ORIGINAL ARTICLE Antimicrobial photodynamic therapy minimizes the deleterious effect of nicotine in female rats with induced periodontitis Erivan Clementino Gualberto Jr.1 & Letícia Helena Theodoro1 & Mariellén Longo1 & Vivian Cristina Noronha Novaes1 & Maria José Hitomi Nagata1 & Edilson Ervolino2 & Valdir Gouveia Garcia1,3,4 Received: 8 April 2015 /Accepted: 13 October 2015 /Published online: 6 November 2015 # Springer-Verlag London 2015 Abstract The aim of this study was to compare the use of antimicrobial photodynamic therapy (aPDT) as an adjunct to scaling and root planing (SRP) in the treatment of experimen- tally induced periodontitis in female rats that were systemical- ly treated with or without nicotine. Female rats (n=180) were divided into two groups: vehicle administration (Veh) and nicotine administration (Nic). Mini-pumps containing either vehicle or nicotine were implanted in the rats 30 days before the induction of experimental periodontitis (EP). EP was in- duced by placing a cotton ligature around the left mandibular first molar. After 7 days, the ligature was removed, and the rats were randomly divided into three treatment subgroups: SRP (only SRP), DL (SRP plus diode laser), and aPDT (SRP plus aPDT). The aPDT consisted of phenothiazine photosensitizer deposition followed by diode laser irradiation. Ten rats from each subgroup were euthanized at 7, 15, and 30 days after treatment. Alveolar bone loss (ABL) in the furcation region was evaluated using histological, histometric, and immuno- histochemical analyses. The rats that were treated with nico- tine showed more ABL compared to those treated with vehicle. In both the Veh and Nic groups, SRP plus aPDT treatment resulted in reduced ABL, smaller numbers of both TRAP- and RANKL-positive cells, and higher numbers of PCNA-positive cells compared to SRP treatment alone. aPDT was an effective adjunctive therapy for the treatment of periodontitis in female rats regardless of whether they re- ceived nicotine. Keywords Alveolar bone loss . Lasers . Nicotine . Periodontitis . Photochemotherapy . RANK ligand Introduction Tobacco smoking is strongly associated with both an in- creased risk of inflammatory disease, such as periodontitis, and increased disease severity [1]. Many of the undesirable effects of tobacco have been attributed to nicotine [2]. Differences in the rate of nicotine metabolism between men and women have been suggested, with women metabolizing nicotine more rapidly than men. This difference explains women’s increased predisposition to addiction and difficulty in quitting smoking [3, 4]. Among the reasons for this differ- ence in metabolism is the influence of estrogen on CYP2A6 [5], the main enzyme responsible for the conversion of nico- tine to its inactive metabolite, cotinine [6]. Additionally, in women, nicotine reduces estrogen levels and leads to the early onset of menopause [7, 8]. Nicotine exerts potent effects on alveolar bone tissue neoformation and resorption, processes that substantially af- fect the progression of periodontal disease and the host re- sponse to treatment. In cells of the osteoblast lineage, nicotine suppresses proliferation and differentiation and inhibits the expression of key angiogenic and osteogenic mediators in a dose-dependent manner [9, 10]. In contrast, in the cells of the * Valdir Gouveia Garcia vgouveia@foa.unesp.br 1 Group for the Research and Study of Laser in Dentistry, Department of Surgery and Integrated Clinic, Division of Periodontics, São Paulo State University (UNESP), Araçatuba, São Paulo, Brazil 2 Department of Basic Science, São Paulo State University (UNESP), Araçatuba, São Paulo, Brazil 3 Dental School of Barretos, University Center of the Educational Foundation of Barretos, Barretos, SP, Brazil 4 Faculdade de Odontologia de Araçatuba-UNESP, Rua José Bonifácio, 1193, 16015-050 Araçatuba, SP, Brazil Lasers Med Sci (2016) 31:83–94 DOI 10.1007/s10103-015-1820-8 http://crossmark.crossref.org/dialog/?doi=10.1007/s10103-015-1820-8&domain=pdf osteoclast lineage, nicotine also stimulates the production of osteoclast-like cells in a dose-dependent manner [11], espe- cially via upregulation of the macrophage colony-stimulating factor (M-CSF) [12]. Nicotine is also able to change another regulatory pathway related to osteoclastogenesis and osteo- clastic activity, the RANK/RANKL/OPG system. RANKL, which is produced predominantly by cells of the osteoblast lineage, interacts with RANK expressed by osteoclast lineage cells, leading to increased osteoclastogenesis and osteoclastic activity [13, 14]. OPG is a decoy receptor for RANKL and prevents its interaction with RANK [15]. Nicotine strongly influences this system by increasing the RANKL/OPG ratio [16, 17]. Mechanical removal of contaminants is usually the first mode of therapy recommended for periodontitis [18]. However, this therapy has limitations that may be due to sev- eral factors, such as tooth anatomy, tissue invasion by peri- odontal pathogens, and potential recolonization from other diseased sites [19]. Antimicrobial photodynamic therapy (aPDT) has recently emerged as an adjunct to scaling and root planing (SRP). aPDT can enhance the effectiveness of peri- odontal treatment through its bactericidal effects and the inac- tivation of bacterial virulence factors and host cytokines that impair periodontal restoration [20]. Studies in male rats that were systemically treated with nicotine [21] and in ovariecto- mized female rats [22] showed that aPDT was able to reduce bone loss and inflammatory immune responses in induced periodontitis. However, the effect of aPDT in intact female rats systemically treated with nicotine and with induced peri- odontitis requires further study. Accordingly, the aim of this study was to assess the effect of aPDT, as an adjunct to SRP, on alveolar bone loss, inflam- matory immune responses, the modulation and recruitment of osteoclasts, and periodontal repair in female rats with experi- mentally induced periodontitis under the effects of systemic nicotine treatment. Materials and methods This study was conducted with adult femaleWistar rats. At the start of the study, the rats were 3 months of age and weighed 266±6.5 g (mean±SD). After an acclimatization period, the estrous cycle was monitored, and the rats that exhibited ab- normal cycles were removed. The remaining rats (n=180) were randomly distributed into two groups: the Veh group (n=90), which was administered vehicle (0.9 % sodium chlo- ride), and the Nic group (n=90), which was administered nic- otine. All protocols were approved by the Institutional Review Board of Araçatuba Dental School, São Paulo State University, Araçatuba, São Paulo, Brazil (no. 2010/005074). For all surgical procedures, the rats were anesthetized with ketamine (70 mg/kg; Vetaset®, Fort Dodge Animal Health, Fort Dodge, IA, USA) and xylazine (6 mg/kg; Rompum, Bayer do Brazil, São Paulo, SP, Brazil) via intramuscular injection. Estrous cycle and dosage of estrogen To confirm regularity, estrous cycles were monitored 2 weeks after the osmotic pump placement and 1 week after the peri- odontal treatment. Changes in vaginal smears during the 4 to 5 days of the estrous cycles were observed in all rats (data not shown) [22]. The estrogen level was measured by radioimmunoassay. Blood was collected from the rats by cardiac puncture at the time of euthanasia. Serum samples were analyzedwith a Coat- A-Count Estradiol kit (17β-estradiol double antibody, KE2D; Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) and measured for 60 s with a gamma counter (Packard Cobra TM II Auto Gamma Counter Packard Bioscience, Meriden, CT, USA). Three standard curves were analyzed, and all sam- ples were compared to their closest standard curve. Standards and samples were analyzed in duplicate. Protocol for the administration of vehicle or nicotine Nicotine and vehicle were administered by osmotic mini- pumps. Thirty days before ligature placement, the rats were anesthetized, and osmotic mini-pumps (Alzet model #2006, Durect Corp., Palo Alto, CA, USA) containing nicotine or vehicle were surgically inserted subcutaneously into the backs of the rats. The pumps delivered the solution at a rate of 0.17 μL/h for a period of up to 40 days. Nicotine tartrate (Sigma Chemical Co., St Louis, MO, USA) was dissolved in a sterile solution of 0.9 % sodium chloride to deliver an aver- age of 6 mg nicotine/kg/day. In both groups, the pumps were replaced after the first 40 days, and the new pumps remained until euthanasia [23]. Experimental periodontitis protocol Thirty days after the mini-pumps were implanted, a cotton ligature in a submarginal position was applied to one left mandibular first molar of each rat in both groups to in- duce experimental periodontitis (EP). The ligatures were removed from all rats after 7 days. The rats in each group (Veh and Nic) were randomly assigned through a computer-generated table to one of three treatment sub- groups: only SRP, SRP plus diode laser (DL), or SRP plus antimicrobial photodynamic therapy (aPDT). Each sub- group had 30 rats. All treatment procedures were per- formed by the same experienced operator (ECGJ). 84 Lasers Med Sci (2016) 31:83–94 SRP treatment SRP was performed with manual curettes (#1–2 Mini Five Gracey curettes, Hu-Friedy, Chicago, IL., USA) using ten distal-mesial traction movements on both the buccal and lingual aspects. The furcation and interproximal regions of each tooth were scaled with the same curettes using cervical- occlusal traction movements [21, 22]. DL treatment The laser used in this study was an indium-gallium- aluminum-phosphorous laser (TheraLase, DMC Equipment, São Carlos, SP, Brazil) with a wavelength of 660 nm and a spot size of 0.0283 cm2. Laser light was applied to the buccal and lingual aspects of the left mandibular first molar perpen- dicularly and in contact with the gingivae. The laser was acti- vated at a power of 0.035 W for 12 s for each buccal and lingual aspect (24 s per tooth), with 0.14 J per point of energy and an energy density of 14.82 J/cm2. Each tooth received a total energy density of 29.64 J/cm2 [22]. aPDT treatment For the aPDT treatment, SRP of the left mandibular first molar was performed. Then, 1 mL of a 7-amino-8-methyl- phenothiazin-3-ylidene-dimethyl-ammonium (phenothiazine; Sigma Chemical Co., St. Louis, MO, USA) solution (100 μg/ mL) was applied, followed by DL treatment. The phenothia- zine solution (1 mL) was slowly dropped into the periodontal pocket around the left mandibular first molar using a syringe and an unbeveled needle (13×0.45 mm). After 1 min of phe- nothiazine application, the laser was applied to each buccal and lingual aspect of the left mandibular first molar perpen- dicularly and in contact with the gingivae. The laser parame- ters were the same as those described above [22]. Experimental periods Ten rats from each treatment subgroup were euthanized 7, 15, and 30 days after the periodontal treatment via a lethal dose of thiopental (150 mg/kg; Cristália, Itapira, SP, Brazil). Their jaws were removed and fixed with 4 % formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 48 h. Laboratory procedures The specimens were demineralized in a solution of 10 % EDTA (Sigma Chemical Co., St Louis, MO, USA), subjected to conventional histological processing, and embedded in paraffin. Semi-serial sections (4 μm) were obtained in the latero-lateral direction so that the mandibular first molars were sectioned along the longitudinal axis. Some sections were stained with he- matoxylin and eosin (HE) or Masson Trichrome (MT). Other sec t ions were subjec ted to the indi rec t immunoperoxidase method with the following primary antibodies: anti-PCNA (VP 980—Vector Laboratories, Burlingame, CA, USA), anti-TRAP (goat anti-TRAP— SC 30833, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-RANKL (goat anti-RANKL—SC 7628, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-OPG (goat anti-OPG—SC 8468, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunohis- tochemical procedure followed the protocol described by Garcia et al. [24]. Histopathological analysis The sections stained with HE were analyzed by a certi- fied histologist blinded to the treatments (EE). The fol- lowing parameters were examined: nature and extension of the inflammatory process; presence of necrotic tissue; presence, extent, and nature of resorption of the bone, cementum, and dentin; state of the vasculature; structure of the extracellular matrix of the periodontal tissues; and cellular pattern of the periodontal tissues. The entire furcation region of the left mandibular first molar was analyzed. Histometric analysis The sections stained with TM were used to assess alve- olar bone loss (ABL) in the furcation region. One trained examiner (ML), who was blinded to the groups and treatments, selected the sections for histometric analysis. Five equidistant sections were selected from each specimen and imaged using a digital camera coupled to a light microscope (AxioStar Plus, Carl Zeiss MicroImaging Gmb, 37030 Gottingen, Germany). Another calibrated examiner (ECGJ), who was blinded to the groups and treatments, conducted the histometric analysis. At ×50 magnification, an image analysis sys- tem (AxioVision 4.8.2, Carl Zeiss MicroImaging GmbH, 07740 Jena, Germany) was used to measure the distance between the outer surface of the cementum and the boundary of the alveolar bone crest to determine the area of ABL (mm2) [21, 22, 24]. ABL was measured three times by the same examiner on different days to reduce variations in the data. The mean values were averaged and compared statistically. Immunohistochemical analysis An examiner who was blinded to the groups and treatments (EE) conducted the immunohistochemical analyses. The Lasers Med Sci (2016) 31:83–94 85 values for each section were measured three times by the same examiner on different days to reduce variations in the data [21, 22, 24]. Immunolabeling for RANKL and OPG was analyzed in the entire furcation region of the mandibular first molar under ×400 magnification. A semi-quantitative analysis was performed. Three histological sections from each an- imal were used, and the immunolabeling criteria followed those of Garcia et al. [24] as follows: score 0—no mark- ing (0 %), score 1—weak marking (<25 % of cells), score 2—moderate marking (<50 % of cells), and score 3— strong intensity (<75 % of cells). Fig. 1 Photomicrographs showing the histological appearance of the furcation region of the left mandibular first molar at 7 days in the Veh (a–f) and Nic (g–l) groups after treatment with SRP (a, d, g, and j), SRP plus DL (b, e, h, and k), and SRP plus aPDT (c, f, i, and l). Abbreviations and symbols: ab, alveolar bone; asterisks, inflammatory infiltrate. Staining: hematoxylin and eosin (HE). Original magnification: a–c and g–i, ×100; d–f and j–l, ×250. Scale bars: a–c and g–i, 250 μm; d–f and j–l, 100 μm 86 Lasers Med Sci (2016) 31:83–94 PCNA- and TRAP-positive cells were analyzed within a 1000×1000 μm portion of the central area of the inter- radicular septum under ×200 magnification. The coronary limit was the bone crest, which was spanned apically for a distance of 1000 μm. Quantitative analysis for TRAP and PCNA was performed in five sections from each animal. Only the TRAP- positive, multinucleated cells were analyzed [24]. Intra-examiner reproducibility Before the histometric and immunohistochemical analyses were performed, the examiner was trained and then he cali- brated the analyses by performing double measurements of 30 specimens with a 1-week interval. Pearson’s correlation coef- ficient revealed a very high correlation (0.95) between the two Fig. 2 Photomicrographs showing the histological appearance of the furcation region of the left mandibular first molar at 15 days in the Veh (a–f) and Nic (g–l) groups after treatment with SRP (a, d, g, and j), SRP plus DL (b, e, h, and k), and SRP plus aPDT (c, f, i, and l). Abbreviations and symbols: ab, alveolar bone; asterisks, inflammatory infiltrate. Staining: hematoxylin and eosin (HE). Original magnification: a–c and g–i, ×100; d–f and j–l, ×250. Scale bars: a–c and g–i, 250 μm; d–f and j–l, 100 μm Lasers Med Sci (2016) 31:83–94 87 sets of measurements for both the histometric and immuno- histochemical analyses. Statistical analysis The results demonstrated that with a sample size of 10 (p<0.05), the power of the study would be 95 %. The hypothesis that neither ABL nor the number of TRAP- and PCNA-positive cells in the furcation region would differ be- tween the groups and time points was tested using a statistical software (Bioestat 5.3, Manaus, AM, Brazil). The normality of the histometric data was analyzed using the Shapiro-Wilk test. The intra- and inter-group analyses were performed with analysis of variance (ANOVA) (p<0.05). When ANOVA Fig. 3 Photomicrographs showing the histological appearance of the furcation region of the left mandibular first molar at 30 days in the Veh (a–f) and the Nic (g–l) groups after treatment with SRP (a, d, g, and j), SRP plus DL (b, e, h, and k), and SRP plus aPDT (c, f, i, and l). Abbreviations and symbols: ab, alveolar bone; asterisks, inflammatory infiltrate. Staining: hematoxylin and eosin (HE). Original magnification: a–c and g–i, ×100; d–f and j–l, ×250. Scale bars: a–c and g–i, 250 μm; d–f and j–l, 100 μm 88 Lasers Med Sci (2016) 31:83–94 detected a significant difference, multiple comparisons were performed using Tukey’s test (p<0.05) for ABL- and TRAP- positive cells. Bonferroni’s test (p<0.05) was used for PCNA- positive cells. Results Hormone radioimmunoassay At the time of euthanasia, the mean serum concentration of 17β-estradiol did not statistically differ between the Veh (45.74 pg/mL) and Nic (43.08 pg/mL) groups. Histological analyses SRP subgroupAt 7 and 15 days, the Veh and Nic groups that were treated with SRP exhibited connective tissue with in- tense inflammatory infiltrate, which was composed mainly of neutrophils, in the furcation regions (Figs. 1a, d, g, j and 2a, d, g, j). After 30 days, the volume of inflammatory infil- trate was reduced in the Veh group, and the connective tissue and bone showed signs of repair (Fig. 3a, d). At the same point in time, the Nic group showed a pattern of tissue disruption similar to the one observed at 15 days (Fig. 3g, j). At all time points, the magnitude of the inflammatory response, tissue disorganization, and ABL level were greater in the Nic group than in the Veh group (Figs. 1g, j; 2g, j; and 3g, j). DL subgroup At 7 days, the Veh group showed a moderate inflammatory infiltrate in the furcation region (Fig. 1b, e). At 15 and 30 days, the inflammatory infiltrate was greatly de- creased, the connective tissue showed a moderate amount of fibroblasts and collagen fibers, and the bone tissue showed fewer signs of active resorption (Figs. 2b, e and 3b, e). The Nic group, compared with the Veh group, showed more in- tense inflammation and tissue destruction after 7 days (Fig. 1h, k). At 15 and 30 days, signs of tissue repair were tangible; however, the delay in repair was clear (Figs. 2h, k and 3h, k). aPDT subgroup At 7 days, both the Veh and Nic groups showed mild infiltration into the connective tissue of the fur- cation region; this infiltrate was rich in fibroblasts and exhib- ited a moderate amount of collagen fibers (Fig. 1c, f, i, l). At 15 days, tissue repair signals and a decrease in inflammation were clear in both groups; however, the tissues were more mature in the Veh group (Fig. 2c, f, i, l). At day 30, the histo- logical results of both the Veh and Nic groups were very similar: the inflammatory infiltrate was very mild in the fi- brous connective tissue, with moderate quantities of fibro- blasts and blood vessels. The bone tissue showed few areas of active resorption and many areas of bone formation (Fig. 3c, f, i, l). Histometric assessment Inter-group assessment demonstrated that, at 7 and 15 days, the Veh group exhibited reduced ABL compared to the Nic group (p<0.05). Intra-group assessment revealed that at 7 and 15 days, the rats in the Veh group that were treated with DL and aPDT exhibited decreased ABL compared with those treated with SRP (p<0.01). In the Nic group, there was less ABL at 7, 15 (p<0.01), and 30 days (p<0.05) in the rats that were treated with aPDT compared with those treated with SRP. At 15 and 30 days (p<0.05), there was less ABL in the rats in the Nic group that were treated with DL compared to those treated with SRP (Fig. 4). Immunohistochemical assessment The immunohistochemical method used to detect PCNA, TRAP, RANKL, and OPG showed high specificity, which was confirmed by the total absence of labeling in the negative control. The immunoreactive cells were brownish in color. Positive immunoreactivity was Fig. 4 Graphic showing the mean±SD of alveolar bone loss (mm2) in the furcation region of the left mandibular first molar according to group, treatment, and time point. Symbols: asterisks, significantly different from SRP treatment, for the same group and period (ANOVA and Tukey, p<0.05); daggers, significantly different from the Veh group, for the same treatment and period (ANOVA and Tukey, p<0.05) Lasers Med Sci (2016) 31:83–94 89 predominantly found in cells located in the connective tissue and bone for PCNA, in osteoclasts for TRAP, and in osteoblasts for RANKL and OPG. PCNA The Veh group showed a higher number of PCNA- positive cells at 7 days compared to the Nic group (p<0.05). At 15 days, the Nic group showed a higher number of PCNA- 90 Lasers Med Sci (2016) 31:83–94 positive cells compared to the Veh group (p<0.05). In the Veh group, the rats that were treated with aPDT showed a greater number of PCNA-positive cells on day 7 compared to those treated with SRP (p<0.05). At 15 days, the rats of both exper- imental groups that were treated with aPDT showed a higher number of PCNA-positive cells than the rats treated with SRP (p<0.05). In the Veh group, the rats that were treated with DL showed a higher number of PCNA-positive cells compared with those treated with SRP at 7 and 15 days (p<0.05). At 15 days, the rats in the Nic group also showed a higher number of PCNA-positive cells than those treated with SRP (p<0.05) (Fig. 5a, b, c, d, e, f, g). TRAPCompared with the Veh group, the Nic group showed a greater number of TRAP-positive cells at all post-treatment time points (p<0.05). In both the Veh and Nic groups, the rats that were treated with SRP plus aPDTshowed a lower number of TRAP-positive cells at 7 days compared to those treated with SRP (p<0.05). In the Veh group, the rats that were treated with DL showed a lower number of TRAP-positive cells at 7 days compared to those treated with SRP (p<0.05) (Fig. 5h, i, j, k, l, m, n). RANKL In the Veh group, moderate immunostaining (score 2) was most prevalent, except at 15 and 30 days after SRP plus aPDT treatment, when low immunostaining (score 1) was prevalent (Fig. 6a, b, c). In the Nic group, high immunostain- ing (score 3) was most prevalent after SRP treatment, and moderate immunostaining (score 2) was observed after SRP plus aPDT. In the subgroup that was treated with SRP plus DL, the immunostaining was predominantly high (score 3) at 7 days and moderate (score 2) at 15 and 30 days (Fig. 6d, e, f). OPG In the Veh and Nic groups, low immunostaining (score 1) was most prevalent, except at 30 days in the SRP plus DL- treated rats of the Veh group and in the rats of both the Veh and Nic groups that were treated with SRP plus aPDT (Fig. 6g, h, i, j, k, l). Discussion In this study, nicotine accelerated ABL by increasing and maintaining the number of osteoclasts. On the other hand, when combined with SRP, aPDT minimized the deleterious effects of nicotine, especially on bone metabolism, by regu- lating the RANK/RANKL/OPG system. The radioimmunoas- say showed that estrogen concentrations were within the phys- iological range [25]. Although the effect of nicotine on estro- gen was not a focus of this study, nicotine did not negatively impact estrogen levels, which conflicts with the results of Raval et al. [26]. More ABL was observed in the rats that were treated with nicotine than in the rats that were treated with vehicle, which corroborates the results of previous studies [21, 27]. This ef- fect was greater at 7 and 15 days. Nicotine inhibits apoptosis in certain cell types, such as osteoclasts; as a result, these cells live longer and are able to continue resorption long after the end of their normal life cycle [11]. Furthermore, nicotine sig- nificantly reduces trabecular bone volume and the trabecular thickness of the mineralized surface, the mineral apposition rate, and new bone formation, leading to an increase in oste- oclast area [28]. Nicotine negatively affected the tissue re- sponse to the periodontal treatments used in this study. The female rats that were treated with aPDT showed re- duced ABL compared with the rats that were treated with SRP alone; this finding was observed in the Veh group at 7 and 15 days and in the Nic group at all time points. However, the ABL effect was statistically significant only in the Veh group at 7 days. The beneficial effects of the use of aPDT as an adjunct to mechanical treatments for periodontal disease are most l ikely due to the act ivi ty of aPDT against periodontopathogenic bacteria [29]. During the process of aPDT, the photosensitizer (PS) absorbs energy directly from the laser and transfers it to oxygen molecules, resulting in the formation of reactive oxygen species (type I reaction) and singlet oxygen (type II reaction) [30, 31]. Gram-positive bac- teria are more susceptible to aPDT, but with the selection of the appropriate PS and wavelength, gram-negative bacteria can also become susceptible [32]. Phenothiazines (which are used as PSs) are cationic molecules composed of a tricyclic aromatic ring. These structural characteristics enable PSs to absorb light at a wavelength of 600–660 nm [33]. The photo- sensitizer toluidine blue O (PS TBO) has been successfully used in aPDT against various types of bacteria [34, 35]. Although it is toxic to bacteria, at a concentration of 100 μg/ �Fig. 5 Immunolabeling for PCNA and TRAP in the furcation region of the left mandibular first molar in different experimental groups. aGraphic showing the mean±SD of the number of PCNA-positive cells according to group, treatment, and time point. b–g Photomicrographs showing PCNA-positive cells (black arrows) in the Veh (b–d) and Nic (e–g) groups after treatment with SRP (b and e), SRP plus DL (c and f), and SRP plus aPDT (d and g) at 7 days. h Graphic showing the mean±SD of the number of TRAP-positive cells according to group, treatment, and time point. i–n Photomicrographs showing TRAP-positive cells (white arrows) in the Veh (i–k) and Nic (l–n) groups after treatment with SRP (i and l), SRP plus DL (j and m), and SRP plus aPDT (k and n) at 7 days. Abbreviations and symbols: ab, alveolar bone; black arrows, TRAP- positive osteoclasts; red arrows, PCNA-positive cells; asterisks, significantly different from SRP treatment for the same group and time point; daggers, significantly different from day 7 for the same group and treatment; double daggers, significantly different from day 15 for the same group and treatment; section sign, significantly different from DL treatment for the same group and time point; double vertical lines, significantly different from the Veh group for the same treatment and time point (PCNA: ANOVA and Bonferroni; TRAP: ANOVA and Tukey, p<0.05). Counterstaining: Fast green (b–g) or Harris hematoxylin (i–n). Original magnification: b–g and i–n, ×1000; scale bars: b–g and i–n, 25 μm Lasers Med Sci (2016) 31:83–94 91 mL, TBO is safe to periodontal tissues. Studies by our re- search group have shown that TBO at this concentration re- duced the inflammatory infiltrate in the periodontal tissues and lowered the inflammatory immune response [24]. In this study, nicotine affected the RANKL/RANK/OPG axis, upregulated RANKL, and downregulated OPG. The RANKL/RANK/OPG axis plays an important role in osteo- clast differentiation and bone remodeling [15, 36]. An in- crease in RANKL leads to the activation of RANK, which in turn leads to increased osteoclastogenesis, the activation of mature osteoclasts and the inhibition of apoptosis [11, 15, 36, 37]. These effects could explain the larger increase of TRAP-positive cells that was observed in the Nic group. Any deregulation of this axis can alter bone metabolism and result in either the loss or gain of bone mass [11, 36]. In a healthy periodontium, RANKL is upregulated, whereas OPG is downregulated in periodontitis; this difference results in a higher RANKL/OPG ratio [37]. The fact that nicotine can modulate osteoclast stimulation may partially explain the rap- id increase of bone loss in the periodontium [11]. In the present study, aPDT affected the RANK/RANKL/ OPG system, resulting in RANKL downregulation, OPG up- regulation, and, consequently, lower numbers of TRAP- positive cells. This effect shows that aPDT reversed the nicotine-induced changes in this system, making it compatible with periodontal health. The histological analysis showed that aPDT decreased the inflammatory infiltrate, which may ex- plain the downregulation of RANKL and upregulation of OPG. These results corroborate those of other studies [21, 22, 24]. The initial response to bacterial infection involves a local inflammatory reaction that activates the innate immune sys- tem [37, 38]. During an inflammatory response, cytokines, chemokines, and other mediators can induce osteoclastogen- esis by increasing the expression of RANKL and decreasing the expression of OPG in osteoblasts/stromal cells [39, 40]; aPDT can impact the gingival inflammation during chronic periodontitis and lead to a specific decrease in antigen- presenting cells [41]. Most likely, the antimicrobial effect of aPDT inhibits the exacerbated inflammatory response to local Fig. 6 Immunostaining for RANKL and OPG in the furcation region of the left mandibular first molar in the different experimental groups. a–f Photomicrographs showing RANKL-positive cells (arrows) in the Veh (a–c) and Nic (d–f) groups after treatment with SRP (a, d), SRP plus DL (b, e), and SRP plus aPDT (c, f) at 7 days. g– l Photomicrographs showing OPG-positive cells (arrows) in the Veh (g–i) and Nic (j–l) groups after treatment with SRP (g, j), SRP plus DL (h, k), and SRP plus aPDT (i, l) at 30 days. Abbreviations and symbols: ab, alveolar bone; black arrows, immunolabeled cells. Counterstaining: Harris hematoxylin. Original magnification ×1000; scale bars 25 μm 92 Lasers Med Sci (2016) 31:83–94 bacterial infections and, consequently, the cascade of events that leads to osteoclastogenesis and bone loss via the RANK/RANKL/OPG axis. In this study, nicotine delayed the peak cell proliferation that is essential for repair. The results showed that there were more PCNA-positive cells at 7 days in the Veh group and at 15 days in the Nic group. However, the rats in both the Veh and Nic groups that were treated with DL and aPDTshowed a high number of PCNA-positive cells. By leading to downreg- ulation of RANKL and, hence, to a small number of TRAP- positive cells, aPDTcreated a more favorable environment for repair. These results are in general agreement with those of Garcia et al. [22], who showed that the number of PCNA- positive cells in the periodontal ligament was significantly higher in rats treated with aPDT compared to SRP alone. Conclusion In this study, aPDTwas an effective adjunct to SRP treatment, as demonstrated by the modulation of the RANK/RANKL/ OPG system, the decrease in the number of osteoclasts, and the high numbers of PCNA-positive cells in the furcation re- gion that were observed in the female rats that were treated with or without nicotine. Thus, aPDT could be considered an effective adjuvant and indicated for the treatment of periodon- titis in women under the systemic effects of nicotine; however, additional experimental studies in animals and clinical trials in women must be performed. Acknowledgments This study was financially supported by the São Paulo Research Foundation (FAPESP), São Paulo, SP, Brazil (FAPESP processes no. 2011/00936-0 and no. 2010/15094-2). 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J Photochem Photobiol B 101:348–354 94 Lasers Med Sci (2016) 31:83–94 Antimicrobial photodynamic therapy minimizes the deleterious effect of nicotine in female rats with induced periodontitis Abstract Introduction Materials and methods Estrous cycle and dosage of estrogen Protocol for the administration of vehicle or nicotine Experimental periodontitis protocol SRP treatment DL treatment aPDT treatment Experimental periods Laboratory procedures Histopathological analysis Histometric analysis Immunohistochemical analysis Intra-examiner reproducibility Statistical analysis Results Hormone radioimmunoassay Histological analyses Histometric assessment Immunohistochemical assessment Discussion Conclusion References