antimicrobial photodynamic effect to treat residual pockets in periodontal patients: a randomized controlled clinical trial. J Clin Periodontol 2015; doi: 10.1111/jcpe.12393.

Abstract Aim: A randomized controlled clinical trial was designed to evaluate the efficacy of the photodynamic therapy (PDT) in the treatment of residual pockets of chronic periodontitis patients. Material and Methods: Thirty-four patients with at least four residual periodontal pockets undergoing maintenance care were included and randomly assigned to test group (PDT, n = 18) or control group (sham procedure, n = 16). The intervention was performed at baseline, 3, 6 and 12 months. Clinical parameters such as pocket probing depth (PPD), clinical attachment level (CAL), bleeding on probing (BoP) and plaque index (PI) were measured before intervention and after 3, 6 and 12 months. Subgingival samples were obtained at baseline, and after 7 days, 3, 6 and 12 months to quantify Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia by realtime polimerase chain reaction (PCR). Results: All clinical variables showed significant improvement during the study, but there was no significant difference between test and control groups. The microbiological analyses showed no differences between groups at any time during the study. Conclusion: Within the limits of this clinical trial and considering the laser and photosensitizer protocol used, PDT failed to demonstrate additional clinical and bacteriological benefits in residual pockets treatment.

Periodontal treatment involves mechanical cleaning of tooth surfaces to remove calculus and dental

biofilm, and a strict control of biofilm prevents re-colonization of the subgingival area (Quirynen et al.

Conflict of interest and source of funding statement This study was supported by Sao Paulo State Research Foundation - FAPESP (#2009/53934-5). M.H.H. and R.D.C.H. are recipients of fellowships from CNPq, Brazil. The authors declare that there are no conflicts of interest in this study. Clinical Trial Gov Registration: NTC01034501 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Key words: chronic periodontitis; microbiology; periodontal pockets; photodynamic therapy; polymerase chain reaction Accepted for publication 13 March 2015

2005). In some cases, scaling and root planing is insufficient to solve periodontal infection (Socransky & Haffajee 2002). The persistence of pathogenic microbiota may provide favourable conditions to subgingival re-colonization and recurrence of periodontal disease (Petersilka et al. 2002). Residual pockets increase the risk of

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additional attachment loss and tooth loss (Matuliene et al. 2008). Therefore, the treatment requires complementary procedures, such as repeated scaling, periodontal surgery or antibiotics (Herrera et al. 2008, Tonetti et al. 2012). To overcome the limitations of scaling and root planing and to reduce the bacterial load, photodynamic therapy (PDT) has been proposed as adjunctive to periodontal treatment. PDT has a light-activated photosensitizer that transfers energy from light to molecular oxygen, generating reactive oxygen species (ROS), which are toxic to bacteria (Meisel & Kocher 2005). Therefore, PDT has been suggested as an alternative to eliminate subgingival microbial species and to promote more effective disinfection of the root surfaces. The effect of PDT on dental biofilm was confirmed by in vitro studies (Fontana et al. 2009). The singlet oxygen, a ROS, degrades polysaccharides from the extracellular matrix molecules of the biofilm (Soukos et al. 2003). Laser photoactivation of dyes, such as methylene blue, toluidine blue O and malachite green, cause death of periodontal pathogenic bacteria, by means of oxygen singlet production in dental biofilm (Chan & Lai 2003, Prates et al. 2007 Qin et al. 2008). Animal studies support these observations of bacterial load reduction, and demonstrated safety to the surrounding tissues, absence of loss of connective tissue and necrosis (Sigusch et al. 2005, Almeida et al. 2007, Prates et al. 2011). PDT as an adjunctive of periodontal treatment has been evaluated by several clinical studies (Braun et al. 2008, Giannelli et al. 2012, Bassir et al. 2013, Luchesi et al. 2013, Betsy et al. 2014), which reported controversial results about the benefit of PDT in the treatment of chronic periodontitis (Atieh 2010, Azarpazhooh et al. 2010, Sgolastra et al. 2013). For periodontal patients under supportive therapy, it has been shown that a single application of PDT reduced the percentage of sites with bleeding on probing (Chondros et al. 2009). Moreover, additional benefits were found in pocket probing depth (PPD), clinical attachment

level (CAL) and bleeding on probing (BoP) when five repeated cycles of PDT were applied (Lulic et al. 2009). Recent studies have shown that better results in the reduction in PPD and BoP were found when a single application of PDT was associated to SRP (Campos et al. 2013), and better reduction in PPD was found when 2 or a single application of PDT was associated to ultrasonic debridement (M€ uller Campanile et al. 2015). PDT has also shown better reduction in periodontopathogens load in maintenance patients, when associated to ultrasonic debridement before intervention, in comparison to SRP (Cappuyns et al. 2012). However, PDT alone has failed to show superior results in clinical and microbial variables when compared with ultrasonic debridement (R€ uhling et al. 2010). Considering that the results of clinical trials evaluating the benefits of antimicrobial PDT are still inconclusive, the aim of this study was to evaluate the clinical and microbiological effects of PDT in treatment of residual pockets of patients with chronic periodontitis subjected to supportive therapy. Material and Methods Study design and sample

This study is a randomized controlled parallel-group clinical trial with 12 months of follow-up. The research protocol was approved by the Ethical Committee of the School of Dentistry of the University of Sao Paulo (SD/USP) (protocol # 211/ 2008), and written informed consent for the enrolment in the study was given by all subjects. This study was performed in accordance to standards of reporting trials – Consort Statement - (Schulz et al. 2010), and registered at clinicaltrials.gov, number NTC01034501, entitled: “Adjunctive photodynamic therapy in treatment of chronic periodontitis”. Patients with chronic periodontitis were recruited from the Dental Clinic of the SD/USP, according to the eligibility criteria. Inclusion criteria were as follows: age between 35 and 75 years old, diagnosis of chronic periodontitis with proximal

attachment loss ≥5 mm in more than 30% of the teeth (Tonetti & Claffey 2005), at least 10 teeth in the mouth, and at least four sites with PPD ≥4 mm at the revaluation exam. Exclusion criteria were as follows: pregnancy or lactation, diabetes, current smoking or smoking within the last 10 years, antibiotic therapy or periodontal treatment in the previous 6 months, long-term use of anti-inflammatory and immunosuppressive medication, need for antibiotics prophylaxis (Wilson et al. 2007), severe occlusal dysfunction, orthodontic treatment, class II or III mobility, endodontic problem. The sample size was calculated considering that a mean difference of 1.0 mm in CAL change between test and control groups (one-tailed) would be clinically relevant. Using a power of 90%, a level of significance of 5% and considering a standard deviation of 1.0 mm in CAL, 17 subjects per group would be necessary. To compensate losses during the follow-up, 19 subjects per group were recruited (10% more than the calculated). Clinical procedures

In the pre-study phase, patients were submitted to oral hygiene instruction (brushing, flossing, inter-dental brushing), scaling and root planing with periodontal curettes (5/6, 11/12, 13/14 Gracey curettes, Hu-FriedyÒ, Rio de Janeiro, RJ, Brazil) and ultrasonic device (MiniPiezonÒ, EMS, Electro Medical System, Nyon, Switzerland) and fillings, extractions, temporary prosthesis, if necessary, were performed in four to six sessions by two periodontists (MFR, PVAC). After 45 days (Segelnick & Weinberg 2006) clinical examination was performed to select the eligible subjects presenting at least four sites with residual pockets (at least one site with PPD ≥5 mm), the enrolment was performed by VFC after clinical examination. The subjects were randomly assigned to test (PDT) or control (sham procedure) group, based in a random computerized generated sequence in blocks of four, by an independent statistician. Allocation concealment was conducted with opaque sealed envelopes, which were opened in a

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Photodynamic therapy in residual pockets numeric sequence, by the intervention operator (MCC) at the time of intervention. The treatment assignments were concealed to subjects and the examiner. The intervention was applied only to sites with PPD ≥4 mm. In the PDT group, the photosensitizer (methylene blue 0.01%, ChiHypofarma, Belo mioluxÒ, Horizonte, MG, Brazil) was applied with a sterile disposable syringe to the bottom of the periodontal pocket in coronal direction, and after 5 minutes the photosensitizer was rinsed with water to remove any excess. The pocket was exposed to a diode laser with wavelength of 660 nm, using an optic fibre tip into the periodontal pocket for 90 s and energy density of 90 J/cm2, 40 mW power, according to the manufacturer’s instructions (Laser HandÒ – MM Optics, S~ao Carlos, SP, Brazil). In the control group, the irrigation of the pocket was carried out with saline solution, and the laser light was not powered on during the supposed irradiation, to guarantee the patient masking. The application time of both photosensitizer and laser light was controlled by a timer, in both groups. A power meter (Laser CheckÒ – Coherent Molectron, Portland, OR, USA), was used every time before laser application to check the energy to be delivered. The treatment was repeated 3, 6 and 9 months after the initial procedure. Before the PDT applications, the supragingival plaque was carefully removed from experimental sites, subgingival plaque was collected and clinical examination was performed, except at the 9th month for data collection. After the PDT, maintenance care was delivered, including the reinforcement of oral hygiene as well as plaque and calculus removal from full mouth. The possible adverse effects, such as oedema, erythema and pain, were analysed by a questionnaire, which included the Visual Analog Scale (VAS) to patient’s evaluation of pain and discomfort in a scale from zero to 100 mm, asked by the intervention operator. Clinical parameters were CAL, PPD, BoP, PI that were evaluated before and after each three-month therapy. Clinical variables were recorded by a blinded, trained and

calibrated examiner – 82% reproducibility for PPD and 90% for CAL – (VFC), using a computerized periodontal probe (Florida Probe SystemÒ, Florida Probe Corporation – Gainesville, FL, USA). Microbiological analysis

Subgingival microbiological sampling was pooled from the four experimental sites (PPD ≥4 mm) at baseline, 7 days, 3, 6 and 12 months. Supragingival plaque was carefully removed with periodontal curettes. A sterile paper point #30 (Tanari Industrial Ltda, S~ ao Paulo, Brazil) was inserted into the bottom of the periodontal pocket for 30 s, controlled by a timer (Hartroth et al. 1999). The paper points were transferred to a sterile microtube (Axygen Scientific, Union City, CA, USA) and stored at 20°C. The subgingival plaque sampling was performed by a single operator (PVAC). DNA was isolated from subgingival samples using an extraction DNA kit (QiaAmp DNA mini kitÒ, Qiagen, Hilden, Germany), according to manufacturer’s instructions. Bacterial detection and quantification were performed using a quantitative real-time PCR (qPCR) TaqMan system (Applied Biosystem, Foster City, CA, USA). Oligonucleotide primers and probes were selected based on species-specific highly conserved regions from the 16S rRNA gene for species: A. actinomycetemcomitans, P. gingivalis, Treponema denticola (T. denticola) and Tannerella forsythia (T. forsythia). To establish a quantitative assay, cloning procedures were performed as previously described (Rodrigues et al. 2012), to obtain positive plasmid controls of the selected species. Tenfold serial dilutions from each plasmid control were run in triplicate to obtain a standard curve. Plasmid standard controls (102 to 108), negative controls (sterilized DNAase-free water), and samples were run in triplicate, in a 20 ll reaction mixture, containing: 10 ll of Taqman FAST Universal PCR Master MixÒ (Applied Biosystem, Foster City, CA, USA), 0.5 ll of the forward and reverse primers (Applied Biosystem), 0.5 ll of probe, 6.5 ll of sterilized DNAase-free water, and 2 ll

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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DNA sample. The amplification was carried out in a 7500 Fast Real-Time PCR equipment using the following cycling conditions: one cycle at 95°C for 20 s, and 40 cycles at 95°C for 3 s and at 60°C for 30 s. The microbiological analyses were conducted blinded to clinical examination and allocation group by a single operator (VFC). Primary and secondary outcomes measures

The primary outcome variable of the study was CAL. Secondary outcome variables were PPD, BoP, PI and level of periodontal pathogens. Statistical analysis

Data were analysed by SPSS version 10.0 (SPSS, IBM, Armonk, NY, USA). Means of test and control groups were calculated. For CAL, PPD, BoP and PI variables, it was used a generalized linear model (ANOVA and Newman–Keuls test) to verify differences between groups and changes during the follow-up. The comparison between groups, related to bacterial quantity, was done by repeated measures ANOVA and the multiple comparisons by Newman– Keuls test. All statistical analyses considered the level of significance at 5%. The analysis strategy of this clinical trial was intention-to-treat analysis. The comparison between groups test and control, in relation to VAS was performed by Student’s t test. Results

The recruitment period occurred from April 2010 to August 2012 (Fig. 1) and the follow-up period was from August 2010 to August 2013. The mean age of the PDT group was similar (48.2  9.5 years) to the control group (51.9  8.1 years) (p > 0.05). There was 61.1% of women in test group, and 37.5% in control group. Mean values of the clinical parameters were similar in both groups at baseline (p > 0.05). Periodontal treatment, conducted in the pre-study phase, resulted in significant improvement of PPD, CAL, BoP and PI in both groups (Table 1). After revaluation a signifi-

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Carvalho et al. 397 not meeting eligibility criteria 262 presented exclusion criteria:

Screening exam n = 483 Pre-study phase

- 63 smokers - 32 quitted smoking less than 10 years - 20 diabetics - 103 sistemic condition which could influence in periodontal treatment - 1 pregnant - 33 had received antibiotics or periodontal treatment in previous 6 months - 6 orthodontic treatment - 2 alcoholics - 2 occlusion dysfunction

Initial periodontal therapy n = 86

135 not presented inclusion criteria

Revaluation Baseline exam n = 38

Randomization

Study phase

Test group

Control Group

3 months n = 19

3 months n= 19 Lost to follow-up n = 1; could not be contacted

6 months n = 18

1 exclusion antibiotics

3 months n = 18

Lost to follow-up n = 2; could not

be contacted

12 months n = 18

3 months n = 16

Fig. 1. Flow chart of participants.

cant gain of CAL was observed after 3, 6 and 12 months, but without significant differences between groups. There was a significant reduction in PPD and BoP at 3, 6 and 12 months, but there was no significant difference between groups at any. Also, there was no significant differences between groups as regards moderate residual pockets (Table 2). Both groups presented a significant reduction in the number of residual pockets without difference between groups (Table 3). There was a significant association between group and presence of Porphyromonas gingivalis at 12 months (p = 0.02), as 88.9% of the test subjects harboured this bacteria, as compared to 50% of the control subjects (Table 4). Table 5 shows that there were no differences between groups at any time of study, for any of the bacterial species. When only sites with BoP and PPD ≥5 mm at baseline were analysed, a significant reduction in PPD and BOP was observed in both

groups. There was not significant CAL gain in both groups. No significant difference between groups was observed in any period (p > 0.05) for any of the outcomes (Table 6). As regards adverse events, PDT group showed a higher VAS score only at the 3-month examination (p = 0.04). Discussion

The aim of this clinical trial was to evaluate the clinical and microbiological effects of PDT in chronic periodontitis residual pockets. This trial was carried out in patients with residual pockets. The results showed that both experimental groups presented PPD and BoP reduction and gain of CAL. Our hypothesis was rejected, as PDT failed to show better results for the treatment of residual pockets, than supragingival plaque control. Areas with limited access to instrumentation, as deep pockets, require complementary therapy. Residual pockets represent greatest difficulty to obtain improvement in results in

comparison to sites not yet treated. Clinical studies with persisting pockets ensure only a slight clinical improvement, an explanation may be that major healing effects were obtained during the cause-related treatment phase (Eickholz et al. 2005), as observed in our results. After revaluation, in the study phase, we also observed improvement in all clinical parameters, but without significant difference between groups (Table 1). The BoP reduced 34.72% in the test group, and 26.55% in the control group (Table 1). Although a sham procedure has been performed in the control group, we observed significant improvement in this group. This effect may have been a residual effect of non-surgical periodontal therapy delivered during the prestudy phase, since the period from this phase until revaluation was relatively short (45 days). Our clinical results (Tables 1, 2 and 6) are consistent with a number of recent clinical investigations that reported treatment of residual pockets. R€ uhling et al. (2010) and Chondros et al. (2009) applied a single session of PDT after ultrasonic debridement, and the only favourable result to PDT was the greater reduction in BoP in one study (Chondros et al. 2009). Similar results were observed when PDT was compared to debridement with curettes (Cappuyns et al. 2012). This study used the same parameters of laser as our study, allowing for a better comparison between results. This study applied four cycles of PDT, in maintenance appointments, one at every 3 month. Lulic et al. (2009) applied five cycles of PDT in a smaller interval, and showed better results of PDT, but the study included only 10 patients. Campos et al. (2013) presented improved results in clinical parameters of PDT after 3 months. M€ uller Campanile et al. (2015) showed greater pocket reduction after two applications of PDT in 27 patients followed up for 3 months. Mongardini et al. (2014) presented an improved pocket reduction, in 1 week of follow-up. This period of observation is considered insufficient for clinical analysis (Segelnick & Weinberg 2006). Therefore, the studies that presented clinical benefits of PDT had some

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Photodynamic therapy in residual pockets

Table 1. Mean, standard deviation (SD) and comparison of pocket probing depth, clinical attachment level, plaque index, bleeding on probing Full mouth Test (n = 18) Control (n = 16) p-values Test (n = 18) Control (n = 16) p-values Test (n = 18) Control (n = 16) p-values Test (n = 18) Control (n = 16) p-values

CAL (mm) PPD (mm) BoP (%) PI (%) Experimental sites

CAL (mm) PPD (mm) BoP (%) PI (%)

Pre-study

Baseline

Test (n = 18) Control (n = 16) p-values Test (n = 18) Control (n = 16) p-values Test (n = 18) Control (n = 16) p-values Test (n = 18) Control (n = 16) p-values

5.56 5.87 0.46 4.79 4.87 0.75 58.33 42.18 0.06 18.05 18.75 0.92

 0.83  1.39  0.47  0.74  29.70  19.83  23.95  29.58

4.17 4.29 0.74 3.58 3.45 0.66 45.42 37.63 0.17 54.21 47.11 0.25

Baseline 3.36† 3.99† 0.09 2.68† 2.78† 0.72 16.00† 12.89† 0.58 18.26† 12.21† 0.33

 1.15  1.18  1.09  0.98  23.07  23.40  23.50  27.82

 0.72  1.37  0.58  0.92  9.97  7.64  8.86  8.22

3 months

6 months

12 months

Difference baseline12 months

4.61†  1.13 4.78†  1.44 0.68 3.70†  0.85 3.39†  0.76 0.76 19.44†  21.95 17.18†  23.66 0.78 12.50  22.08 10.93  18.18 0.97

4.44†  1.06 4.78†  1.44 0.92 3.41†  0.93 3.48†  0.70 0.8 31.94†  29.46 23.43†  19.29 0.55 16.67  21.00 09.33  12.50 0.85

4.60†  1.11 4.33†  1.21 0.79 3.55†  0.92 3.23†  0.77 0.75 23.61†  21.82 15.63†  22.13 0.86 12.50  17.68 6.25  19.36 0.82

0.96 1.54 0.09 1.24 1.64 0.21 34.72 26.55 0.44 5.55 12.5 0.93

*Significant inter-group difference 5% (Newman–Keuls test). † Significant intra-group difference related to baseline (Newman–Keuls test). Table 2. Clinical variables in test and control groups with moderate residual pockets (pocket probing depth 4–6 mm at re-evaluation exam) Variables

CAL (mm) PPD (mm)

Baseline

Test (n = 18) Control (n = 16) p-values Test (n = 18) Control (n = 16) p-values

5.37 5.76 0.33 4.60 4.76 0.53

 0.68  1.15  0.40  0.50

3 months

4.45† 4.71† 0.79 3.53† 3.31† 0.8

 1.07  1.33  0.76  0.71

6 months

4.26† 4.81† 0.73 3.31† 3.49† 0.88

12 months

 0.95  1.43  0.85  0.69

4.33† 4.50† 0.9 3.32† 3.24† 0.98

 1.03  1.43  0.96  0.77

Difference baseline12 months 1.04 1.26 0.5 1.28 1.52 0.48

*Significant inter-group difference 5% (Newman–Keuls test). † Significant intra-group difference related to baseline (Newman–Keuls test). Table 3. Frequency distribution of pockets ≥4 mm and ≥5 mm, according to treatment group Baseline n pockets ≥4 mm n pockets ≥5 mm

Test Control p-values (chi-square) Test Control p-values (chi-square)

76 76 – 39 48 0.18

12 months

p-value (McNemar)

31 25 0.39 14 8 0.24

Antimicrobial photodynamic effect to treat residual pockets in periodontal patients: a randomized controlled clinical trial.

A randomized controlled clinical trial was designed to evaluate the efficacy of the photodynamic therapy (PDT) in the treatment of residual pockets of...
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