UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” Campus de Araçatuba CAMILA AYUMI IVANAGA Efeitos fotodinâmicos da Curcumina no tratamento de bolsas residuais de pacientes portadores de periodontite crônica e Diabetes Mellitus tipo 2: estudo de boca dividida randomizado ARAÇATUBA 2019 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” Campus de Araçatuba CAMILA AYUMI IVANAGA Efeitos fotodinâmicos da Curcumina no tratamento de bolsas residuais de pacientes portadores de periodontite crônica e Diabetes Mellitus tipo 2: estudo de boca dividida randomizado Dissertação apresentada à Faculdade de Odontologia de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, como parte dos requisitos para a obtenção do título de Mestre em Periodontia. Orientadora: Profa. Assoc. Leticia Helena Theodoro Co-orientador: Prof. Titular Valdir Gouveia Garcia ARAÇATUBA 2019 Catalogação na Publicação (CIP) Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Ivanaga, Camila Ayumi. I93e Efeitos fotodinâmicos da curcumina no tratamento de bolsas residuais de pacientes portadores de periodontite crônica e Diabetes mellitus tipo 2 : estudo de boca dividida randomizado / Camila Ayumi Ivanaga. - Araçatuba, 2019 65 f. : il.; tab. Dissertação (Mestrado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientadora: Profa. Leticia Helena Theodoro Coorientador: Prof. Valdir Gouveia Garcia 1. Diabetes mellitus 2. Raspagem dentária 3. Curcumina 4. Fotoquimioterapia I.T. Black D6 CDD 617.64 Claudio Hideo Matsumoto CRB-8/5550 2 À toda minha família, expresso minha imensa gratidão por todo amor e suporte em cada etapa de minha vida. O apoio de vocês me fortifica e me impulsiona a sempre perseguir meus sonhos e desbravar novas oportunidades. Dedico a vocês este trabalho com todo meu amor. 3 AGRADECIMENTOS ESPECIAIS À minha mãe, Alessandra Takebe Carleial, agradeço pelo amor imensurável dedicado a mim. Saiba que tenho imensa admiração por sua força e determinação. Você é meu maior exemplo de que com empenho, organização e perseverança, não há limites para nossos sonhos, tudo pode ser concretizado e todos os obstáculos superados. Ao meu pai, Carlos Alberto Ivanaga, agradeço por todo amor e dedicação ao meu cuidado. Você é meu melhor amigo, sempre com uma palavra de conforto ou prestes a me fazer sorrir em qualquer momento. Aos meus avós maternos, Ryoko Takebe e Choituiro Takebe¸ agradeço por me acolherem como filha e sempre me proporcionarem um ambiente repleto de amor e carinho. À minha avó, Ryoko Takebe, agradeço especialmente por tudo que sempre fez e faz por mim, por não medir esforços em prol de minha felicidade e de meus sonhos. Sou imensamente grata por ter o privilégio de conviver com a senhora e por todos nossos momentos juntas. Você é um exemplo para mim de integridade e caráter imensuráveis. 4 Aos meus avós paternos, Maria Kimio Ivanaga e José Carlos Ivanaga, agradeço pela família maravilhosa que formamos e por nossa união. À minha tia, Marcela Yuri Ivanaga e Eliane Takebe, e a meu tio, Marcelo Takebe, agradeço por serem companheiros tão leais a mim. A conexão que temos é inabalável e sou muito grata pela nossa amizade. À minha irmã, Isadora Peres Ivanaga, agradeço por todos nossos momentos, pelo amor que me proporciona e por me permitir voltar a ser criança e sorrir alegremente quando estamos juntas. Minha pequena, saiba que tenho um amor incondicional por você e nunca medirei esforços para continuarmos sempre unidas. Ao meu namorado, Felipe Ferreira Passagem, agradeço pelo companheiro inigualável que é para mim. Saiba que sou grata por nossa amizade e por todos os momentos que juntos compartilhamos desde a faculdade. Ao meu padrasto, Fabio Vilarim Carleial, agradeço pelo companheirismo, amizade e disposição em sempre me ajudar a todos os momentos. Sou grata pela família que você, minha mãe e eu formamos. 5 A todos vocês, expresso meu mais profundo amor e agradeço por sempre se fazerem presentes em minha vida. Vocês são minha base e meus maiores exemplos de vida. Amo muito vocês! 6 AGRADECIMENTOS À minha orientadora, Profa. Leticia Helena Theodoro, agradeço pela orientação, paciência e todo suporte à condução do meu projeto de pesquisa. Sou muito grata por todo conhecimento clínico e teórico que adquiri com a senhora ao longo dessa jornada, além do seu apoio que foi fundamental nesse processo. Ao meu co-orientador, Prof. Valdir Gouveia Garcia, agradeço por contribuir de forma tão expressiva com a elaboração deste trabalho e enriquecê-lo com seu vasto conhecimento e experiência profissional. À aluna, Beatriz Goulart, agradeço pelo auxílio e disposição em contribuir com todas as etapas clínicas durante a execução deste trabalho. Sua colaboração foi de fundamental importância nesse processo. À doutoranda, Marta Aparecida Alberton Nuernberg, agradeço primeiramente pela amizade. Sou muito grata por todo auxílio a mim dispensado, por sua disposição em ajudar e ensinar, sempre de forma tão humilde e paciente. Você é um exemplo de profissionalismo, competência e integridade. À doutoranda, Daniela Maria Janjacomo Miessi, agradeço pelo atendimento clínico prestado aos pacientes deste estudo e por contribuir de forma tão expressiva com a execução deste trabalho. Aos meus queridos colegas do curso de mestrado do programa de Pós- graduação em odontologia, Natália Campos, Natália Januário, Breno, Guilherme, Henrique, João, David, Marina e Fred, agradeço pela amizade e companheirismo que tornaram essa jornada mais leve e prazeirosa. 7 Ao Professor Juliano Almeida Milanezi, agradeço por compartilhar sua vasta experiência clínica e teórica. A possibilidade de ter sido orientada pelo senhor durante a clínica de pós-graduação foi uma experiência incrível que aumentou ainda mais minha admiração pela área de Periodontia. À Professora Maria José Hitomi Nagata, agradeço por todo conhecimento que adquiri com a senhora durante o curso de mestrado. A senhora é uma inspiração para mim. Agradeço a todos os Professores e funcionários da Faculdade de Odontologia de Araçatuba – UNESP pela receptividade. Agradeço à Faculdade de Odontologia de Araçatuba – UNESP pela oportunidade de realizar meu mestrado e por possibilitar a realização desta pesquisa. 8 “The pessimist sees difficulty in every opportunity. The optimist sees opportunity in every difficulty.” Winston Churchill 9 Ivanaga, CA. Efeitos fotodinâmicos da Curcumina no tratamento de bolsas residuais de pacientes portadores de periodontite crônica e Diabetes Mellitus tipo 2: estudo de boca dividida randomizado. [Dissertação] – Universidade Estadual Paulista (Unesp), Faculdade de Odontologia, Araçatuba, 2019. RESUMO Introdução: A presença de bolsas residuais representa um fator de risco preditor de progressão da doença periodontal. A presença de Diabetes Mellitus (DM) aumenta a prevalência de periodontite e influencia negativamente na capacidade de reparo tecidual. O objetivo do estudo foi avaliar a eficácia clínica da terapia fotodinâmica antimicrobiana (aPDT) com curcumina e LED, como terapia coadjuvante à raspagem e alisamento radicular (RAR), no tratamento de bolsas residuais de pacientes com DM tipo 2. Métodos: Para este estudo clínico controlado randomizado de boca dividida, vinte e cinco pacientes foram selecionados. Em cada paciente, todas as bolsas residuais com profundidade de sondagem (PS) ≥5 mm e sangramento à sondagem (SS), por quadrante, foram aleatoriamente alocados para receber: 1) RAR (grupo RAR); 2) RAR e irrigação com solução de curcumina (grupo CUR); 3) RAR e irradiação com LED (grupo LED); 4) RAR e terapia fotodinâmica antimicrobiana (grupo aPDT). Para a aPDT, utilizou-se solução de curcumina (100 mg/L) seguida de irradiação com LED (InGaN, 465 - 485 nm, 100 mW/cm2, 60 segundos). Os parâmetros clínicos de PS, recessão gengival (RG), nível de inserção clínica (NIC), SS e índice de placa visível (IP) foram avaliados no início (baseline), 3 e 6 meses após os tratamentos. Resultados: Na comparação intergrupo, não houve diferença estatisticamente significante nos valores médios dos parâmetros clínicos avaliados (PS, RG, NIC, SS e IP) no início do estudo (baseline), aos 3 e 6 meses (p > 0,05). De forma semelhante, não houve diferença na média de redução de PS e ganho de NIC aos 3 e 6 meses comparados ao início (baseline) (p > 0,05). A análise intragrupo revelou que em todos os grupos de tratamento houve redução da PS e SS aos 3 e 6 meses (p < 0,05). Todos os grupos demonstram redução do IP, mas no grupo LED só foi estatisticamente significante aos 6 meses (p < 0,05). Nenhum grupo apresentou diferença na RG nos períodos avaliados (p > 0,05). Apenas nos grupos aPDT e LED houve melhora significativa do NIC 10 aos 3 meses (aPDT 4,95  2,33; LED 4,41  1,98) em comparação aos dados iniciais (aPDT 6,71  1,85; LED 6,85  1,61) (p < 0,05). Conclusão: aPDT ou irradiação com LED, como coadjuvantes à RAR, promoveram benefícios clínicos a curto prazo no tratamento de bolsas residuais de pacientes portadores de diabetes tipo 2. Apesar disso, os benefícios clínicos poderiam ser relacionados apenas ao efeito fotobiomodulador após irradiação tecidual com LED. Palavras-chave: Periodontite. Diabetes Mellitus. Raspagem Dentária. Fotoquimioterapia. Curcumina. 11 Ivanaga, CA. Photodynamic effects of Curcumin in the treatment of residual pockets in patients with chronic periodontitis and type 2 Diabetes Mellitus: a randomized and controlled split mouth clinical trial. [Dissertation] – São Paulo State University (Unesp), School of Dentistry, Araçatuba, 2019. ABSTRACT Introduction: Residual pockets represent a risk factor for periodontal disease progression, which is exacerbated by Diabetes Mellitus (DM) by increasing the prevalence of periodontal disease and negatively influencing healing capacity. The present study aimed to evaluate the clinical efficacy of antimicrobial photodynamic therapy (aPDT) with curcumin and LED, as an adjunctive therapy to scaling and root planing (SRP), in the treatment of residual pockets in patients with type 2 DM. Methods: A randomized and controlled split-mouth clinical trial was conducted with twenty-five patients. In each patient, all residual pockets with probing depth (PD) ≥5 mm and bleeding on probing (BOP), per quadrant, were randomly allocated to receive: 1) SRP (SRP group); 2) SRP and irrigation with curcumin solution (CUR group); 3) SRP and LED irradiation (LED group); 4) SRP and aPDT (aPDT group). The aPDT was performed with curcumin solution (100 mg/L) followed by LED irradiation (InGaN, 465 - 485 nm, 100 mW/cm2, 60 seconds). Clinical parameters of PD, gingival recession (GR), clinical attachment level (CAL), BOP and visible plaque index (PI) were evaluated at baseline, 3 and 6 months post-therapies. Results: In an intergroup comparison, the mean values for PD, GR, CAL, BOP and PI were not different at baseline, 3 and 6 months (p > 0.05). Similarly, the mean difference in the reduction of PD and CAL gain between baseline and 3 or 6 months were not statistically different (p > 0.05). The intragroup comparison showed reduction in PD and BOP in all treatment groups at 3 and 6 months (p < 0.05). All treatment groups showed reduction in PI, but in the LED group the difference was statistically significant only at 6 months (p < 0.05). Mean GR did not differ in any intervention group throughout the study (p > 0.05). Only aPDT and LED groups showed CAL gain at 3 months (aPDT 4.95  2.33, LED 4.41  1.98) in comparison to baseline data (aPDT 6.71  1.85, LED 6.85  1.61) (p < 0.05). 12 Conclusion: aPDT or LED irradiation, as adjunctive therapies to SRP, may yield short- term clinical benefits in the treatment of residual pockets in patients with type 2 diabetes. However, the clinical improvements may be related to the photobiomodulatory effects of LED irradiation. Keywords: Periodontitis. Diabetes Mellitus. Periodontal debridement. Photochemotherapy. Curcumin. 13 LISTA DE FIGURAS Figure 1 Flowchart of the study design. 44 14 LISTA DE TABELAS Table 1 Subject characteristics at baseline: number of patients, test sites, age and HbA1c. 45 Table 2 Clinical outcomes at baseline, 3 and 6 months. 46 Table 3 Data (mean difference and standard deviation) for reduction in PD and CAL gain between baseline and 3 months, and baseline and 6 months post-treatment, in mm. 47 15 LISTA DE ANEXOS Appendix A Certificado do Comitê de Ética em Pesquisa. 48 Appendix B Guide for authors (Manuscript submission - “Photodiagnosis and Photodynamic Therapy”). 51 Appendix C CONSORT 2010 checklist. 63 16 LISTA DE ABREVIATURAS E SIGLAS AGEs Advanced glycation end-products ANOVA Analysis of variance aPDT Antimicrobial photodynamic therapy BOP Bleeding on probing CAL Clinical attachment level COX-2 Cyclooxygenase-2 CUR Curcumin DM Diabetes Mellitus DMSO Dimethyl sulfoxide GR Gingival recession HbA1c Glycated hemoglobin ICC Intraclass correlation coefficient IL-1 Interleukin-1 InGaN Gallium and indium nitride J/cm² Joules per square centimeter LED Light emitting diode mg/day Milligram per day mg/g Milligram per gram mg/L Milligram per liter mm Millimeters mW/cm² Milliwatts per square centimeter NF-B Nuclear-B factor nm Nanometers OPG Osteoprotegerin PBM Photobiomodulation PD Probing depth PI Visible plaque index RAGE Receptor for advanced glycation end-products RANKL Receptor activator of nuclear factor kappa-B ligand 17 RCT Randomized controlled clinical trial ROS Reactive oxygen species SPT Supportive periodontal therapy SRP Scaling and root planing TNF- Tumor necrosis factor- 18 anuscrito para ublicação 1 1 Normalização Segundo a Revista “Photodiagnosis and Photodynamic Therapy”. 19 SUMMARY 1. Introduction ................................................................................................................. 22 2. Material and Methods ................................................................................................. 25 2.1 Study design ............................................................................................................. 25 2.2 Sample size calculation ............................................................................................ 25 2.3 Study population ....................................................................................................... 25 2.4 Treatment protocol ................................................................................................... 26 2.5 Oral hygiene program ............................................................................................... 27 2.6 Clinical parameters .................................................................................................. 28 2.7 Intra-examiner calibration ........................................................................................ 28 2.8 Statistical analysis ................................................................................................... 28 3. Results ....................................................................................................................... 29 3.1 Adverse effects ........................................................................................................ 29 3.2 Clinical outcomes ……………………………………….….……………………………. 29 4. Discussion ................................................................................................................. 30 5. Conclusion ................................................................................................................. 36 References ................................................................................................................... 37 Appendix ....................................................................................................................... 48 20 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” Campus de Araçatuba Title Photodynamic effects of Curcumin in the treatment of residual pockets in patients with chronic periodontitis and type 2 Diabetes Mellitus: a randomized and controlled split- mouth clinical trial. Authors Camila Ayumi Ivanagaa, Valdir Gouveia Garciab, Leticia Helena Theodoroc* a Department of Surgery and Integrated Clinic, Division of Periodontology; São Paulo State University (Unesp), School of Dentistry; Araçatuba, São Paulo, Brazil. b Full Professor, Department of Surgery and Integrated Clinic, Division of Periodontology; São Paulo State University (Unesp), School of Dentistry; Araçatuba, São Paulo, Brazil. c Adjunct Professor, Department of Surgery and Integrated Clinic, Division of Periodontology; São Paulo State University (Unesp), School of Dentistry; Araçatuba, São Paulo, Brazil. *Corresponding author Letícia Helena Theodoro José Bonifácio Street, 1193 Araçatuba, São Paulo, Brazil Zip Code 16015-050 Phone number: +55 (18) 3636-2860 Email: letheodoro@foa.unesp.br 21 Highlights  aPDT and LED irradiation promoted CAL gain at 3 months evaluation.  The photobiomodulation effect may be responsible for the clinical improvements.  Only LED irradiation may yield faster healing in compromised individuals. 22 1. Introduction Diabetes Mellitus (DM) is the most prevalent chronic metabolic disorder characterized by higher than normal blood glucose levels due to deficient management of insulin by the organism. The state of chronic hyperglycemia leads to increased levels of advanced glycation end-products (AGEs). AGEs act directly on cells, causing proinflammatory effects and oxidative stress [1]. On the other hand, AGEs may interact with its receptor, named receptor for advanced glycation end-products (RAGE), present on different cell surfaces, altering cell function. This interaction increases proinflammatory cytokine levels, interfering with tissue repair through reduced bone turnover and collagen synthesis [2, 3]. A correlation between DM and periodontal disease is evidenced by the literature [1, 2, 3, 4], supporting a risk up to 3 folds for individuals with diabetes to develop periodontitis [2], and an increased prevalence and severity of periodontal disease for those with poor glycaemic control [2, 3]. Periodontitis refers to a multifactorial inflammatory disease [5] associated with dysbiotic biofilms [6]. Periodontal tissue destruction is mainly related to an inappropriate host immune-inflammatory response [4, 6], influenced by genetic, epigenetic and environmental factors, such as tobacco, alcohol consumption and diabetes [6]. The conventional mechanical debridement through scaling and root planing (SRP) is an effective approach to treat periodontal disease [7, 8]. In fact, limitations inherent to the technique may fail to eliminate microorganisms from anatomical structures or soft tissue, which may act as reservoirs of periodontal pathogens, enabling the re- colonization of previously treated sites [9]. Residual pockets represent a risk factor for the progression of periodontitis, especially sites with PD 6 mm after initial therapy [10, 11] or multiple sites with PD 5 mm [10]. Therefore, supportive periodontal therapy (SPT) helps prevent disease recurrence and early identification of diseased sites, reducing the probability of tooth loss [12]. SRP for treatment of residual pockets demonstrates feasibility preserving clinical attachment level (CAL) [7], although the literature suggests that no positive predictable results may be expected by repeating the treatment [13, 14], and the effectiveness of SRP substantially decreases in sites with probing depth (PD) 5 mm [8]. 23 Moreover, considering that the state of chronic hyperglycemia impairs tissue repair in patients with diabetes, studies have evaluated adjunctive therapies to SRP, including antimicrobial photodynamic therapy (aPDT) [15, 16, 17, 18, 19]. The aPDT has lethal effects on microorganisms through the damage caused by reactive oxygen species (ROS) (type I reaction) or by singlet oxygen (type II reaction) [20]. The main advantages of aPDT include its broad spectrum of action (bacteria, fungi and protozoa) with minimal effects to the host tissue, and absence of selection of photoresistant strains even after repeated applications [21]. Natural substances with biological properties have been evaluated, and clinical studies appointed to the therapeutic use of curcumin (CUR) as a photosensitizer in aPDT [22, 23, 24], in the form of solution for subgingival irrigation [25] or as gel for local application [26]. CUR is derived from the rhizome of Curcuma longa, commonly known as turmeric, and exhibits antioxidant, anti-inflammatory and anticancer effects [27], antimicrobial property. There is evidence that CUR effects are potentiated in the presence of light, and the phototoxicity is related to the free radicals and ROS produced, with restricted local effects even at low concentrations (≤5 µM) [28]. CUR has a broad spectrum of light absorption that ranges from 300-500 nanometers (nm) (maximum absorption 430- 435 nm). In vitro studies have demonstrated the biocompatibility of CUR through the absence of cytotoxic effects on fibroblasts [29], capacity to reduce the viability of the periodontal pathogen Aggregatibacter Actinomycetencomitans, with strengthened effects when associated with a Light Emitting Diode (LED) [30, 31]. In addition, an in vivo study reported that aPDT with CUR and LED, as a monotherapy, was effective in controlling alveolar bone loss and reducing the expression of RANKL (receptor activator of nuclear factor kappa-B ligand) in rats with induced periodontitis [32]. Few studies have evaluated CUR as a photosensitizer in aPDT for the treatment of periodontitis [22]. Therefore, this study aimed to investigate the photodynamic effects of CUR associated with LED as a light source, and as an adjunctive therapy to SRP in the treatment of residual pockets in patients with type 2 DM under SPT. The hypothesis of the study is that the association of aPDT with CUR and LED promotes a significant clinical improvement compared with 24 conventional mechanical debridement, for treatment of residual pockets in patients with type 2 DM. 25 2. Material and Methods 2.1 Study design The study was designed as a split-mouth, blinded-examiner, randomized and controlled clinical trial (RCT). This clinical trial was approved by the local Ethics Committee of the São Paulo State University (Unesp), School of Dentistry, Araçatuba (CAAE: 69463517.8.0000.5420), registered at the “International Clinical Trials Registry Platform – UTN” (Protocol UTN U1111-1205-0218) and in the Brazilian platform for clinical trials “Registro Brasileiro de Ensaios Clínicos – REBEC” (RBR-4tq9yq). The study was conducted according to the Consort Statement [33] for clinical trials. 2.2 Sample size calculation The sample size was calculated at 90% power to detect a significant difference of 1 mm on CAL among groups, the primary outcome variable of the study, considering a 5% significance level and 1 mm standard deviation. A minimum sample of 21 patients would be required. However, considering the possibility of patient loss to follow-up, a total of 25 patients were included in the study [34]. 2.3 Study population Twenty-five patients with medical diagnosis of type 2 DM (glycated hemoglobin (HbA1c) ≥ 6.5%), exhibiting chronic periodontitis and under SPT were recruited from patients referred to the São Paulo State University (Unesp), School of Dentistry of Araçatuba (SP, Brazil). Initially, a detailed anamnesis was performed, and patients were informed about the potential benefits and risks of their participation in the study. Blood tests were requested to confirm HbA1c level and to evaluate fasting blood glucose. Patients were considered eligible if they met the following criteria: 1) age range 30-70 years [35]; 2) medical diagnosis of type 2 DM (HbA1c ≥ 6.5%) [36]; 3) history of chronic periodontitis treated in the previous 3 to 6 months after cause-related therapy; 4) at least one residual pocket per quadrant with PD ≥5 mm, bleeding on probing (BOP) and CAL ≥3 mm; 5) at least 15 teeth, excluding third molars [37]. The exclusion criteria included [38]: 1) current smokers or regular smoking 12 months prior to participation in the study; 2) patients with anemia; 3) active cancer; 4) use of antibiotics within the 26 previous 6 months; 5) use of anti-inflammatory drugs within the previous 6 months; 7) pregnancy; 8) patients undergoing orthodontic treatment. Informed consent was obtained from all participants. 2.4 Treatment protocol The participants were submitted to clinical examination performed by a blinded examiner (CAI). Individuals presenting at least one residual pocket (PD ≥5 mm and BOP) per quadrant were selected. The experimental sites from each quadrant were randomly assigned to receive SRP (SRP group), irrigation with CUR solution (CUR group), LED irradiation (LED group) or aPDT with CUR and LED (aPDT group). In the pre-study phase, the professional responsible for the patients’ treatment (DMJM) conducted the randomization procedure. Each treatment group was randomly assigned as group A, B, C or D, and then allocated to the four quadrants by an online randomization system (www.sealedenvelope.com). The twenty-five generated combinations were maintained in opaque sealed envelopes with no identification. According to the combination of the envelope, each quadrant randomly received the following treatments: SRP group, a single session of SRP [35, 39] was performed using an ultrasonic device and periodontal curettes (Gracey Curettes, Hu-Friedy Co., Chicago, IL), and irrigation with 1 mL of saline solution; CUR group, a single session of SRP was performed using an ultrasonic device and periodontal curettes, and irrigation with 1 mL of CUR solution 100 mg/L for 1 minute; LED group, a single session of SRP was performed using an ultrasonic device and periodontal curettes, irrigation with 1 mL of saline solution and LED irradiation; aPDT group, a single session of SRP was performed using an ultrasonic device and periodontal curettes, irrigation with 1 mL of CUR solution 100 mg/L and LED irradiation after 1 minute. The 100 mg/L CUR solution was obtained from the solubilization of CUR (Curcuma longa, 4-Hydroxy-3-methoxyphenyl1,6-heptadiene3,5dione, Diferuloylmethane,Diferulylmethane C1386 Sigma Aldrich, MO, USA) in 99.9% of absolute ethanol and 0.1% of dimethyl sulfoxide (DMSO). A solution with 0.15% was used to obtain the final solution with a concentration of 100 mg/L in distilled water [24] 27 (Apothicário Manipulation Pharmacy, Araçatuba, SP, Brazil). The experimental sites in the aPDT group were irrigated with 1 mL of CUR solution using a syringe and an insulin needle (13 X 0.45 mm) (Becton Dickson Ind. Ltda., Curitiba, PR, Brasil). After 1 minute, the LED tip was positioned perpendicular to the long axis of the tooth on the buccal or lingual face, depending on the site location, for 60 seconds. The irradiation was performed with a gallium and indium nitride LED (InGaN; Kon-lux Kondortech Dental Equipments Ltd., São Carlos, SP, Brazil) at a wavelength ranging from 465-485 nm and power density of 600 mW/cm². The LED tip used has a spot size of 0.78 cm², power density of 100 mW/cm² measured by a power meter (Power meter Demetron Research Corp. Danbury, CT, EUA) for 60 seconds, with a total energy density of 7.69 J /cm². Initially, all sites received SRP under local anesthesia and only after that, a combination with the treatment groups was revealed. All clinical procedures were performed by a single operator, who is a specialist in Periodontics (DMJM). Patients were instructed not to discuss with the examiner about the treatments received. The randomization code was not broken until all data were collected and tabulated by the examiner (CAI). 2.5 Oral hygiene program The participants were informed about the etiology of periodontal disease and instructed regarding oral hygiene. The baseline clinical evaluation was performed 15 days after this procedure. After the treatment of residual pockets, all subjects were recruited at 30 days for clinical evaluation to detect any alterations such as periodontal abscess, erythema, edema, pruritus, sensitivity or increase in tooth mobility, which may be related to the therapy. In addition, all patients were engaged in an oral hygiene program monthly up to 180 days post-treatment, for reinforcement of oral hygiene and professional prophylaxis with rubber cup and prophylactic paste [40]. 28 2.6 Clinical parameters The following clinical parameters were evaluated at site level: visible plaque index (PI) [41], PD, BOP, gingival recession (GR) and CAL [42]. GR was measured from the cemento-enamel junction to the gingival margin and BOP was classified as present, if bleeding was detected during the 30 seconds after probing. The clinical parameters were measured using a UNC 15 periodontal probe (PCPUNC-15, Hu-Friedy, Chicago, IL, USA). A single examiner (CAI), blinded to the therapies, assessed the clinical parameters at baseline and at 3 and 6 months posttreatment. 2.7 Intra-examiner calibration In the pre-study phase, 2 non-study individuals were selected for intra- examiner calibration, and 170 sites were evaluated. Duplicate measurements of PD and CAL were assessed within 1 week. The intra-rater agreement for PD and CAL variables were obtained by intraclass correlation coefficient (ICC). The calibration was considered satisfactory for PD (0.8528) and CAL (0.859). 2.8 Statistical analysis The primary outcome variable was the mean CAL value. Average and standard deviation values for the clinical parameters of PD, GR and CAL were obtained to compare treatment protocols and evaluation periods. Data of PI and BOP were transformed into percentages [40], considered at site level. Statistical analysis was performed with the software BioEstat 5.3 (BioEstat 5.3, BioEstat Software, Manaus, AM, Brazil), considering a 5% significance level. The initial data analysis was performed to evaluate if a parametric or non- parametric distribution was reached (Shapiro-Wilk test). All variables were submitted to intergroup comparison by Kruskal-Wallis test. The intragroup comparisons at different evaluation periods were performed by Analysis of variance (ANOVA) using a mixed-model approach for variables that reached a normal distribution, followed by Tukey’s test. Data presenting non-normal distribution were analyzed by Friedman’s test in intragroup comparison. 29 3. Results A total of 25 patients were included in the study, including 16 male and 7 female patients. Two patients were excluded during follow-up: one did not complete the 90-day evaluation (female) and the other one was excluded from the 180-day evaluation (male), both related to antibiotic therapy for systemic impairment. Data from these patients were excluded from the statistical analysis. Patient recruitment started in May 2017 and was completed by the end of March 2018. The patient recruitment process is described in figure 1. Treatment modalities were performed in a total of 332 sites, but only 290 sites were considered for final evaluation. Table 1 presents characteristics of subjects at baseline, number of sites treated, mean age and HbA1c level. 3.1 Adverse effects Patients presented no adverse effects related to the therapy, nor pain or discomfort after treatment procedures. 3.2 Clinical outcomes In the intergroup comparison, no significant differences (p > 0.05) were identified in the clinical parameters evaluated (PD, GR, CAL, PI and BOP) during all study periods. The mean difference in the reduction of PD and CAL gain were not statistically significant in the intergroup comparison between baseline and 3 months (PD: p =0.79; CAL: p =0.31), and baseline and 6 months (PD: p =0.82; CAL: p =0.77). The intragroup comparison revealed a reduction in PD and BOP in all treatment groups at 3 and 6 months compared to baseline (p < 0.05). No differences in GR were observed in any group throughout the study (p > 0.05). A significant difference in CAL was detected only in the aPDT and LED groups at 3 months (aPDT=4.95  2.33 mm; LED=4.41  1.98 mm) compared with baseline (aPDT=6.71  1.85 mm; LED=6.85  1.61) (p < 0.05). Analysis of PI in aPDT, CUR and SRP groups revealed a reduction from baseline to 3 and 6 months (p < 0.05), but in the LED group this difference was significant only at 6 months compared to baseline (p < 0.05). Clinical outcomes at baseline, 3 and 6 months are presented in table 2. Data of the mean difference in PD reduction and CAL gain are presented in table 3. 30 4. Discussion The current clinical investigation revealed that all treatment modalities were effective in reducing the mean PD and BOP in residual pockets at 3 and 6 months. The parameter of PI reduced from baseline to 3 and 6 months in all treatment groups, but a statistically significant difference was not identified in the LED group at 3 months follow- up. Specifically, the association of aPDT to SRP promoted a significant CAL gain at 3 months, and similar clinical results were observed in the LED group. Thus, a single session of aPDT with CUR and LED or LED irradiation as adjunctive therapies may yield short-term (3 months) clinical benefits in the treatment of residual pockets in patients with type 2 DM. According to split-mouth clinical trials in normoglycemic individuals, the adjunctive treatment of residual pockets with a single session of aPDT also demonstrated similar clinical results as SRP performed alone, evidenced by PD reduction and CAL gain at the 3-month evaluation period [43, 44, 45]. Nevertheless, two of these studies considered only single-rooted teeth as study sites [44, 45] and one reinstituted subgingival debridement at 3 and 6 months follow-up [43]. Goh et al., 2017 reported no significant differences between conventional debridement or association with aPDT, with significant improvement in mean PD reduction and CAL gain at 6 months of evaluation [43]. However, these authors noticed that the adjunctive therapy provided a faster resolution at 3 months post-therapy, which may be beneficial for patients with an impaired tissue repair [43]. It is well established that diabetes is an important modifying factor of periodontitis [46]. It is pertinent to mention that in the present study the mean value of HbA1c was 8.73%  1.82, which categorizes patients as patients with decompensated diabetes. Individuals with DM have a deficient healing capacity, mainly related to the increased levels of AGEs, as an effect of chronic hyperglycemia. The impairment in tissue repair may be associated with the imbalance of RANKL/ OPG (osteoprotegerin) ratio, which increases bone destruction [1], in association with a decrease in osteoblast differentiation [3, 47]. In addition, the collagen turnover is altered, exhibiting lower collagen synthesis by fibroblasts [2, 3], and increased susceptibility to degradation by matrix metalloproteinases [3]. 31 In this context, few studies have evaluated the clinical efficacy of aPDT as an adjunctive therapy to SRP, in the nonsurgical treatment of chronic periodontitis in patients with type 2 DM [15, 16, 17, 18, 19]. Overall, the studies did not evidence additional benefits in the parameters of PD and CAL, when a single session of aPDT was performed [15, 16, 17, 18]. However, differences in the study protocols must be taken into account. In the study of Castro Dos Santos et al., 2016, the similar results at 6 months evaluation following ultrasonic debridement or association with aPDT, may be related to the location of the study sites in single rooted teeth, which may have favorable clinical outcomes only with mechanical debridement [17]. Similarly, the results of Macedo et al., 2014 at 3 months follow-up, possibly have been influenced by the concomitant antibiotic therapy instituted for both treatment groups (SRP X SRP+aPDT), with systemic 100 mg/day doxycycline for 2 weeks, after initial dose of 200 mg [18]. Regarding these clinical trials in individuals with DM, those who compared systemic doxycycline (100 mg/day) versus aPDT as adjunctive therapies to SRP, identified that both treatments were effective and improved the clinical parameters of PD and CAL at 3 months [16, 19]. Al-Zahrani et al., 2009, in addition to the comparison of systemic doxycycline or aPDT as adjunctive therapies, also evaluated SRP as a monotherapy, and did not evidence differences between treatment modalities [16]. According to Ramos et al., 2016, although both systemic doxycycline or multiple sessions of aPDT (0, 3, 7 and 14 days) effectively reduced PD, GR and CAL (p < 0.05) and aPDT therapy reduced moderate pockets (5-6 mm) in single rooted teeth at 3 months, the authors suggested that adjunctive aPDT may be an alternative to systemic antibiotics [19]. Thus, considering the comparable outcomes with systemic antibiotic therapy and aPDT, this approach may be feasible to avoid the indiscriminate use of antibiotics, mainly related to the public concern on selection of resistant bacterial strains. It is estimated that a non-surgical treatment for periodontitis results in mean BOP reduction of 45% from baseline level, mean PD reduction of 1.29 mm and CAL gain of 0.55 mm in moderate pockets (PD 4-6 mm) [8]. On the other hand, mean PD reduction of 2.16 mm and 1.19 mm CAL gain are expected in deep pockets (PD 7 mm), with remarkable results at 1 to 3 months post-therapy, according to the review by Cobb, 2003 [8]. However, in the present study, no distinction was made between moderate and deep 32 pockets, and the residual pockets were considered site-specific per quadrant. Approximately 73.57%  2.91 of the test sites corresponded to moderate pockets (PD 5- 6 mm), and only 26.11%  3.48 to deep pockets (PD 7 mm). Accordingly, data reported by this investigation regarding the mean reduction of PD at 3 months are comparable with the values estimated for moderate pockets, as reported by Cobb, 2003 [8] (aPDT 1.38  1.10; CUR 1.29  1.18; SRP 1.09  0.80; LED 1.31  0.93). In a similar manner, the percentage of BOP significantly reduced at 3 months (aPDT 42.60  44.23; CUR 37.03  39.38; SRP 48.26  38.53; LED 35.38  36.81) and 6 months (aPDT 34.99  40.33; CUR 37.33  36.06; SRP 30.64  34.50; LED 39.32  39.60), compared with baseline (100%). In contrast, different data for the mean difference in CAL gain were found in this study at 3 months (aPDT 1.76  1.29; CUR 1.25  1.34; SRP 1.09  1.19; LED 1.43  1.15). Nevertheless, only the aPDT and LED group promoted a significant CAL gain at 3 months compared with baseline. The antimicrobial property of aPDT is related to the photooxidation of biomolecules, such as lipids, proteins and nucleic acids, through type I or type II reaction. When the photosensitizer at a ground state is illuminated by a compatible light source, it becomes highly energized (triplet state) and two different reactions may occur. In type I reaction, the excited photosensitizer reacts with an organic molecule, and the free radicals species generated may interact with endogenous molecular oxygen to produce ROS (hydrogen peroxide, superoxide and hydroxyl radicals), that cause damage to the cell membrane. Type II reaction involves the direct interaction with molecular oxygen to produce singlet oxygen, which may interfere with several microbial structures [20]. Moreover, it is important to mention that the LED therapy alone may be beneficial to tissue repair as a consequence of its photobiomodulatory effect, associated with photon absorption by cells that trigger intracellular mechanisms that will lead to an increased cell proliferation and survival, and protein synthesis [48]. Furthermore, the anti-inflammatory properties are related to the reduction of edema, oxidative stress in cells, and level of pro- inflammatory cytokines [48]. In the present investigation, only aPDT and LED groups resulted in significant CAL gain, although restricted to the 3-month evaluation (aPDT=4.95  2.33 mm; LED=4.41  1.98 mm), in comparison to baseline (aPDT=6.71  1.85 mm; LED=6.85  33 1.61) (p < 0.05). Thus, it may be presumed that the similar outcomes are mainly related to the photobiomodulatory effects promoted by the LED light, than a real effect of the aPDT. It is known that the photobiomodulation (PBM) effects when using a low-level laser or LED may accelerate tissue repair in periodontal disease [48]. Therefore, the PBM by the LED may yield a faster tissue repair, which can be advantageous to individuals with DM. A recent systematic review evidenced that when low-level laser therapy is associated with SRP, a reduction in PD may be expected only in the short-term (1 to 2 months), considering the methodological weakness of the few clinical studies assessed [49]. Specifically, the clinical trial of Demirturk-Gocgun et al, 2016 in patients with type 2 DM, evidenced a significant reduction only in BOP from deep pockets following low-level laser therapy at 1, 2 and 7 days after SRP, but restricted to 1 month post-therapy [50]. It is well established that there is a “biphasic dose response”, mainly related to energy density (J/cm²) for PBM effects, characterized by opposite effects when the dose exceeds the optimal value [48]. An in vivo study reported that LED irradiation at a wavelength of 660 nm may lead to faster periodontal healing through a decrease in tissue inflammation, stimulating collagen synthesis and new bone apposition in rats with induced periodontitis [51]. Remarkable results were obtained with an energy density of 10 J/cm² [51]. In the present study, the LED (InGaN) was used as a light source at a wavelength ranging from 465-485 nm and total energy density of 7.69 J /cm². It is important to mention that several factors may interfere with the efficacy of aPDT, such as the photosensitizer and its concentration, pre-irradiation time, light source and irradiation parameters [20]. In this clinical trial, CUR was investigated as a photosensitizer in aPDT, resulting in significant CAL gain at 3 months, like the LED group. Additionally, a recent in vivo study demonstrated that the monotherapy with aPDT (CUR and LED), CUR irrigation and LED irradiation were effective in controlling alveolar bone loss in rats with induced periodontitis, but improved results were obtained in the aPDT group [32]. CUR is a lipophilic molecule that alters membrane permeabilization in a similar manner in gram-positive and gram-negative bacteria, which may explain its antimicrobial property [52]. The anti-inflammatory effect may be associated with the inhibition of the 34 nuclear-B factor pathway (NF-B) that is related to the expression of proinflammatory cytokines, such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1) [53] and cyclooxygenase-2 (COX-2) [54] in a dose-dependent manner. In the present investigation, CUR as an adjunctive therapy did not provide clinical advantages compared with SRP alone. To the best of our knowledge, only one clinical study assessed the efficacy of aPDT with CUR 10 mg/g and LED, as an adjunctive therapy to SRP in the treatment of sites with PD 5 mm, in individuals with chronic periodontitis [22]. Sreedhar et al., 2015 compared four treatment groups in a split-mouth design: 1) SRP; 2) SRP + CUR gel for 5 min; 3) SRP + aPDT at day 0; 4) SRP + aPDT at 0, 7 and 21 days. The authors reported that all treatment modalities were effective in reducing PI, sulcus bleeding index and CAL at 3 months follow-up, but multiple sessions of aPDT provided greater results, with a mean difference of 0.76 mm in CAL gain [22]. In contrast, the present study revealed that a single session of aPDT promoted a mean difference of 1.76  1.29 in CAL gain, in the same evaluation period. However, differences between study protocols must be considered. Sreedhar et al., 2015 also adopted the split-mouth design, but the treatment allocation was pre- established according to quadrants [22]. Differently, the treatment allocation in this study was performed after debridement of all quadrants, through combinations in opaque sealed envelopes with no identification. Moreover, Sreedhar et al., 2015 evaluated CUR gel (10 mg/g), 5 minutes of pre-irradiation, followed by LED irradiation with a wavelength of 470 nm and power density of 620 mW/cm² for 5 minutes [22]. In our study, a CUR solution (100 mg/L) was used with 1 minute of pre-irradiation, followed by LED irradiation (465- 485 nm) with power density of 100 mW/cm², for 60 seconds (total energy density of 7.69 J /cm²). To guarantee the validity of studies with split-mouth design, several requirements must be met, including treatment randomization, blinding of professionals, adequate statistical analysis and sample size calculation [55]. Potential problems related to the study design implicates the difficulty in patient recruitment presenting similar disease patterns among quadrants [56]. Considering the small sample size necessary for this type of study, losses to follow-up are more relevant and must be considered in sample 35 size calculation [55]. In this investigation, two patients were excluded from the statistical analysis related to antibiotic therapy for systemic impairment, but the sample calculation was performed considering potential loss to follow-up. Indeed, as the study aimed to evaluate residual pockets during SPT, mean PD, CAL, GR, BOP and PI were not statistically different between groups at baseline (p < 0.05), which may not limit the external validity of the results by restricting patient recruitment [56]. Moreover, the applicability of the split-mouth design is based on the principle that the influence of inter-subject characteristics are subtracted, which increases the power of the study [55, 56], although the therapies and their effects must be localized [55]. Considering the local effects of aPDT, mainly related to the short lifespan of singlet oxygen as a result of type II reaction, the cellular damage on bacteria, protozoa, viruses and fungi are restricted to the therapy site [20]. Accordingly, the applicability of the split-mouth design in this clinical trial was feasible to evaluate the local effects of aPDT that show low risk of carry-over effects, and to reduce inter-subject variance, considering the variability of the host immune response in diabetic individuals. Therefore, this split-mouth clinical trial revealed that aPDT using CUR as photosensitizer and LED as light source, or LED irradiation as adjunctive therapies to SRP were effective in reducing PD and promoting CAL gain in residual pockets of patients with DM, after 3 months. Although the interference of the level of HbA1c was not the object of the present study, the association of these conservative approaches to conventional debridement may yield an improved tissue response in patients with decompensated diabetes. In addition, these results indicate that the clinical advantages may be obtained only with LED irradiation through cellular and molecular PBM effects. The LED therapy features easy application and low cost, which may be relevant in patients with DM that exhibit healing capacity impairment. Moreover, reassessment visits occurred monthly and supragingival prophylaxis was performed, reinforcing the importance of periodic maintenance in patients with chronic periodontitis. 36 5. Conclusion The conventional mechanical debridement associated with a single session of aPDT (CUR 100 mg/L and LED) or LED irradiation, for the treatment of residual pockets in patients with type 2 DM, promoted significant CAL gain in the short-term (3 months). However, aPDT did not demonstrate clinical advantages over LED irradiation. It is assumed that the PBM effects through LED irradiation are responsible for the faster tissue repair observed in this study, and this conservative therapeutic approach may be important to improve the healing capacity in these patients. 37 References [1] J.J. TAYLOR, P.M. PRESHAW PM, E. 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KUMARI, et al., Bactericidal activity of curcumin I is associated with damaging of bacterial membrane, PLoS ONE. 10 (3) (2015) e0121313. [53] D. CHEN, M. NIE, M.W. FAN, Z. BIAN, Anti-inflammatory activity of curcumin in macrophages stimulated by lipopolysaccharides from Porphyromonas gingivalis, Pharmacology. 82 (4) (2008) 264-9. [54] P. HU, P. HUANG, M.W. CHEN, Curcumin attenuates cyclooxygenase-2 expression via inhibition of the NF-kB pathway in lipopolysaccharide-stimulated human gingival fibroblasts, Cell Biol Int. 37 (5) (2013) 443–8. [55] A.A. ANTCZAK-BOUCKOMS, J.F. TULLOCH, C.S. BERKEY. Split-mouth and cross-over designs in dental research, J Clin Periodontol. 17 (1990) 446-53. [56] E. LESAFFRE, B. PHILSTROM, I. NEEDLEMAN, H. WORTHINGTON, The design and analysis of split-mouth studies: what statisticians and clinicians should know, Stat Med. 28 (2009) 3470-82. 44 Figure 1 - Flowchart of the study design. Assessed for eligibility (n = 100) Patient enrollment (n = 25) Quadrant randomization aPDT group CUR group LED group SRP group 3 months follow up (n = 24) 6 months follow up (n = 23) Patient loss (n = 1) - Antibiotic therapy Patient loss (n = 1) - Antibiotic therapy Not meeting inclusion criteria (n= 75) 45 Table 1 - Subject characteristics at baseline: number of patients, test sites, age and HbA1c. Study patients (n) 25 Male (n) 17 Female (n) 8 Test sites (n) 332 Age (M  SD) 55.0  10.2 HbA1c (%) (M  SD) 8.73  1.82 n sample number; M  SD mean value and standard deviation; HbA1c glycated hemoglobin. 46 Table 2 - Clinical outcomes at baseline, 3 and 6 months. Groups Baseline (M  SD) 3 Months (M  SD) 6 Months (M  SD) PD (mm) aPDT CUR SRP LED 5.71  0.92 5.71  0.74 5.67  0.78 5.61  0.77 4.33  1.78* 4.41  1.07* 4.58  1.24* 4.29  1.19* 4.47  1.40* 4.68  1.22* 4.70  1.37* 4.55  1.33* GR (mm) aPDT CUR SRP LED 1.30  1.27 1.14  1.32 0.99  1.27 1.43  1.34 0.88  1.18 1.19  1.44 1.03  1.33 1.29  1.20 1.30  1.35 1.12  1.35 0.79  1.12 1.37  1.30 CAL (mm) aPDT CUR SRP LED 6.71  1.85 6.68  1.86 6.63  1.66 6.85  1.61 4.95  2.33* 5.42  2.26 5.54  2.19 4.41  1.98* 5.46  1.98 5.78  2.17 5.44  1.99 5.70  1.88 BOP (%) aPDT CUR SRP LED 100 100 100 100 42.60  44.23† 37.03  39.38† 48.26  38.53† 35.38  36.81† 34.99  40.33† 37.33  36.06† 30.64  34.50† 39.32  39.60† PI (%) aPDT CUR SRP LED 68.24  38.23 69.34  38.44 75.61  32.22 64.53  36.38 33.03  43.52† 31.57  37.82† 45.11  42.14† 42.49  44.16 29.78  41.18† 34.06  38.43† 38.75  40.22† 34.70  39.60† M  SD mean and standard deviation. * Significant intragroup difference from baseline by ANOVA test (p < 0.05). † Significant intragroup difference from baseline by Friedman test (p < 0.05). 47 Table 3 - Data (mean difference and standard deviation) for reduction in PD and CAL gain between baseline and 3 months, and baseline and 6 months post-treatment, in mm. Evaluation periods Groups P-value** aPDT* CUR* SRP* LED* PD 0 – 3 month (mm) 1.38  1.10 1.29  1.18 1.09  0.80 1.31  0.93 0.79 0 – 6 month (mm) 1.23  0.91 1.02  0.96 0.96  0.89 1.05  1.13 0.82 CAL 0 – 3 month (mm) 1.76  1.29 1.25  1.34 1.09  1.19 1.43  1.15 0.31 0 – 6 month (mm) 1.24  1.03 0.89  1.13 1.18  1.33 1.14  1.29 0.77 * Mean difference  standard deviation. ** p-value for aPDT vs CUR vs SRP vs LED by ANOVA test (p < 0.05). 48 APPENDIX Appendix A – Certificado do Comitê de Ética em Pesquisa. 49 50 51 Appendix B - Guide for authors (Manuscript submission - “Photodiagnosis and Photodynamic Therapy”). 52 EDITORIAL BOARD . Editor-in-Chief R. Allison, 21st Century Oncology, Greenville, North Carolina, USA Emeritus Editor K. Moghissi, Yorkshire Laser Centre, Goole, UK Associate Editors H. Barr, Gloucester, UK R. Boyle, Hull, UK K. Dixon, Goole, UK M. Eljamel, Lanark, UK L. Freitag, Hemer, Germany K. Furukawa, Tokyo, Japan R. Hamblin, Boston, Massachusetts, USA C. Hopper, Bromley, UK Z. Huang, Lakewood, Colorado, USA H. Kato, Tokyo, Japan T. Mang, Orchard Park, New York, USA H. Moseley, Dundee, UK L. Turnbull, Hull, UK H. Walt, Zurich, Switzerland H. Wolfson, Jacksonville, Florida, USA Editorial Board M. Adamek, Katowice, Poland A. Akopov, St. Petersburg, Russian Federation D. Allan, Manchester, UK E. Allan, Knutsford, UK P. Barber, Dundee, UK A. Batlle, Buenos Aires, Brazil K. Berg, Oslo, Norway G. Downie, Mount Pleasant, Texas, USA R Fekrazad, Tehran, Iran L. Gasparyan, Helsinki, Finland G. Gasser, Zurich, Switzerland P. Hillemanns, Hannover, Germany T. Horvath, Brno, Czech Republic S. Ibbotson, Dundee, UK P. Jichlinski, Lausanne, Switzerland A. Juzeniene, Oslo, Norway N. Kashef, Tehran, Iran H. Kostron, Innsbruck, Austria S. Lecleire, Rouen, France M. Leroy, Suresnes, France L. Li, Guangzhou, China A. Makela, Helsinki, Finland S. Mordon, Loos, France T. Nakamura, Tochigi, Japan D. Robinson, Rotterdam, Netherlands P Ross, Columbus, Ohio, USA A. Sieron, Bytom, Poland W. Stummer, Munster, Germany I.B. Tan, Amsterdam, Netherlands A. Thorpe, Leeds, UK R. Waidelich, Munich, Germany M. Wainwright, Liverpool, England, UK X. Wang, Shanghai, China A. Woong-Shick, Kangham, The Republic of Korea P. Ziolkowski, Wroclaw, Poland AUTHOR INFORMATION PACK 13 Jan 2019 www.elsevier.com/locate/pdpdt 2 53 GUIDE FOR AUTHORS . INTRODUCTION Scope Photodiagnosis and Photodynamic Therapy is an international journal for the dissemination of scientific knowledge and clinical developments of Photodiagnosis and Photodynamic Therapy in all medical specialities. The journal publishes original articles, review articles, case presentations, "how- to-do-it" articles, Letters to the Editor, short communications and relevant images with short descriptions. All submitted material is subject to a strict peer review process. Types of manuscript Research Papers should report original clinical studies or research not previously published or being considered for publication elsewhere. Work in Progress may also be submitted. See below for the standard layout. Submission of a manuscript to this journal gives the publisher the right to publish that paper if it is accepted. Manuscripts may be edited to improve clarity and expression. Review articles, including institutional reviews of recent developments are welcome, and will undergo peer review. Reviews should have an abstract of up to 250 words. Editorials Although most Editorials in the journal are commissioned, authors may contact the Editor-in-Chief to request submission of their own Editorial. Correspondence. Readers are encouraged to write about any topic that relates to photodiagnosis or photodynamic therapy, clinical, scientific, educational, social or economic. Letters should be no longer than 500 words and may include discussions on material previously printed in the Journal. Case report will be considered if formatted as a research letter with 2 figures maximum. Maximum length is up to 1000 words with up to 6 references and 2 tables or figures. There should be no Abstract and no headings. Short Communications should not exceed 1000 words and should consist of a background section (not to exceed 100 words), aims (not to exceed 50 words), methods (not to exceed 250 words), results (not to exceed 250 words) and conclusion (not to exceed 250 words). An abstract of 150-200 words should also be provided. The editorial team reserves the right to decide which tables/figures submitted are necessary. No abstract is necessary. Submission checklist You can use this list to carry out a final check of your submission before you send it to the journal for review. Please check the relevant section in this Guide for Authors for more details. Ensure that the following items are present: One author has been designated as the corresponding author with contact details: • E-mail address • Full postal address All necessary files have been uploaded: Manuscript: • Include keywords • All figures (include relevant captions) • All tables (including titles, description, footnotes) • Ensure all figure and table citations in the text match the files provided • Indicate clearly if color should be used for any figures in print Graphical Abstracts / Highlights files (where applicable) Supplemental files (where applicable) Further considerations • Manuscript has been 'spell checked' and 'grammar checked' • All references mentioned in the Reference List are cited in the text, and vice versa AUTHOR INFORMATION PACK 13 Jan 2019 www.elsevier.com/locate/pdpdt 3 54 55 Copyright Upon acceptance of an article, authors will be asked to complete a 'Journal Publishing Agreement' (see more information on this). 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Learn more about Elsevier's pricing policy: https://www.elsevier.com/openaccesspricing. Green open access Authors can share their research in a variety of different ways and Elsevier has a number of green open access options available. We recommend authors see our open access page for further information. Authors can also self-archive their manuscripts immediately and enable public access from their institution's repository after an embargo period. This is the version that has been accepted for publication and which typically includes author-incorporated changes suggested during submission, peer review and in editor-author communications. Embargo period: For subscription articles, an appropriate amount of time is needed for journals to deliver value to subscribing customers before an article becomes freely available to the public. This is the embargo period and it begins from the date the article is formally published online in its final and fully citable form. Find out more. This journal has an embargo period of 12 months. Elsevier Researcher Academy Researcher Academy is a free e-learning platform designed to support early and mid-career researchers throughout their research journey. The "Learn" environment at Researcher Academy offers several interactive modules, webinars, downloadable guides and resources to guide you through the process of writing for research and going through peer review. Feel free to use these free resources to improve your submission and navigate the publication process with ease. Language (usage and editing services) Please write your text in good English (American or British usage is accepted, but not a mixture of these). Authors who feel their English language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientific English may wish to use the English Language Editing service available from Elsevier's WebShop. Submission Our online submission system guides you stepwise through the process of entering your article details and uploading your files. The system converts your article files to a single PDF file used in the peer- review process. Editable files (e.g., Word, LaTeX) are required to typeset your article for final publication. All correspondence, including notification of the Editor's decision and requests for revision, is sent by e-mail. Referees Referees Please submit, with the manuscript, the names, addresses and e-mail addresses of three potential referees who are willing to review the article (please obtain confirmation from the referees before submitting your manuscript). Note that the editor retains the sole right to decide whether or not the suggested reviewers are used. Revised version of the manuscript On the basis of the comments of the referees and editors, Authors may be asked to revise their manuscript. 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Permitted reuse is defined by your choice of one of the following Creative Commons user licenses: Creative Commons Attribution-NonCommercial-NoDerivs (CC-BY-NC-ND): for non-commercial purposes, lets others distribute and copy the article, and to include in a collective work (such as an anthology), as long as they credit the author(s) and provided they do not alter or modify the article. Elsevier has established agreements with funding bodies. This ensures authors can comply with funding body open access requirements, including specific user licenses, such as CC-BY. Some authors may also be reimbursed for associated publication fees. https://www.elsevier.com/fundingbodies If you need to comply with your funding body policy you can apply for the CC-BY license after your manuscript is accepted for publication. To provide open access, this journal has a publication fee which needs to be met by the authors or their research funders for each article published open access. Your publication choice will have no effect on the peer review process or acceptance of submitted articles. The open access publication fee for this journal is $USD 2,500 excluding taxes. Learn more about Elsevier's pricing policy https://www.elsevier.com/openaccesspricing Layout of manuscript Divide the manuscript into the following sections: Title page, Structured Abstract, Key words (3-6), Introduction, Materials and Methods, Results, Discussion, Acknowledgments, References. The editors will consider the use of other sections if more suitable for certain manuscripts. AUTHOR INFORMATION PACK 13 Jan 2019 www.elsevier.com/locate/pdpdt 7 58 Essential title page information • Title. Concise and informative. Titles are often used in information-retrieval systems. Avoid abbreviations and formulae where possible. • Author names and affiliations. Please clearly indicate the given name(s) and family name(s) of each author and check that all names are accurately spelled. You can add your name between parentheses in your own script behind the English transliteration. Present the authors' affiliation addresses (where the actual work was done) below the names. Indicate all affiliations with a lower- case superscript letter immediately after the author's name and in front of the appropriate address. Provide the full postal address of each affiliation, including the country name and, if available, the e- mail address of each author. • Corresponding author. Clearly indicate who will handle correspondence at all stages of refereeing and publication, also post-publication. This responsibility includes answering any future queries about Methodology and Materials. Ensure that the e-mail address is given and that contact details are kept up to date by the corresponding author. • Present/permanent address. If an author has moved since the work described in the article was done, or was visiting at the time, a 'Present address' (or 'Permanent address') may be indicated as a footnote to that author's name. The address at which the author actually did the work must be retained as the main, affiliation address. Superscript Arabic numerals are used for such footnotes. The Structured Abstract, of no more than 250 words, should be written with particular care since this will be the only part of the article studied by some readers. The preferred subheadings are: Background, Methods, Results and Conclusions. The Introduction should be brief and set out the purposes for which the study has been performed along with relevant previous studies only where essential. The Materials and Methods should be sufficiently detailed so that readers and reviewers can understand precisely what has been done without studying the references directly. 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Data references This journal encourages you to cite underlying or relevant datasets in your manuscript by citing them in your text and including a data reference in your Reference List. Data references should include the following elements: author name(s), dataset title, data repository, version (where available), year, and global persistent identifier. Add [dataset] immediately before the reference so we can properly identify it as a data reference. The [dataset] identifier will not appear in your published article. Reference management software Most Elsevier journals have their reference template available in many of the most popular reference management software products. These include all products that support Citation Style Language styles, such as Mendeley and Zotero, as well as EndNote. Using the word processor plug-ins from these products, authors only need to select the appropriate journal template when preparing their article, after which citations and bibliographies will be automatically formatted in the journal's style. If no template is yet available for this journal, please follow the format of the sample references and citations as shown in this Guide. If you use reference management software, please ensure that you remove all field codes before submitting the electronic manuscript. More information on how to remove field codes. Users of Mendeley Desktop can easily install the reference style for this journal by clicking the following link: http://open.mendeley.com/use-citation-style/photodiagnosis-and-photodynamic-therapy When preparing your manuscript, you will then be able to select this style using the Mendeley plug- ins for Microsoft Word or LibreOffice. Reference formatting There are no strict requirements on reference formatting at submission. References can be in any style or format as long as the style is consistent. Where applicable, author(s) name(s), journal title/ book title, chapter title/article title, year of publication, volume number/book chapter and the article number or pagination must be present. Use of DOI is highly encouraged. The reference style used by the journal will be applied to the accepted article by Elsevier at the proof stage. Note that missing data will be highlighted at proof stage for the author to correct. If you do wish to format the references yourself they should be arranged according to the following examples: Reference style Text: Indicate references by number(s) in square brackets in line with the text. The actual authors can be referred to, but the reference number(s) must always be given. Example: '..... as demonstrated [3,6]. Barnaby and Jones [8] obtained a different result ....' List: Number the references (numbers in square brackets) in the list in the order in which they appear in the text. Examples: Reference to a journal publication: AUTHOR INFORMATION PACK 13 Jan 2019 www.elsevier.com/locate/pdpdt 10 61 62 63 Appendix C – CONSORT 2010 checklist. 64