Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Departamento de Qumica, Faculdade de Filosoa, Cincias e Letras de Ribeiro Preto-FFCLRP, Universidade de So Paulo, 14040-901 Ribeiro Preto-SP, Brazil
Universit Paris Descartes, Hpital Europen Georges Pompidou, 56 rue Leblanc, 75737 Paris Cedex 15, France
Service dOdontologie, Hpital Louis Mourier, rue des Renouillers, 92700 Colombes, France
d
Departamento de Cirurgia, Traumatologia Buco-Maxilo-Facial e Periodontia, Faculdade de Odontologia-FORP, Universidade de So Paulo, 14040-904 Ribeiro Preto-SP, Brazil
e
Departamento de Biologia Celular e Molecular e Bioagentes Patognicos, Faculdade de Medicina-FMRP, Universidade de So Paulo, 14049-900 Ribeiro Preto-SP, Brazil
b
c
a r t i c l e
i n f o
Article history:
Received 23 March 2010
Received in revised form 6 August 2010
Accepted 10 August 2010
Available online 17 August 2010
Keywords:
PDT
Periodontal disease
Antigen-presenting cell
Dendritic cell
Phthalocyanine
a b s t r a c t
The aim of this study was to evaluate the effects of the photodynamic therapy (PDT) on the inammatory
inltrate and on the collagen network organization in human advanced chronic periodontitis. Two different drug delivery systems (DDS) were tested (liposomes and nanoemulsions) to determine if the effects of
PDT 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) or
in the group nanoemulsions (group N, n = 8) in order to compare the effects of each DDS. Seven days
before extractions one tooth of each patient was treated with PDT using phthalocyanine derivatives as
photosensitizers and the contralateral tooth was taken as control. In group L the density of gingival
collagen 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 delivery
system; the number of macrophages was signicantly decreased (p < 0.05) in group L while the number
of Langerhans cells was signicantly decreased in group N (p < 0.02). These ndings demonstrate that
PDT presents an impact on gingival inammatory phenomenon during chronic periodontitis and leads
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 initiated and maintained by the bacterial plaque and its metabolic
products which trigger the local inltration of inammatory cells
associated with the degradation of extracellular matrix molecules
[1,2]. Neither mechanical plaque removal nor ushing or rinsing
with disinfectants allows the total eradication of bacterial reservoirs within periodontal pockets. Current therapeutic strategies
for periodontitis that use antimicrobial agents such as tetracyclines
and metronidazole suffer from one major drawback: the difculty
to maintain therapeutic concentrations of the agent in the peri Corresponding author at: Universit Paris Descartes, Hpital Europen Georges
Pompidou, 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),
Bernard.coulomb@parisdescartes.fr (B. Coulomb), atedesco@usp.br (A.C. Tedesco).
1011-1344/$ - see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2010.08.007
S. Sguier et al. / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354
the pocket avoiding damage to adjacent host tissues [19] and disruption of the normal microora [20]. However, there is a lack of
in vivo studies evaluating the effect of PDT on the gingival inammatory cells and on the matrix macromolecules as collagen bers
during human periodontal diseases.
The aim of this study was to evaluate the potential efciency of
PDT on a limited number of patients presenting advanced chronic
human periodontitis. Two drug delivery systems (DDS) were tested
(liposomes and nanoemulsions) in case of each DDS interacts differently with a tissular target. The effect of PDT on the inammatory cells, on the density of the collagen network and on the
expression of metalloproteinases was evaluated from gingival
biopsies a week after the treatment.
2. Materials and methods
2.1. Patient population
The experimental protocol was reviewed and approved by the
Institutions Human Research Committee and the protocol was approved on June 21, 2007 (protocol 2007.1.487.58.7, Ribeiro Preto,
So Paulo University, Brazil) and the experiments were undertaken
with the understanding and written consent of each subject. Sixteen patients (6 females, 10 males, aged 5065) presenting two
teeth with a clinical diagnosis of advanced chronic periodontitis,
ultimate degree of tooth mobility (mobility IV, horizontal and axial
mobilities) and periodontal indication for extraction were selected.
For each selected patient, both teeth presented the same degree of
gingival inammation and the equivalent tooth mobility. Diagnosis
of chronic periodontitis was established on the basis of clinical and
radiographic criteria (bone resorption) according to the classication system for periodontal diseases and conditions [21]. The patients included in this study had neither other oral or systemic
diseases, nor any overt immunological abnormalities and did not
take any preoperatory medication.
2.2. Study design
The study was performed using the split-mouth design. A total
of 16 pairs of contralateral maxillary or mandibulary teeth were included. In each contralateral pair, one tooth was assigned as control whereas the other tooth was treated with photodynamic
therapy (PDT). No subgingival mechanical therapy (scaling and
root planing) was performed prior to PDT. All patients were treated
by the same operator and the extractions of both teeth (control and
treated) were performed 7 days after treatment by PDT. Gingival
tissue samples, which otherwise would have been discarded, were
obtained during surgery under local anaesthesia, avoiding local
anaesthetic inltration into the biopsy site, and deformation or
compression of the samples. Gingival samples of control and treated teeth were obtained from the inner part of the ap (that was in
contact 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 as
photosensitizers (NzPC and AlClPC) and two different drug delivery
systems were used: liposomes (n = 8, group L) and nanoemulsions
(n = 8, group N). The photosensitizer was applied on each surface of
the tooth by placing the applicator at the bottom of the periodontal
pocket and was continuously deposited in a coronal direction. This
application was done three times with 5 min of waiting between
each application and laser exposure was done 15 min after the last
application of the photosensitizer. The laser used in this study was
an Eagle Diode Laser (Quantum Technology, Brazil) with a wave-
349
350
S. Sguier et al. / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354
Fig. 1. Schematic representation showing the technical procedure of photodynamic therapy. (A) Application of the photosensitizer by placing the applicator in the
periodontal pocket. (B) Application of the laser using an Eagle Diode Laser (Quantum Technology, Brazil) with a wavelength of 670 nm, a uence rate of 0.5 W/cm2 delivered
during 40 s (10 s for each face of the tooth) and a total uence of 12.7 J/cm2.
Dako, Glostrup, Denmark), anti-CD8 (Suppressor/cytotoxic T lymphocytes, dilution 1:200, Dako), anti-CD4 (helper T lymphocytes,
dilution 1:100, Dako), anti-CD68 (monocytes/macrophages, dilution 1:200, Dako), anti-CD1a (Langerhans cells, dilution 1:100,
Dako) using an avidinbiotinimmunoperoxidase technique as
previously 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 polyacrylamide
gels (10 cm height, 1.5 mm thickness) (Millipore, Saint Quentin
en Yvelines, France) contained 1 mg/mL of gelatin [25] dispersed
in buffered solution consisting of 2.5 mL gel 1.5 M TrisHCl pH
8.8; 100 lL of sodium dodecyl sulfate (SDS) 10%, 4 mL polyacrylamide and 4 mL of distilled water pH 8.8 stacking gel contained
4% polyacrylamide in 0.125 M Tris, pH 6.8. Gels were polymerized
by adding 50 lL of 10% ammonium persulfate and 10 lL of 0.1%
TEMED. Samples (5 lL of conditioned medium) were half diluted
in 1 mol/L Tris pH 6.8 containing 50% glycerol and 0.4% bromophenol blue, and gels were run under Laemmli conditions (40 mA, 1 h).
Following electrophoresis, gels were washed twice in 200 mL of
2.5% Triton X-100 in distilled water under constant mechanical
stirring, 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 stained
with 0.25% Coomassie brilliant blue G-250 (50% methanol, 10%
acetic acid) and destained appropriately (40% methanol, 10% acetic
acid). Proteinase activity was evident as clear (unstained) zones. Finally the gels were incubated for one hour in 5% methanol, 7.5%
acetic acid and kept under cellophane as previously described [2].
351
S. Sguier et al. / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354
Fig. 2. Histological staining of collagen bers with sirius red F3Ba in control and treated gingival samples of patients treated by photodynamic therapy using liposomes
(group L). (A) Control gingival section of a patient of group L showing stained collagen bers (arrow) strongly destroyed and degraded during the inammatory phenomenon.
(B) Treated gingival section of a patient of group L showing an increased of the area fraction of the collagen bers (arrow) a week after the treatment by photodynamic
therapy. E, epithelium; CT, connective tissue; Original magnication 10.
Table 1
Mean number (SD) of inammatory cell populations (number of cells/mm2) in control and treated gingival samples by PDT in the group liposomes (group L) and the group
nanoemulsions (group N).
CD45+
(cells/mm2)
CD1a+
(cells/mm2)
CD68+
(cells/mm2)
CD8+
(cells/mm2)
CD4+
(cells/mm2)
Group L
Controls
Treatment
P-value
319 225
218 124
NS
138 85
115 71
NS
585 121
300 127
S (p = 0.042)
122 76
107 83
NS
157 111
131 66
NS
Group N
Controls
Treatment
P-value
178 47
161 115
NS
150 56
81 39
S (p = 0.019)
351 113
305 105
NS
96 29
102 96
NS
230 104
161 78
NS
The CD45+ cells, CD4+ cells, CD8+ cells and CD1a+ cells were quantied in the epithelium, the CD68+ cells were quantied in the connective tissue. Signicance of differences
(one-tailed Student t-test for paired series) between control and treated gingival samples in each group.
NS = not signicant, S = signicative difference (p < 0.05).
of patients with severe periodontitis using two drug delivery systems (DDS, liposomes and nanoemulsions) and gingival biopsies
were collected 1 week after the clinical treatment by PDT.
The phthalocyanine dyes used belong to a second generation of
dyes with important production of reactive oxygen species, mainly
singlet oxygen [28,29]. The main advance in the use of phthalocyanine as photosensitizer is the fact that this family of dyes could
act by the two classical PDT mechanisms of radical production
(type I) or singlet oxygen (type II) according to Foote [30]. The
association with the specic DDS allows a good biodistribution of
phthalocyanine dyes and an excellent stability.
Many studies [3,31] have shown that periodontopathogenic
bacteria are susceptible to lethal photosensitization and PDT has
been reported in the literature to be effective in eradicating various
micro-organisms using different photosensitizers, different wavelengths of light, and different light sources [32]. Recent clinical
and microbiological studies using PDT for periodontitis were performed to evaluate the effectiveness of PDT as a primary mode of
treatment or as an adjunct to non-surgical treatment of scaling
and root planning (SRP) compared to a conventional non-surgical
SRP treatment. For some authors [33,34] the adjuvant application
of PDT seems appropriate to reduce inammatory symptoms and
to successfully treat infection with Fusobacterium nucleatum; for
other [35] PDT as an independent treatment or as an adjunct to
SRP was not superior (changes in clinical attachment level, probing
depth, gingival recession, full-mouth plaque or bleeding scores) to
control treatment of SRP. However, there is a lack of histological
352
S. Sguier et al. / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354
Fig. 3. Immunohistochemical staining of CD68+ macrophages in control and treated gingival samples of patients treated by photodynamic therapy using liposomes (group L).
(A) Control gingival section of a patient of group L showing a large number of immunolabelled CD68+ macrophages in the upper gingival connective tissue (arrows). (B)
Treated gingival section of a patient of group L showing a decreased number of immunolabelled CD68+ macrophages in the upper gingival connective tissue (arrows). E,
epithelium; CT, connective tissue; Original magnication 20.
Fig. 4. Immunohistochemical staining of CD1a+ Langerhans cells in control and treated gingival samples of patients treated by photodynamic therapy using nanoemulsions
(group N). (A) Control gingival section of a patient of group N showing numerous CD1a+ Langerhans cells in the gingival epithelium (arrows). (B) Treated gingival section of a
patient of group N showing a decreased number of immunolabelled CD1a+ Langerhans cells in the gingival epithelium (arrows). E, epithelium; CT, connective tissue; Original
magnication 10.
Fig. 5. Gelatin zymogram revealing gelatinase activities in the supernatant of control (C) and treated (T) gingival explants by photodynamic therapy using liposomes. As
shown in zymogram, heterogenic gelatinase activities were observed between patients (n = 8) and no reproducible differences could be detected between control and treated
marginal gingiva of each patient.
and biochemical studies evaluating the effect of PDT on the gingival inammatory cells, on the matrix macromolecules as collagen
bers and on the expression of the metalloproteinases (MMPs)
during human periodontal diseases.
Our aim was to evaluate the consequence of PDT on the inammatory inltrate during human chronic periodontitis. Several studies reveal that PDT has a signicant impact on neutrophils [36,37]
but other studies suggest a pro-tolerogenic effects of PDT on dendritic cells [38] or describe immunosuppressive effects of phthalocyanine PDT mediated by CD4+ and CD8+ T cells [39]. Then, it
seems that PDT has an impact on different inammatory cell populations and it appears that the composition and the extension of
the inammatory inltrate differ according the time (15 min to
72 h) of cell analysis after PDT as suggested by Prignano et al.
S. Sguier et al. / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354
353
354
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
S. Sguier et al. / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 348354
incorporated into liposomes for photodynamic therapy use, J. Nanosci.
Nanotechnol. 8 (2008) 32083215.
J.P. Longo, S.P. Lozzi, A.R. Simioni, P.C. Morais, A.C. Tedesco, R.B. Azevedo,
Photodynamic therapy with aluminum-chloro-phthalocyanine induces
necrosis and vascular damage in mice tongue tumors, J. Photochem.
Photobiol. B 94 (2009) 143146.
N. Kmerik, H. Nakanishi, A.J. MacRobert, B. Henderson, P. Speight, M. Wilson,
In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photosensitization in an animal model, Antimicrob. Agents Chemother. 47 (2003)
932940.
M. Wilson, Lethal photosensitisation of oral bacteria and its potential
application in the photodynamic therapy of oral infections, Photochem.
Photobiol. Sci. 3 (2004) 412418.
R.R. de Oliveira, H.O. Schwartz-Filho, A.B. Novaes Jr., M. Taba Jr., Antimicrobial
photodynamic therapy in the non-surgical treatment of aggressive
periodontitis: a preliminary randomized controlled clinical study, J.
Periodontol. 78 (2007) 965973.
B.W. Sigusch, A. Ptzner, V. Albrecht, E. Glockmann, Efcacy of photodynamic
therapy on inammatory signs and two selected periodontopathogenic species
in a beagle dog model, J. Periodontol. 76 (2005) 11001105.
P. Chondros, D. Nikolidakis, N. Christodoulides, R. Rssler, N. Gutknecht, A.
Sculean, Photodynamic therapy as adjunct to non-surgical periodontal
treatment in patients on periodontal maintenance: a randomized controlled
clinical trial, Lasers Med. Sci. 24 (2009) 681688.
Y.L. Qin, X.L. Luan, L.J. Bi, Y.Q. Sheng, C.N. Zhou, Z.G. Zhang, Comparison of
toluidine blue-mediated photodynamic therapy and conventional scaling
treatment for periodontitis in rats, J. Periodontal Res. 43 (2008) 162167.
X.L. Luan, Y.L. Qin, L.J. Bi, C.Y. Hu, Z.G. Zhang, J. Lin, C.N. Zhou, Histological
evaluation of the safety of toluidine blue-mediated photosensitization to
periodontal tissues in mice, Lasers Med. Sci. 24 (2009) 162166.
G.C. Armitage, Development of a classication system for periodontal diseases
and conditions, Ann. Periodontol. 4 (1999) 16.
A.D. Bangham, M.M. Standish, J.C. Watkins, Diffusion of univalent ions across
the lamellae of swollen phospholipids, J. Mol. Biol. 13 (1965) 238252.
A.C. Pelegrino, M. Carolina, A.F. Gotardo, A.R. Simioni, M.D. Assis, A.C. Tedesco,
Photophysical properties of crowned porphyrins, Photochem. Photobiol. 81
(2005) 771776.
F.L. Primo, M.V. Bentley, A.C. Tedesco, Photophysical studies and in vitro skin
permeation/retention
of
Foscan/nanoemulsion
(NE)
applicable
to
photodynamic therapy skin cancer treatment, J. Nanosci. Nanotechnol. 8
(2008) 340347.
C. Heussen, E.D. Dowdle, Electrophoretic analysis of plasminogen activators in
polyacrylamide gels containing sodium dodecyl sulfate and copolymerized
substrates, Anal. Biochem. 102 (1980) 196202.
S. Seguier, G. Godeau, M. Leborgne, G. Pivert, N. Brousse, Immunohistologic
and morphometric analysis of cytotoxic T lymphocytes in gingivitis, J.
Periodontol. 70 (1999) 13831391.