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

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Impact of photodynamic therapy on inammatory cells during human

chronic periodontitis
Sylvie Sguier a,b,c,, Sergio L.S. Souza d, Anna C.V. Sverzut d, Andreza R. Simioni a, Fernando L. Primo a,
Agns Bodineau b,c, Vani M.A. Corra e, Bernard Coulomb b, Antonio C. Tedesco a
a

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

odontal pocket for a sufcient length of time to ensure eradication

of the bacteria present [3,4].
To supplement the armament of antibacterial measures and decrease the inammatory process, in recent years different attempts
were made to introduce photodynamic therapy (PDT) as a new
treatment of chronic periodontitis [38]. PDT is based on the injection, ingestion, or topical application of photosensitizers dyes (usually using drug delivery systems (DDS) such as liposomes or
nanoemulsions) followed by visible light activation. These photosensitizers are chemical compounds that absorb light at a specic
wavelength and are able to produce several reactive oxygen species (ROS) that activate biologic systems [913]. In particular, a
large number of micro-organisms (including oral species) have
been reported to be killed by PDT [14,15] and virulence factors
(lipopolysaccharides and proteases) have also been shown to be
reduced by photosensitization. Because it is easy to access the periodontal pocket, periodontitis would be very amenable to treatment
by PDT [1518]. Hence, the photosensitizer can be placed directly
into the pocket which can then be irradiated either through the
thin gingival tissues or via an optical ber placed directly into

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

length 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.
2.4. Preparation of photosensitizers
Liposomes have been used as drug delivery systems since the
1960s [22]. NzPC is a silicon (IV) phthalocyanine derivative (metal
phthalocyanine) with two axial ligands substituted in the macrocycle, belong to the second generation of photoactive agents used in
PDT. The incorporation of NzPC into the phospholipids bilayer of La-phosphatidylcholine and dimethyl-dioctadecylammoniumbromide (DDAB) was carried out according to the modied injection
method described by Pelegrino et al. [23]. Basically, 360 lL of an
ethanolic solution, which was 68.6 mmol/L phosphatidylcholine,
0.19 mmol/L DDAB and 50 lL in NzPC, was injected with a syringe
into 5 mL PBS, pH 7.4. The injection was performed at 56 C, under
magnetic stirring and at ow rate 1 lL/s.
Nanoemulsion was obtained by spontaneous emulsication
process as described by Primo et al. [24]. The natural soy phospholipids Epikuron 170 (7.5%) and Miglyol 812 N oil (250 lL) were obtained from Hulls Inc. (Puteaux, France) and initially dissolved in
10 mL of spectroscopic acetone at 55 C under magnetic stirrer.
In the same time the biopolymer Poloxamer 188 (SigmaAldrich
Co., St. Louis, MO, USA) at 7.5% was dissolved in ultra-pure water
to obtain an aqueous phase. In the sequence the organic phase
was added slowly in aqueous medium in the emulsication step
for 20 min. After total homogenization, the acetone was removed
by reduced pressure at approximately 6070 C for a nal volume
of 10 mL of nanoemulsion. Chloroaluminum phthalocyanine (SigmaAldrich Co., St. Louis, MO, USA) was dissolved directly in Miglyol 812 N oil at 55 C for nal concentration of 0.05 mg/mL in
nanoemulsion. Its incorporation in the nanoemulsion was accomplished on organic phase in emulsication process as previously
described [24]. The photophysical, photochemical and photobiological properties and biological responses of the phthalocyanine
derivates were similar as revealed in previous study from our
group and allow the comparison between them [912].
2.5. Tissue preparation
Seven days after the treatment by PDT, for each patient, the
control and treated teeth were removed and their marginal surrounding periodontal tissues were immediately divided into three
parts. The rst part was xed in 4% phosphate buffered formalin,
pH 7.4 and processed by routine laboratory techniques for parafn
embedding. The second part was immediately oriented in melting
isopentane (Tissue-Tek OCT Compound, Sakura, Zoeterwoude,
Netherlands) so that the sulcular/gingival epithelium and the
underlying connective tissue would be present in the same section,
and snap-frozen in liquid nitrogen. These two parts were used for
histological and immunohistochemistry studies. The third part was
maintained during 48 h in Hanks medium at 37 C under an atmosphere of 95% air, 5% CO2 and biochemical studies were performed
with conditioned media from these organ cultures kept at 80 C
until 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, then
xed in acetone for 10 min and kept at 80 C until the day of
the study. For all specimens staining of collagen bers was performed with sirius red F3Ba according to Junqueiras technique
(35). For immunohistochemistry, a panel of monoclonal antibodies
was used: anti-CD45 (Leukocyte common antigen, dilution 1:100,

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].

2.8. Quantitative determination of area fraction of collagen bers,

number of immunolabelled cell populations and zymogram lysis bands
by computer-assisted image analysis
The evaluation of the area fraction (AA%) of collagen bers was
determined in the whole of connective tissue visualized on the section stained with sirius red. Histological sections were observed
under a microscope (Zeiss Model Axioskop, Germany) using a
10 objective equipped with a video camera (Diagnostic, USA).
Captured images were transferred to a microcomputer in which a
software (Samba, Tribvn, France) calculated the area fraction
(AA%) occupied by the collagen bers.

The number of immunolabelled cells per unit area (number of

cells/mm2) was determined using the same computer-assisted image analysis and the immunolabelled cells were counted using 10
or 20 objectives either in the gingival epithelium or in the connective tissue, according to the cell density. As a matter of fact, a
previous study [26] has shown that, as in the connective tissue,
the number of inammatory cells in the gingival epithelium could
reect the severity of the inammatory phenomenon. The CD45+
cells, CD4+ cells, CD8+ cells and CD1a+ cells were quantied in
the epithelium, the CD68+ cells were quantied in the connective
tissue. For all the gingival samples ve elds per tissue section,
randomly selected, were analyzed. Thus, the number of immunolabelled cell subsets for each sample represents the mean of the ve
countings.
The surface area and the color intensity of zymogram lysis
bands were analyzed using Image J software (Image J; http:/rsb.info.nih.gov/ij/index.html).
2.9. Statistical analysis
The means of area fractions occupied by collagen bundles
(AA%), cell numbers and amounts of matrix metalloproteinases
(MMPs) between the control and treated teeth were compared
using the one-tailed Student t-test (paired series). The criterion
for statistical signicance was dened as a level of p < 0.05. The results are given as mean and standard deviation (mean SD) to describe 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) as
drug delivery system, the area fraction (AA%) of collagen bers
(66 19%) was signicantly increased (p < 0.02) in gingival samples when compared with controls (35 21%) (Fig. 2).
In patients (n = 8) treated by PDT using nanoemulsions (group
N), the area fraction of collagen bers (56 23%) was not signicantly different when compared with group C (44 23%).
3.2. Inammatory cell populations
For CD45+ cells, CD8+ cells and CD4+ cells, no signicant differences were observed between controls and treated teeth with

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).

either liposomes or nanoemulsions (Table 1). In both control

groups (for groups L and N) the numbers of cell populations were
in accordance with those reported previously in gingival samples
with chronic periodontitis [27].
In patients treated by PDT using liposomes (group L), the number of CD68+ macrophages was signicantly decreased (p < 0.05) in
gingival connective tissue (300 cells/mm2 187) when compared
with controls (585 cells/mm2 321) (Table 1 and Fig. 3).
In patients treated by PDT using nanoemulsions (group N), the
number of CD1a+ Langerhans cells was signicantly (p < 0.02) decreased in gingival epithelium (81 cells/mm2 39) when compared
with controls (150 cells/mm2 56) (Fig. 4).
3.3. Zymography
Whatever the drug delivery system (groups L and N), both pro
and active forms of MMP-2 and MMP-9 were present in control
and in gingival samples treated by PDT. Nevertheless, after quantitative analysis of zymogram lysis bands by computer-assisted image analysis, no differences could be detected between control and
treated 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

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

[40]. In the present study, the analysis of inammatory inltrate

was performed 7 days after PDT and at that time, the number of
some inammatory cell populations was decreased.
Our ndings showed that Antigen-Presenting Cells (APC, macrophages and Langerhans cells) located in the inammatory inltrate
and known as one of the rst immune barrier against pathogens, are
particularly sensitive to PDT. As a matter of fact, the number of macrophages was signicantly decreased after PDT using liposomes, and
the number of Langerhans cells (dendritic cells family, APC) was also
signicantly decreased after PDT using nanoemulsions.
A previous study showed that PDT has immunomodulatory
activity in various mouse models and dendritic cells treated by
PDT showed a reduced capacity to stimulate the proliferation of
alloreactive T cells [41]. The ability of PDT to down-regulate autoimmune processes appears to be related to its capacity to inuence
the immunostimulatory attributes of APC [38,42]. The PDT could
act on APC in two ways: rst, by decreasing their number, and second, by reducing their capacity of activation of T lymphocytes. As a
nal result, PDT could reduce the inammatory phenomenon since
it is well known that APC are able on phagocytose and endocytose
processes and thus, are cells specialized in the incorporation of foreign elements such as liposomes or nanoemulsions.
Interestingly, in our study, PDT targeted different cell populations depending upon the drug delivery system used. Concerning
Langerhans cells in group N (nanoemulsions), our results suggested that nanoemulsions could lead to their migration towards
the gingival connective tissue in order to realize the antigenic presentation. Concerning macrophages in group L (liposomes), our
ndings 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 constitutes the major component of the gingival connective tissue and
plays a key role in its architecture due to its bers organization.
During the periodontal diseases, inammatory cells including macrophages produce and release proteases (such as metalloproteinases) and cytokines which, together, generate periodontal tissue
destruction and a degradation of collagen bers. In addition, macrophages are strongly implicated in the turnover of matrix macromolecules by phagocytose of collagen bers. In patients treated by
PDT using liposomes, the density of collagen bers was signicantly increased in gingival samples when compared with controls.
This result could be linked to the signicant decrease in the number of the macrophages in this group.
Pathogenic bacteria and inammatory cells produce a number
of enzymes and potent proteases capable of degrading host tissues
leading to the periodontal destruction and the degradation of collagen bers observed during periodontitis. Nevertheless, studies on
the effect of photosensitizers in combination with light in this context have, in general, focused on the effects of this potential therapeutic modality on the viability of the micro-organisms or on the
reduction of bacterial virulence factors [43]. It is well known by
experiments on beagle dogs [17] and humans [18] that PDT procedure induces a signicant reduction of periodontopathogens-infected sites.
Thus, in the present study we also analyzed by zymography the
activities of the gelatinases (MMP-2 and MMP-9) since the active
form of MMP-9 has been proposed as a marker for the clinical
severity of periodontal diseases a previous study [2]. In our conditions, the activities of the gelatinases were not signicantly different between control and treated teeth with either liposomes or
nanoemulsions. It is likely that the experimental period of 1 week
between the clinical treatment by PDT and the teeth extraction
was unadapted to evidence eventual changes in the MMPs expression. Furthermore, it seems strongly possible that 7 days after PDT
there is a re-colonisation of periodontal pocket by bacterial
elements.

353

In conclusion, our ndings demonstrate that the treatment of

chronic periodontitis by PDT leads to a specic decrease of antigen-presenting cells populations according to the drug delivery
system. Thus, PDT might be considered as an effective coadjuvant
treatment for chronic periodontal diseases by supplementing the
antibacterial mechanical measures. Furthermore, the use of liposomes has the advantage to reduce the degradation of the gingival
extracellular matrix. These results are thus encouraging and now
justify to initiate an additional study with an increased number
of patients including an analysis of periodontal pocket re-colonisation 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 conict of interest including any nancial, personal or other relationships with other people or organizations within 3 years of
beginning our submitted work that could inappropriately inuence, or be perceived to inuence, our work.
This work was supported by grants from Fundao de Amparo a
Pesquisa do Estado de So Paulo-FAPESP, So Paulo, Brasil; Conselho Nacional de Desenvolvimento Cientco e Tecnlogico-CNPq,
Braslia, Brasil. This work was also supported by grants from Paris
Descartes University, France.
This collaboration between Paris Descartes University and Sao
Paulo University is supported by CAPES/COFECUB No. 523/06.
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 FAPESP fellowships.
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 breakdown
during periodontitis linked to inammatory cells and expression of matrix
metalloproteinases and tissue inhibitors of metalloproteinases in human
gingival tissue?, J Periodontol. 72 (2001) 13981406.
[3] P. Meisel, T. Kocher, Photodynamic therapy for periodontal diseases: state of
the art, J. Photochem. Photobiol. B 79 (2005) 159170.
[4] G. Jori, Photodynamic therapy of microbial infections: state of the art and
perspectives, 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-induced
periodontitis 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 dental
furcations, J. Periodontol. 79 (2008) 10811088.
[7] N. Christodoulides, D. Nikolidakis, P. Chondros, J. Becker, F. Schwarz, R. Rossler,
A. Sculean, Photodynamic therapy as an adjunct to non-surgical periodontal
treatment: a randomized, controlled clinical trial, J. Periodontol. 79 (2008)
16381644.
[8] A. Braun, C. Dehn, F. Krause, S. Jepsen, Short-term clinical effects of adjunctive
antimicrobial photodynamic therapy in periodontal treatment: a randomized
clinical trial, J. Clin. Periodontol. 35 (2008) 877884.
[9] C.N. Lunardi, J.C. Rotta, A.C. Tedesco,
Synthesis, photophysical and
photobiological study of synergic photosensitizer: zinc-phthalocyanine with
2+
Ca 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 lament
behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa
cells, Cell Biol. Int. 32 (2008) 10241028.
[11] S.M.T. Nunes, F.S. Sguila, A.C. Tedesco, Photophysical studies of zinc
phthalocyanines and chloroaluminium phthalocyanines incorporated into
liposomes in the presence of additives, Braz. J. Med. Biol. Res. 37 (2004)
273284.
[12] A.R. Simioni, M.M. Pelisson, M. Beltrame Jr., A.C. Tedesco, Photophysical and
photobiological studies of a silicon tribenzonaphthoporphyrazinato

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.

[27] S. Seguier, G. Godeau, N. Brousse, Collagen bres and inammatory cells in

healthy and diseased human gingival tissues: a comparative and quantitative
study by immunohistochemistry and automated image analysis, J. Periodontol.
71 (2000) 10791085.
[28] M. Idowu, T. Nyokong, Photophysical and photochemical properties of zinc
and 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 as
biodegradable 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 Fusobacterium
nucleatum-infected periodontitis patients, J. Periodontol. 81 (2010) 975981.
[34] M.A. Atieh, Photodynamic therapy as an adjunctive treatment for chronic
periodontitis: a meta-analysis, Lasers Med. Sci. 25 (2010) 605613.
[35] A. Azarpazhooh, P.S. Shah, H.C. Tenenbaum, M.B. Goldberg, The effect of
photodynamic therapy for periodontitis: a systematic review and metaanalysis, J. Periodontol. 81 (2010) 414.
[36] P.C. Kousis, B.W. Henderson, P.G. Maier, S.O. Gollnick, Photodynamic therapy
enhancement 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 therapy
with TH9402 on dendritic cells, J. Clin. Apher. 23 (2008) 8291.
[39] N. Yusuf, S.K. Katiyar, C.A. Elmets, The immunosuppressive effects of
phthalocyanine photodynamic therapy in mice are mediated by CD4+ and
CD8+ T cells and can be adoptively transferred to naive recipients, Photochem.
Photobiol. 84 (2008) 366370.
[40] F. Prignano, T. Lotti, A. Spallanzani, S. Berti, V. de Giorgi, S. Moretti, Sequential
effects 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 murine
splenic 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 two
virulence factors of Gram-negative bacteria, Photochem. Photobiol. 72 (2000)
676680.