merzomFCLPar

Departamento de Biologia Celular e Molecular e Bioagentes Patognicos, Faculdade de Medicina-FMRP, Universidade de So Paulo, 14049-900 Ribeiro Preto-SP, Brazil

a r t i c l e i n f o

and metronidazole suffer from one major drawback: the difcultyto maintain therapeutic concentrations of the agent in the peri-

wavelength and are able to produce several reactive oxygen spe-cies (ROS) that activate biologic systems [913]. In particular, alarge number of micro-organisms (including oral species) havebeen reported to be killed by PDT [14,15] and virulence factors(lipopolysaccharides and proteases) have also been shown to bereduced by photosensitization. Because it is easy to access the peri-odontal pocket, periodontitis would be very amenable to treatmentby PDT [1518]. Hence, the photosensitizer can be placed directlyinto the pocket which can then be irradiated either through thethin gingival tissues or via an optical ber placed directly into

Corresponding author at: Universit Paris Descartes, Hpital Europen GeorgesPompidou, 56 rue Leblanc, 75737 Paris Cedex 15, France. Tel.: +33 (0) 1 53 98 80 76;fax: +33 (0) 1 53 98 79 58.

E-mail addresses: sylvie.seguier@parisdescartes.fr (S. Sguier), scombati@forp.usp.br (S.L.S. Souza), cesve@terra.com.br (A.C.V. Sverzut), dezasimi@yahoo.com.br (A.R. Simioni), ferprimo@usp.br (F.L. Primo), Agnes.mobarak-bodineau@parisdescartes.fr (A. Bodineau), vanimariaac@yahoo.com.br (V.M.A. Corra),

Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354

Contents lists availab

Journal of Photochemistry an

journal homepage: www.elseBernard.coulomb@parisdescartes.fr (B. Coulomb), atedesco@usp.br (A.C. Tedesco).to a specic decrease of antigen-presenting cells populations according to the drug delivery system used. 2010 Elsevier B.V. All rights reserved.

1. Introduction

It is well known that inammatory periodontal diseases are ini-tiated and maintained by the bacterial plaque and its metabolicproducts which trigger the local inltration of inammatory cellsassociated with the degradation of extracellular matrix molecules[1,2]. Neither mechanical plaque removal nor ushing or rinsingwith disinfectants allows the total eradication of bacterial reser-voirs within periodontal pockets. Current therapeutic strategiesfor periodontitis that use antimicrobial agents such as tetracyclines

odontal pocket for a sufcient length of time to ensure eradicationof the bacteria present [3,4].

To supplement the armament of antibacterial measures and de-crease the inammatory process, in recent years different attemptswere made to introduce photodynamic therapy (PDT) as a newtreatment of chronic periodontitis [38]. PDT is based on the injec-tion, ingestion, or topical application of photosensitizers dyes (usu-ally using drug delivery systems (DDS) such as liposomes ornanoemulsions) followed by visible light activation. These photo-sensitizers are chemical compounds that absorb light at a specicArticle history:Received 23 March 2010Received in revised form 6 August 2010Accepted 10 August 2010Available online 17 August 2010

Keywords:PDTPeriodontal diseaseAntigen-presenting cellDendritic cellPhthalocyanine1011-1344/$ - see front matter 2010 Elsevier B.V. Adoi:10.1016/j.jphotobiol.2010.08.007a b s t r a c t

The aim of this study was to evaluate the effects of the photodynamic therapy (PDT) on the inammatoryinltrate and on the collagen network organization in human advanced chronic periodontitis. Two differ-ent drug delivery systems (DDS) were tested (liposomes and nanoemulsions) to determine if the effects ofPDT could differ according to the DDS used.Sixteen patients presenting two teeth with chronic advanced periodontitis and important tooth

mobility with clinical indication of extraction were included in the group liposomes (group L, n = 8) orin the group nanoemulsions (group N, n = 8) in order to compare the effects of each DDS. Seven daysbefore extractions one tooth of each patient was treated with PDT using phthalocyanine derivatives asphotosensitizers and the contralateral tooth was taken as control. In group L the density of gingivalcollagen bers (66 19%) was signicantly increased (p < 0.02) when compared to controls (35 21%).Concerning the antigen-presenting cells, PDT had differential effects depending on the drug deliverysystem; the number of macrophages was signicantly decreased (p < 0.05) in group L while the numberof Langerhans cells was signicantly decreased in group N (p < 0.02). These ndings demonstrate thatPDT presents an impact on gingival inammatory phenomenon during chronic periodontitis and leadsService dOdontologie, Hpital Louis Mourier, rue des Renouillers, 92700 Colombes, FrancedDepartamento de Cirurgia, Traumatologia Buco-Maxilo-Facial e Periodontia, Faculdade de Odontologia-FORP, Universidade de So Paulo, 14040-904 Ribeiro Preto-SP, BrazileImpact of photodynamic therapy on inachronic periodontitis

Sylvie Sguier a,b,c,, Sergio L.S. Souza d, Anna C.V. SvAgns Bodineau b,c, Vani M.A. Corra e, Bernard CoulaDepartamento de Qumica, Faculdade de Filosoa, Cincias e Letras de Ribeiro Preto-FbUniversit Paris Descartes, Hpital Europen Georges Pompidou, 56 rue Leblanc, 75737cll rights reserved.matory cells during human

ut d, Andreza R. Simioni a, Fernando L. Primo a,b b, Antonio C. Tedesco a

RP, Universidade de So Paulo, 14040-901 Ribeiro Preto-SP, Brazilis Cedex 15, France

le at ScienceDirect

d Photobiology B: Biology

vier .com/locate / jphotobiol

anaesthetic inltration into the biopsy site, and deformation or

nd Pcompression of the samples. Gingival samples of control and trea-ted teeth were obtained from the inner part of the ap (that was incontact with the root) in the buccal marginal gingiva.

2.3. Photodynamic therapy (PDT) and laser treatment (Fig. 1)

The PDT was performed using phthalocyanine derivatives asphotosensitizers (NzPC and AlClPC) and two different drug deliverysystems were used: liposomes (n = 8, group L) and nanoemulsions(n = 8, group N). The photosensitizer was applied on each surface ofthe tooth by placing the applicator at the bottom of the periodontalpocket and was continuously deposited in a coronal direction. Thisapplication was done three times with 5 min of waiting betweenthe pocket avoiding damage to adjacent host tissues [19] and dis-ruption of the normal microora [20]. However, there is a lack ofin vivo studies evaluating the effect of PDT on the gingival inam-matory cells and on the matrix macromolecules as collagen bersduring human periodontal diseases.

The aim of this study was to evaluate the potential efciency ofPDT on a limited number of patients presenting advanced chronichuman periodontitis. Two drug delivery systems (DDS) were tested(liposomes and nanoemulsions) in case of each DDS interacts dif-ferently with a tissular target. The effect of PDT on the inamma-tory cells, on the density of the collagen network and on theexpression of metalloproteinases was evaluated from gingivalbiopsies a week after the treatment.

2. Materials and methods

2.1. Patient population

The experimental protocol was reviewed and approved by theInstitutions Human Research Committee and the protocol was ap-proved on June 21, 2007 (protocol 2007.1.487.58.7, Ribeiro Preto,So Paulo University, Brazil) and the experiments were undertakenwith the understanding and written consent of each subject. Six-teen patients (6 females, 10 males, aged 5065) presenting twoteeth with a clinical diagnosis of advanced chronic periodontitis,ultimate degree of tooth mobility (mobility IV, horizontal and axialmobilities) and periodontal indication for extraction were selected.For each selected patient, both teeth presented the same degree ofgingival inammation and the equivalent tooth mobility. Diagnosisof chronic periodontitis was established on the basis of clinical andradiographic criteria (bone resorption) according to the classica-tion system for periodontal diseases and conditions [21]. The pa-tients included in this study had neither other oral or systemicdiseases, nor any overt immunological abnormalities and did nottake any preoperatory medication.

2.2. Study design

The study was performed using the split-mouth design. A totalof 16 pairs of contralateral maxillary or mandibulary teeth were in-cluded. In each contralateral pair, one tooth was assigned as con-trol whereas the other tooth was treated with photodynamictherapy (PDT). No subgingival mechanical therapy (scaling androot planing) was performed prior to PDT. All patients were treatedby the same operator and the extractions of both teeth (control andtreated) were performed 7 days after treatment by PDT. Gingivaltissue samples, which otherwise would have been discarded, wereobtained during surgery under local anaesthesia, avoiding local

S. Sguier et al. / Journal of Photochemistry aeach application and laser exposure was done 15 min after the lastapplication of the photosensitizer. The laser used in this study wasan Eagle Diode Laser (Quantum Technology, Brazil) with a wave-length of 670 nm, a uence rate of 0.5 W/cm2 delivered during40 s (10 s for each face of the tooth) and a total uence of 12.7 J/cm2.

2.4. Preparation of photosensitizers

Liposomes have been used as drug delivery systems since the1960s [22]. NzPC is a silicon (IV) phthalocyanine derivative (metalphthalocyanine) with two axial ligands substituted in the macrocy-cle, belong to the second generation of photoactive agents used inPDT. The incorporation of NzPC into the phospholipids bilayer of L-a-phosphatidylcholine and dimethyl-dioctadecylammoniumbro-mide (DDAB) was carried out according to the modied injectionmethod described by Pelegrino et al. [23]. Basically, 360 lL of anethanolic solution, which was 68.6 mmol/L phosphatidylcholine,0.19 mmol/L DDAB and 50 lL in NzPC, was injected with a syringeinto 5 mL PBS, pH 7.4. The injection was performed at 56 C, undermagnetic stirring and at ow rate 1 lL/s.

Nanoemulsion was obtained by spontaneous emulsicationprocess as described by Primo et al. [24]. The natural soy phospho-lipids Epikuron 170 (7.5%) and Miglyol 812 N oil (250 lL) were ob-tained from Hulls Inc. (Puteaux, France) and initially dissolved in10 mL of spectroscopic acetone at 55 C under magnetic stirrer.In the same time the biopolymer Poloxamer 188 (SigmaAldrichCo., St. Louis, MO, USA) at 7.5% was dissolved in ultra-pure waterto obtain an aqueous phase. In the sequence the organic phasewas added slowly in aqueous medium in the emulsication stepfor 20 min. After total homogenization, the acetone was removedby reduced pressure at approximately 6070 C for a nal volumeof 10 mL of nanoemulsion. Chloroaluminum phthalocyanine (Sig-maAldrich Co., St. Louis, MO, USA) was dissolved directly in Mi-glyol 812 N oil at 55 C for nal concentration of 0.05 mg/mL innanoemulsion. Its incorporation in the nanoemulsion was accom-plished on organic phase in emulsication process as previouslydescribed [24]. The photophysical, photochemical and photobio-logical properties and biological responses of the phthalocyaninederivates were similar as revealed in previous study from ourgroup and allow the comparison between them [912].

2.5. Tissue preparation

Seven days after the treatment by PDT, for each patient, thecontrol and treated teeth were removed and their marginal sur-rounding periodontal tissues were immediately divided into threeparts. The rst part was xed in 4% phosphate buffered formalin,pH 7.4 and processed by routine laboratory techniques for parafnembedding. The second part was immediately oriented in meltingisopentane (Tissue-Tek OCT Compound, Sakura, Zoeterwoude,Netherlands) so that the sulcular/gingival epithelium and theunderlying connective tissue would be present in the same section,and snap-frozen in liquid nitrogen. These two parts were used forhistological and immunohistochemistry studies. The third part wasmaintained during 48 h in Hanks medium at 37 C under an atmo-sphere of 95% air, 5% CO2 and biochemical studies were performedwith conditioned media from these organ cultures kept at 80 Cuntil the day of the study.

2.6. Histology and Immunohistochemistry

Serial sections (6 lm thick) were obtained from all specimens.Frozen sections were air-dried at room temperature for 2 h, thenxed in acetone for 10 min and kept at 80 C until the day ofthe study. For all specimens staining of collagen bers was per-

hotobiology B: Biology 101 (2010) 348354 349formed with sirius red F3Ba according to Junqueiras technique(35). For immunohistochemistry, a panel of monoclonal antibodieswas used: anti-CD45 (Leukocyte common antigen, dilution 1:100,

en Yvelines, France) contained 1 mg/mL of gelatin [25] dispersed

icTec

nd Pin buffered solution consisting of 2.5 mL gel 1.5 M TrisHCl pH8.8; 100 lL of sodium dodecyl sulfate (SDS) 10%, 4 mL polyacryl-amide and 4 mL of distilled water pH 8.8 stacking gel contained4% polyacrylamide in 0.125 M Tris, pH 6.8. Gels were polymerizedby adding 50 lL of 10% ammonium persulfate and 10 lL of 0.1%TEMED. Samples (5 lL of conditioned medium) were half dilutedin 1 mol/L Tris pH 6.8 containing 50% glycerol and 0.4% bromophe-Dako, Glostrup, Denmark), anti-CD8 (Suppressor/cytotoxic T lym-phocytes, dilution 1:200, Dako), anti-CD4 (helper T lymphocytes,dilution 1:100, Dako), anti-CD68 (monocytes/macrophages, dilu-tion 1:200, Dako), anti-CD1a (Langerhans cells, dilution 1:100,Dako) using an avidinbiotinimmunoperoxidase technique aspreviously described in the literature [2].

2.7. Zymography

Electrophoreses were carried out using a mini protean II system(Biorad, Marnes la coquette, France). Ten per cent polyacrylamidegels (10 cm height, 1.5 mm thickness) (Millipore, Saint Quentin

Fig. 1. Schematic representation showing the technical procedure of photodynamperiodontal pocket. (B) Application of the laser using an Eagle Diode Laser (Quantumduring 40 s (10 s for each face of the tooth) and a total uence of 12.7 J/cm2.350 S. Sguier et al. / Journal of Photochemistry anol blue, and gels were run under Laemmli conditions (40 mA, 1 h).Following electrophoresis, gels were washed twice in 200 mL of2.5% Triton X-100 in distilled water under constant mechanicalstirring, and incubated in 100 mM TrisHCl, 5 mM CaCl2, 0.005%Brij-35, 0.001% NaN3 pH 8.0 for 648 h at 37 C. Gels were stainedwith 0.25% Coomassie brilliant blue G-250 (50% methanol, 10%acetic acid) and destained appropriately (40% methanol, 10% aceticacid). Proteinase activity was evident as clear (unstained) zones. Fi-nally the gels were incubated for one hour in 5% methanol, 7.5%acetic acid and kept under cellophane as previously described [2].

2.8. Quantitative determination of area fraction of collagen bers,number of immunolabelled cell populations and zymogram lysis bandsby computer-assisted image analysis

The evaluation of the area fraction (AA%) of collagen bers wasdetermined in the whole of connective tissue visualized on the sec-tion stained with sirius red. Histological sections were observedunder a microscope (Zeiss Model Axioskop, Germany) using a10 objective equipped with a video camera (Diagnostic, USA).Captured images were transferred to a microcomputer in which asoftware (Samba, Tribvn, France) calculated the area fraction(AA%) occupied by the collagen bers.The number of immunolabelled cells per unit area (number ofcells/mm2) was determined using the same computer-assisted im-age analysis and the immunolabelled cells were counted using 10or 20 objectives either in the gingival epithelium or in the con-nective tissue, according to the cell density. As a matter of fact, aprevious study [26] has shown that, as in the connective tissue,the number of inammatory cells in the gingival epithelium couldreect the severity of the inammatory phenomenon. The CD45+cells, CD4+ cells, CD8+ cells and CD1a+ cells were quantied inthe epithelium, the CD68+ cells were quantied in the connectivetissue. For all the gingival samples ve elds per tissue section,randomly selected, were analyzed. Thus, the number of immunola-belled cell subsets for each sample represents the mean of the vecountings.

The surface area and the color intensity of zymogram lysisbands were analyzed using Image J software (Image J; http:/rsb.in-fo.nih.gov/ij/index.html).

2.9. Statistical analysis

therapy. (A) Application of the photosensitizer by placing the applicator in thehnology, Brazil) with a wavelength of 670 nm, a uence rate of 0.5 W/cm2 delivered

hotobiology B: Biology 101 (2010) 348354The means of area fractions occupied by collagen bundles(AA%), cell numbers and amounts of matrix metalloproteinases(MMPs) between the control and treated teeth were comparedusing the one-tailed Student t-test (paired series). The criterionfor statistical signicance was dened as a level of p < 0.05. The re-sults are given as mean and standard deviation (mean SD) to de-scribe the dispersion of the data.

3. Results

3.1. Area fraction (AA%) of collagen bers

In patients (n = 8) treated by PDT using liposomes (group L) asdrug delivery system, the area fraction (AA%) of collagen bers(66 19%) was signicantly increased (p < 0.02) in gingival sam-ples when compared with controls (35 21%) (Fig. 2).

In patients (n = 8) treated by PDT using nanoemulsions (groupN), the area fraction of collagen bers (56 23%) was not signi-cantly different when compared with group C (44 23%).

3.2. Inammatory cell populations

For CD45+ cells, CD8+ cells and CD4+ cells, no signicant differ-ences were observed between controls and treated teeth with

reatb

ea fr

rol a

nd PFig. 2. Histological staining of collagen bers with sirius red F3Ba in control and t(group L). (A) Control gingival section of a patient of group L showing stained collagen(B) Treated gingival section of a patient of group L showing an increased of the artherapy. E, epithelium; CT, connective tissue; Original magnication 10.

Table 1Mean number (SD) of inammatory cell populations (number of cells/mm2) in contnanoemulsions (group N).S. Sguier et al. / Journal of Photochemistry aeither liposomes or nanoemulsions (Table 1). In both controlgroups (for groups L and N) the numbers of cell populations werein accordance with those reported previously in gingival sampleswith chronic periodontitis [27].

In patients treated by PDT using liposomes (group L), the num-ber of CD68+ macrophages was signicantly decreased (p < 0.05) ingingival connective tissue (300 cells/mm2 187) when comparedwith controls (585 cells/mm2 321) (Table 1 and Fig. 3).

In patients treated by PDT using nanoemulsions (group N), thenumber of CD1a+ Langerhans cells was signicantly (p < 0.02) de-creased in gingival epithelium (81 cells/mm2 39) when comparedwith controls (150 cells/mm2 56) (Fig. 4).

3.3. Zymography

Whatever the drug delivery system (groups L and N), both proand active forms of MMP-2 and MMP-9 were present in controland in gingival samples treated by PDT. Nevertheless, after quanti-tative analysis of zymogram lysis bands by computer-assisted im-age analysis, no differences could be detected between control andtreated gingival samples, either in group L or N (Fig. 5).

4. Discussion

In the present study, the application of photodynamic therapy(PDT) was tested on diseased gingival tissues of a limited number

CD45+ CD1a+(cells/mm2) (cells/mm2)

Group LControls 319 225 138 85Treatment 218 124 115 71P-value NS NS

Group NControls 178 47 150 56Treatment 161 115 81 39P-value NS S (p = 0.019)

The CD45+ cells, CD4+ cells, CD8+ cells and CD1a+ cells were quantied in the epithelium(one-tailed Student t-test for paired series) between control and treated gingival samplNS = not signicant, S = signicative difference (p < 0.05).ed gingival samples of patients treated by photodynamic therapy using liposomesers (arrow) strongly destroyed and degraded during the inammatory phenomenon.action of the collagen bers (arrow) a week after the treatment by photodynamic

nd treated gingival samples by PDT in the group liposomes (group L) and the group

hotobiology B: Biology 101 (2010) 348354 351of patients with severe periodontitis using two drug delivery sys-tems (DDS, liposomes and nanoemulsions) and gingival biopsieswere collected 1 week after the clinical treatment by PDT.

The phthalocyanine dyes used belong to a second generation ofdyes with important production of reactive oxygen species, mainlysinglet oxygen [28,29]. The main advance in the use of phthalocy-anine as photosensitizer is the fact that this family of dyes couldact by the two classical PDT mechanisms of radical production(type I) or singlet oxygen (type II) according to Foote [30]. Theassociation with the specic DDS allows a good biodistribution ofphthalocyanine dyes and an excellent stability.

Many studies [3,31] have shown that periodontopathogenicbacteria are susceptible to lethal photosensitization and PDT hasbeen reported in the literature to be effective in eradicating variousmicro-organisms using different photosensitizers, different wave-lengths of light, and different light sources [32]. Recent clinicaland microbiological studies using PDT for periodontitis were per-formed to evaluate the effectiveness of PDT as a primary mode oftreatment or as an adjunct to non-surgical treatment of scalingand root planning (SRP) compared to a conventional non-surgicalSRP treatment. For some authors [33,34] the adjuvant applicationof PDT seems appropriate to reduce inammatory symptoms andto successfully treat infection with Fusobacterium nucleatum; forother [35] PDT as an independent treatment or as an adjunct toSRP was not superior (changes in clinical attachment level, probingdepth, gingival recession, full-mouth plaque or bleeding scores) tocontrol treatment of SRP. However, there is a lack of histological

CD68+ CD8+ CD4+(cells/mm2) (cells/mm2) (cells/mm2)

585 121 122 76 157 111300 127 107 83 131 66S (p = 0.042) NS NS

351 113 96 29 230 104305 105 102 96 161 78NS NS NS

, the CD68+ cells were quantied in the connective tissue. Signicance of differenceses in each group.

nd P352 S. Sguier et al. / Journal of Photochemistry aand biochemical studies evaluating the effect of PDT on the gingi-val inammatory cells, on the matrix macromolecules as collagenbers and on the expression of the metalloproteinases (MMPs)during human periodontal diseases.

Our aim was to evaluate the consequence of PDT on the inam-matory inltrate during human chronic periodontitis. Several stud-ies reveal that PDT has a signicant impact on neutrophils [36,37]

Fig. 4. Immunohistochemical staining of CD1a+ Langerhans cells in control and treated g(group N). (A) Control gingival section of a patient of group N showing numerous CD1a+ Lpatient of group N showing a decreased number of immunolabelled CD1a+ Langerhans cemagnication 10.

Fig. 5. Gelatin zymogram revealing gelatinase activities in the supernatant of control (shown in zymogram, heterogenic gelatinase activities were observed between patients (nmarginal gingiva of each patient.

Fig. 3. Immunohistochemical staining of CD68+ macrophages in control and treated ging(A) Control gingival section of a patient of group L showing a large number of immunoTreated gingival section of a patient of group L showing a decreased number of immunepithelium; CT, connective tissue; Original magnication 20.hotobiology B: Biology 101 (2010) 348354but other studies suggest a pro-tolerogenic effects of PDT on den-dritic cells [38] or describe immunosuppressive effects of phthalo-cyanine PDT mediated by CD4+ and CD8+ T cells [39]. Then, itseems that PDT has an impact on different inammatory cell pop-ulations and it appears that the composition and the extension ofthe inammatory inltrate differ according the time (15 min to72 h) of cell analysis after PDT as suggested by Prignano et al.

ingival samples of patients treated by photodynamic therapy using nanoemulsionsangerhans cells in the gingival epithelium (arrows). (B) Treated gingival section of alls in the gingival epithelium (arrows). E, epithelium; CT, connective tissue; Original

C) and treated (T) gingival explants by photodynamic therapy using liposomes. As= 8) and no reproducible differences could be detected between control and treated

ival samples of patients treated by photodynamic therapy using liposomes (group L).labelled CD68+ macrophages in the upper gingival connective tissue (arrows). (B)olabelled CD68+ macrophages in the upper gingival connective tissue (arrows). E,

[7] N. Christodoulides, D. Nikolidakis, P. Chondros, J. Becker, F. Schwarz, R. Rossler,

nd P[40]. In the present study, the analysis of inammatory inltratewas performed 7 days after PDT and at that time, the number ofsome inammatory cell populations was decreased.

Our ndings showed that Antigen-Presenting Cells (APC, macro-phages and Langerhans cells) located in the inammatory inltrateand known as one of the rst immune barrier against pathogens, areparticularly sensitive to PDT. As amatter of fact, the number ofmac-rophageswas signicantlydecreased after PDTusing liposomes, andthe number of Langerhans cells (dendritic cells family, APC)was alsosignicantly decreased after PDT using nanoemulsions.

A previous study showed that PDT has immunomodulatoryactivity in various mouse models and dendritic cells treated byPDT showed a reduced capacity to stimulate the proliferation ofalloreactive T cells [41]. The ability of PDT to down-regulate auto-immune processes appears to be related to its capacity to inuencethe immunostimulatory attributes of APC [38,42]. The PDT couldact on APC in two ways: rst, by decreasing their number, and sec-ond, by reducing their capacity of activation of T lymphocytes. As anal result, PDT could reduce the inammatory phenomenon sinceit is well known that APC are able on phagocytose and endocytoseprocesses and thus, are cells specialized in the incorporation of for-eign elements such as liposomes or nanoemulsions.

Interestingly, in our study, PDT targeted different cell popula-tions depending upon the drug delivery system used. ConcerningLangerhans cells in group N (nanoemulsions), our results sug-gested that nanoemulsions could lead to their migration towardsthe gingival connective tissue in order to realize the antigenic pre-sentation. Concerning macrophages in group L (liposomes), ourndings showed a decrease in the number of this cell population,associated with an increase in the density of the gingival collagen.Among the matrix macromolecules, collagen quantitatively consti-tutes the major component of the gingival connective tissue andplays a key role in its architecture due to its bers organization.During the periodontal diseases, inammatory cells including mac-rophages produce and release proteases (such as metalloprotein-ases) and cytokines which, together, generate periodontal tissuedestruction and a degradation of collagen bers. In addition, mac-rophages are strongly implicated in the turnover of matrix macro-molecules by phagocytose of collagen bers. In patients treated byPDT using liposomes, the density of collagen bers was signi-cantly increased in gingival samples when compared with controls.This result could be linked to the signicant decrease in the num-ber of the macrophages in this group.

Pathogenic bacteria and inammatory cells produce a numberof enzymes and potent proteases capable of degrading host tissuesleading to the periodontal destruction and the degradation of col-lagen bers observed during periodontitis. Nevertheless, studies onthe effect of photosensitizers in combination with light in this con-text have, in general, focused on the effects of this potential thera-peutic modality on the viability of the micro-organisms or on thereduction of bacterial virulence factors [43]. It is well known byexperiments on beagle dogs [17] and humans [18] that PDT proce-dure induces a signicant reduction of periodontopathogens-in-fected sites.

Thus, in the present study we also analyzed by zymography theactivities of the gelatinases (MMP-2 and MMP-9) since the activeform of MMP-9 has been proposed as a marker for the clinicalseverity of periodontal diseases a previous study [2]. In our condi-tions, the activities of the gelatinases were not signicantly differ-ent between control and treated teeth with either liposomes ornanoemulsions. It is likely that the experimental period of 1 weekbetween the clinical treatment by PDT and the teeth extractionwas unadapted to evidence eventual changes in the MMPs expres-

S. Sguier et al. / Journal of Photochemistry asion. Furthermore, it seems strongly possible that 7 days after PDTthere is a re-colonisation of periodontal pocket by bacterialelements.A. Sculean, Photodynamic therapy as an adjunct to non-surgical periodontaltreatment: a randomized, controlled clinical trial, J. Periodontol. 79 (2008)16381644.

[8] A. Braun, C. Dehn, F. Krause, S. Jepsen, Short-term clinical effects of adjunctiveantimicrobial photodynamic therapy in periodontal treatment: a randomizedclinical trial, J. Clin. Periodontol. 35 (2008) 877884.

[9] C.N. Lunardi, J.C. Rotta, A.C. Tedesco, Synthesis, photophysical andphotobiological study of synergic photosensitizer: zinc-phthalocyanine withCa2+ chelating agent, Curr. Org. Chem. 11 (2007) 647654.

[10] M. Maftoum-Costa, K.T. Naves, A.L. Oliveira, A.C. Tedesco, N.S. da Silva, C.Pacheco-Soares, Mitochondria, endoplasmic reticulum and actin lamentbehavior after PDT with chloroaluminum phthalocyanine liposomal in HeLacells, Cell Biol. Int. 32 (2008) 10241028.

[11] S.M.T. Nunes, F.S. Sguila, A.C. Tedesco, Photophysical studies of zincphthalocyanines and chloroaluminium phthalocyanines incorporated intoliposomes in the presence of additives, Braz. J. Med. Biol. Res. 37 (2004)References

[1] M.A. Listgarten, Pathogenesis of periodontitis, J. Clin. Periodontol. 13 (1986)418425.

[2] S. Seguier, B. Gogly, A. Bodineau, G. Godeau, N. Brousse, Is collagen breakdownduring periodontitis linked to inammatory cells and expression of matrixmetalloproteinases and tissue inhibitors of metalloproteinases in humangingival tissue?, J Periodontol. 72 (2001) 13981406.

[3] P. Meisel, T. Kocher, Photodynamic therapy for periodontal diseases: state ofthe art, J. Photochem. Photobiol. B 79 (2005) 159170.

[4] G. Jori, Photodynamic therapy of microbial infections: state of the art andperspectives, J. Environ. Pathol. Toxicol. Oncol. 25 (2006) 505520.

[5] J.M. de Almeida, L.H. Theodoro, A.F. Bosco, M.J. Nagata, M. Oshiiwa, V.G. Garcia,Inuence of photodynamic therapy on the development of ligature-inducedperiodontitis in rats, J. Periodontol. 78 (2007) 566575.

[6] J.M. de Almeida, L.H. Theodoro, A.F. Bosco, M.J. Nagata, M. Oshiiwa, V.G. Garcia,In vivo effect of photodynamic therapy on periodontal bone loss in dentalfurcations, J. Periodontol. 79 (2008) 10811088.Acknowledgements

We thank CAPES for nancial support CAPES/COFECUB 523/06,FAPESP (Fundao de Amparo Pesquisa do Estado de So Paulo)for nancial support, 07/55319-0 and 08/53719-4 A.R.S. (09/51729-5) and F.L. Primo (09/15363-6), were the recipient of FA-PESP fellowships.In conclusion, our ndings demonstrate that the treatment ofchronic periodontitis by PDT leads to a specic decrease of anti-gen-presenting cells populations according to the drug deliverysystem. Thus, PDT might be considered as an effective coadjuvanttreatment for chronic periodontal diseases by supplementing theantibacterial mechanical measures. Furthermore, the use of lipo-somes has the advantage to reduce the degradation of the gingivalextracellular matrix. These results are thus encouraging and nowjustify to initiate an additional study with an increased numberof patients including an analysis of periodontal pocket re-colonisa-tion by bacterial elements with time after PDT.

5. Conict of interest and source of funding statement

The authors declare that they have no actual or potential con-ict of interest including any nancial, personal or other relation-ships with other people or organizations within 3 years ofbeginning our submitted work that could inappropriately inu-ence, or be perceived to inuence, our work.

This work was supported by grants from Fundao de Amparo aPesquisa do Estado de So Paulo-FAPESP, So Paulo, Brasil; Conse-lho Nacional de Desenvolvimento Cientco e Tecnlogico-CNPq,Braslia, Brasil. This work was also supported by grants from ParisDescartes University, France.

This collaboration between Paris Descartes University and SaoPaulo University is supported by CAPES/COFECUB No. 523/06.

hotobiology B: Biology 101 (2010) 348354 353273284.[12] A.R. Simioni, M.M. Pelisson, M. Beltrame Jr., A.C. Tedesco, Photophysical and

photobiological studies of a silicon tribenzonaphthoporphyrazinato

incorporated into liposomes for photodynamic therapy use, J. Nanosci.Nanotechnol. 8 (2008) 32083215.

[13] J.P. Longo, S.P. Lozzi, A.R. Simioni, P.C. Morais, A.C. Tedesco, R.B. Azevedo,Photodynamic therapy with aluminum-chloro-phthalocyanine inducesnecrosis and vascular damage in mice tongue tumors, J. Photochem.Photobiol. B 94 (2009) 143146.

[14] N. Kmerik, H. Nakanishi, A.J. MacRobert, B. Henderson, P. Speight, M. Wilson,In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photo-sensitization in an animal model, Antimicrob. Agents Chemother. 47 (2003)932940.

[15] M. Wilson, Lethal photosensitisation of oral bacteria and its potentialapplication in the photodynamic therapy of oral infections, Photochem.Photobiol. Sci. 3 (2004) 412418.

[16] R.R. de Oliveira, H.O. Schwartz-Filho, A.B. Novaes Jr., M. Taba Jr., Antimicrobialphotodynamic therapy in the non-surgical treatment of aggressiveperiodontitis: a preliminary randomized controlled clinical study, J.Periodontol. 78 (2007) 965973.

[17] B.W. Sigusch, A. Ptzner, V. Albrecht, E. Glockmann, Efcacy of photodynamictherapy on inammatory signs and two selected periodontopathogenic speciesin a beagle dog model, J. Periodontol. 76 (2005) 11001105.

[18] P. Chondros, D. Nikolidakis, N. Christodoulides, R. Rssler, N. Gutknecht, A.Sculean, Photodynamic therapy as adjunct to non-surgical periodontaltreatment in patients on periodontal maintenance: a randomized controlledclinical trial, Lasers Med. Sci. 24 (2009) 681688.

[19] Y.L. Qin, X.L. Luan, L.J. Bi, Y.Q. Sheng, C.N. Zhou, Z.G. Zhang, Comparison oftoluidine blue-mediated photodynamic therapy and conventional scalingtreatment for periodontitis in rats, J. Periodontal Res. 43 (2008) 162167.

[20] X.L. Luan, Y.L. Qin, L.J. Bi, C.Y. Hu, Z.G. Zhang, J. Lin, C.N. Zhou, Histologicalevaluation of the safety of toluidine blue-mediated photosensitization toperiodontal tissues in mice, Lasers Med. Sci. 24 (2009) 162166.

[21] G.C. Armitage, Development of a classication system for periodontal diseasesand conditions, Ann. Periodontol. 4 (1999) 16.

[22] A.D. Bangham, M.M. Standish, J.C. Watkins, Diffusion of univalent ions acrossthe lamellae of swollen phospholipids, J. Mol. Biol. 13 (1965) 238252.

[23] A.C. Pelegrino, M. Carolina, A.F. Gotardo, A.R. Simioni, M.D. Assis, A.C. Tedesco,

[27] S. Seguier, G. Godeau, N. Brousse, Collagen bres and inammatory cells inhealthy and diseased human gingival tissues: a comparative and quantitativestudy by immunohistochemistry and automated image analysis, J. Periodontol.71 (2000) 10791085.

[28] M. Idowu, T. Nyokong, Photophysical and photochemical properties of zincand aluminium phthalocyanines in the presence of magnetic uid, J.Photochem. Photobiol. A: Chem. 188 (2007) 200206.

[29] A.R. Simioni, C. Vaccari, M.I. Re, A.C. Tedesco, PHBHV/PCL microspheres asbiodegradable drug delivery systems (DDS) for photodynamic therapy (PDT), J.Mater. Sci. 45 (2008) 580584.

[30] C.S. Foote, Denition of type-I and type-II photosensitized oxidation,Photochem. Photobiol. 54 (1991) 659663.

[31] R. Malik, A. Manocha, D.K. Suresh, Photodynamic therapy a strategic review,Indian J. Dent. Res. 21 (2010) 285291.

[32] M.A. Biel, Photodynamic therapy of bacterial and fungal biolm infections,Meth. Mol. Biol. 635 (2010) 175194.

[33] B.W. Sigusch, M. Engelbrecht, A. Volpel, A. Holletschke, W. Pster, J. Schutze,Full-mouth antimicrobial photodynamic therapy in Fusobacteriumnucleatum-infected periodontitis patients, J. Periodontol. 81 (2010) 975981.

[34] M.A. Atieh, Photodynamic therapy as an adjunctive treatment for chronicperiodontitis: a meta-analysis, Lasers Med. Sci. 25 (2010) 605613.

[35] A. Azarpazhooh, P.S. Shah, H.C. Tenenbaum, M.B. Goldberg, The effect ofphotodynamic therapy for periodontitis: a systematic review and meta-analysis, J. Periodontol. 81 (2010) 414.

[36] P.C. Kousis, B.W. Henderson, P.G. Maier, S.O. Gollnick, Photodynamic therapyenhancement of antitumor immunity is regulated by neutrophils, Cancer Res.67 (2007) 1050110510.

[37] M.C. Morgan, R.M. Rashid, The effect of phototherapy on neutrophils, Int.Immunopharmacol. 9 (2009) 383388.

[38] R. Broady, J. Yu, M.K. Levings, Pro-tolerogenic effects of photodynamic therapywith TH9402 on dendritic cells, J. Clin. Apher. 23 (2008) 8291.

[39] N. Yusuf, S.K. Katiyar, C.A. Elmets, The immunosuppressive effects ofphthalocyanine photodynamic therapy in mice are mediated by CD4+ andCD8+ T cells and can be adoptively transferred to naive recipients, Photochem.Photobiol. 84 (2008) 366370.

354 S. Sguier et al. / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354Photophysical properties of crowned porphyrins, Photochem. Photobiol. 81(2005) 771776.

[24] F.L. Primo, M.V. Bentley, A.C. Tedesco, Photophysical studies and in vitro skinpermeation/retention of Foscan/nanoemulsion (NE) applicable tophotodynamic therapy skin cancer treatment, J. Nanosci. Nanotechnol. 8(2008) 340347.

[25] C. Heussen, E.D. Dowdle, Electrophoretic analysis of plasminogen activators inpolyacrylamide gels containing sodium dodecyl sulfate and copolymerizedsubstrates, Anal. Biochem. 102 (1980) 196202.

[26] S. Seguier, G. Godeau, M. Leborgne, G. Pivert, N. Brousse, Immunohistologicand morphometric analysis of cytotoxic T lymphocytes in gingivitis, J.Periodontol. 70 (1999) 13831391.[40] F. Prignano, T. Lotti, A. Spallanzani, S. Berti, V. de Giorgi, S. Moretti, Sequentialeffects of photodynamic treatment of basal cell carcinoma, J. Cutan. Pathol. 36(2009) 409416.

[41] D.E. King, H. Jiang, G.O. Simkin, M.O.K. Obochi, J.G. Levy, D.W.C. Hunt,Photodynamic alteration of the surface receptor expression pattern of murinesplenic dendritic cells, Scand. J. Immunol. 49 (1999) 184192.

[42] D.W.C. Hunt, J.G. Levy, Immunomodulatory aspects of photodynamic therapy,Exp. Opin. Invest. Drugs 7 (1998) 5764.

[43] N. Kmerik, M. Wilson, S. Poole, The effect of photodynamic action on twovirulence factors of Gram-negative bacteria, Photochem. Photobiol. 72 (2000)676680.

Impact of photodynamic therapy on inflammatory cells during human chronic periodontitisIntroductionMaterials and methodsPatient populationStudy designPhotodynamic therapy (PDT) and laser treatment (Fig. 1)Preparation of photosensitizersTissue preparationHistology and ImmunohistochemistryZymographyQuantitative determination of area fraction of collagen fibers, number of immunolabelled cell populations and zymogram lysis bands by computer-assisted image analysisStatistical analysis

ResultsArea fraction (AA%) of collagen fibersInflammatory cell populationsZymography

DiscussionConflict of interest and source of funding statementAcknowledgementsReferences