Archives of Oral Biology 110 (2020) 104600

Contents lists available at ScienceDirect

Archives of Oral Biology

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

Treatment of experimental periodontitis with chlorhexidine as adjuvant to T

scaling and root planing
Nubia Rosa Priettoa, Thiago Marchi Martinsa, Carolina dos Santos Santinonib,*,
Natália Marcumini Polaa, Edilson Ervolinoc, Amália Machado Bielemanna,
Fábio Renato Manzolli Leited
a
Graduate Program in Dentistry, School of Dentistry, Federal University of Pelotas, Pelotas, Brazil
b
Dental School of Presidente Prudente, Graduate Program in Dentistry (GPD - Master's Degree), University of Western Sao Paulo, Presidente Prudente, Brazil
c
Department of Basic Sciences, Dental School of Araçatuba, São Paulo State University, São Paulo, Brazil
d
Section of Periodontology, Department of Dentistry and Oral Health, Aarhus University, Aarhus, Denmark

A R T I C LE I N FO A B S T R A C T

Keywords: Objectives: To assess whether subgingival irrigation with 0.12 % or 0.2 % chlorhexidine (CHX) immediately after
Periodontitis scaling and root planing (SRP) enhances periodontal tissue repair compared to irrigation with saline solution
Periodontal diseases (control).
Rats Materials and methods: Periodontitis was ligature-induced in rat molars for 7 days. Animals were distributed into
Wound healing
three groups: 1) SRP group, SRP and irrigation with 0.9 % saline (n = 30); 2) SRP + 0.12 % CHX group, SRP and
Dental scaling
Chlorhexidine
irrigation with 0.12 % CHX (n = 30); 3) SRP + 0.2 % CHX group, SRP and irrigation with 0.2 % CHX (n = 30).
Animals were killed at 7, 15, and 30 days after treatment. Furcation region was histometrically analyzed to
determine the bone area. Immunohistochemical reactions were performed for receptor activator of nuclear
factor-kB ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP).
Results: Both chlorhexidine groups presented less inflammation and improved tissue repair along the entire
experiment when compared with the SRP group. In the histometric analysis at 7, 15 and 30 days, SRP group
(4.58 ± 2.51 mm2, 4.21 ± 1.25 mm2, 3.49 ± 1.48 mm2), showed statistically less bone area than groups
SRP + 0.12 % CHX (1.86 ± 1.11 mm2; 0.79 ± 0.27 mm2; 0.34 ± 0.14 mm2) and SRP + 0.2 % CHX
(1.14 ± 0.51 mm2; 0.98 ± 0.40 mm2; 0.41 ± 0.21 mm2). Both chlorhexidine concentrations modulated the
expression of TRAP, RANKL and OPG.
Conclusions: Subgingival irrigation with chlorhexidine contributed for a quicker shift from a proinflammatory
destructive profile to healing of periodontal tissues.

1. Introduction 2002) and the ratio between their activity and inhibition influences the
healing outcome (Tüter et al., 2002). Different approaches to modulate
Periodontitis is an inflammatory condition, which gradually leads to host inflammatory response have been suggested to induce a more
destruction of the supportive tissues of teeth. The host inflammatory balanced periodontal healing. The idea is to inhibit inflammatory
response triggered by bacteria compounds or other causes of period- pathways leading to tissue breakdown and to trigger those associated
ontal diseases, e.g. diabetes, smoking, among others, is the major re- with cell proliferation and differentiation (Gokhale & Padhye, 2013).
sponsible for the tissue destruction (Nicu & Loos, 2016). Extracellular Therefore, MMP inhibition would be appreciated after periodontal
matrix components, e.g. collagen, fibronectin, and proteoglycans, treatment. Tetracyclines, bisphosphonates and chlorhexidine have been
maintain the structural integrity of the anchoring apparatus. Extra- shown to inhibit the activity of several MMP family members (Gendron,
cellular matrix components degradation causes irreversible loss of Grenier, Sorsa, & Mayrand, 1999; Golub, Sorsa, & Lee, 1995; Teronen,
connective tissue and alveolar bone (Kornman, Page, & Tonetti, 1997). Konttinen, & Lindqvist, 1997).
Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, are Chlorhexidine (CHX) is an antimicrobial agent with broad spectrum
strategic regulators of the healing process (Tüter, Kurtis, & Serdar, antimicrobial activity. This cationic bis-biguanide reduces dental

⁎
Corresponding author at: Universidade do Oeste Paulista – UNOESTE, José Bongiovani, 700 – Cidade Universitária, Presidente Prudente, SP, 19050-920, Brazil.
E-mail address: carolsantinoni@msn.com (C.d.S. Santinoni).

https://doi.org/10.1016/j.archoralbio.2019.104600
Received 9 August 2019; Received in revised form 3 October 2019; Accepted 3 November 2019
0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
N.R. Prietto, et al. Archives of Oral Biology 110 (2020) 104600

biofilm proliferation, therefore its use in subgingival irrigation. CHX biofilm accumulation and consequent periodontitis (De Almeida,
presents proteolytic activity of certain periodontal pathogens (Grenier, Ervolino, & Bonfietti, 2015). The ligature was maintained for 7 days
1993) and antioxidative capacity (Firatli, Unal, Onan, & Sandalli, 1994) and SRP was performed immediately after ligature removal by the same
through inhibition of superoxide anion generation in red and white trained operator using 10 distal–medial traction movements of a curette
blood cells (Gabler, Bullock, & Creamer, 1987; Goultschin & Levy, (−2 Min. Five curette, Hu-Friedy, Chicago, IL) over the buccal and
1986). Chlorhexidine scavenge long-lived nitrogen-chlorine oxidants lingual surfaces of the treated area (De Almeida et al., 2015). The in-
capable of degrading protein sulfhydryl groups linked to endogenous terproximal and furcation areas were scraped using cervical–occlusal
autoactivation of neutrophil collagenase (Weiss, 1989; Weiss, Peppin, traction movements of the same curette.
Ortiz, Ragsdale, & Test, 1985). Due to the role of neutrophils in the
protection of periodontal tissues against bacteria invasion but also in 2.3. Subgingival irrigation
the degradation of the extracellular matrix, a substance that controls
bacteria proliferation and collagenase activity would be interesting. Irrigation was performed just once, immediately after SRP. Group
Recently, a systematic review compared treatment of patients with control received irrigation with 1 mL of 0.9 % saline solution. Groups
periodontitis who underwent mechanical periodontal therapy (scaling treated with chlorhexidine received 1 mL of 0.12 % chlorhexidine so-
and root planing - SRP) combined or not with CHX as an adjunct (da lution (Maquira Produtos Odontológicos, Maringa, Paraná, Brazil) or
Costa, Amaral, Barbirato, Leao, & Fogacci, 2017). The authors con- 1 mL of 0.2 % chlorhexidine solution (Maquira Produtos Odontológicos,
sidered clinical parameters such as probing depth (PD) and clinical Maringa, Paraná, Brazil). All the solutions were inserted slowly into the
attachment level (CAL) as primary outcomes. Groups treated with CHX periodontal pocket, using a 1 mL syringe and insulin needle without
plus SRP had slightly better results than did those treated only with SRP bevel.
(da Costa et al., 2017).
Pondering lack of studies that evaluate the effects of chlorhexidine 2.4. Laboratorial processing
on periodontitis healing, the purpose of this study was to compare the
efficacy chlorhexidine plus conventional mechanical scaling root At 7, 15, or 30 days after treatment, the animals were euthanized
planing (SRP) on alveolar bone loss in a model of acute experimental with an overdose of thiopental (150 mg/kg) (Cristália, Itapira, São
periodontitis in rats. Paulo, Brazil). Mandibles were dissected and fixed in 4 % formaldehyde
for 48 h. Specimens were demineralized in 10 % EDTA, washed, de-
2. Materials and methods hydrated in graded alcohol concentrations, cleared in xylene, and em-
bedded in paraffin. The paraffin blocks were serially cut in a mesio-
2.1. Experimental model distal direction along the long axis of the teeth to generate 5 μm-thick
longitudinal sections. After excluding the first and the last sections
The experimental protocol was approved by the Ethics Committee where the furcation region was evident, five equidistant sections of
on Animal Experimentation (protocol 1587) of the Federal University of each specimen block were selected and stained with hematoxylin and
Pelotas. The Animal Research: Reporting of in vivo Experiments eosin (HE) for histologic and histometric analysis (Fernandes et al.,
(ARRIVE) guidelines were used for reporting. 2009). Other histologic sections were subjected to the indirect im-
The sample size was determined to recognize a significant difference munoperoxidase method. The histological sections were deparaffinized
of 20 % among groups in a scenario with 10 % of standard deviation, and rehydrated through a graded series of ethanol. For antigen re-
power set at 80 % power and 95 % confidence interval. Data from trieval, the slides were incubated in phosphate buffer solution (0.1 M,
changes in the area of alveolar bone (our primary outcome) after li- pH 7.4) at 95 °C for 10 min. At the end of each step of the im-
gature-induced periodontitis from a previous study were used (Messora munohistochemical reaction, the histological slides were washed with a
et al., 2016). A sample size of eight animals per group was required. phosphate buffer solution (0.1 M, pH 7.4). Subsequently, the slides
Considering potential attrition rates due to potential tooth loss, ligature were immersed in 3 % hydrogen peroxide for 1 h and then 1 % albumin
loss, anesthesia complications, among others, 10 animals per experi- bovine sérum for 12 h to block the endogenous peroxidase and non-
mental time were used. specific sites, respectively. The slides containing samples of each of the
Ninety 2-month-old rats (Rattus norvegicus albinus, Wistar) weighing experimental groups were divided into three batches and each batch
130–160 g were obtained from the Central Animal Facility of the was incubated with one of the following primary antibodies (Santa Cruz
Federal University of Pelotas. The rats were kept in individual cages Biotechnology, Santa Cruz, CA): anti-TRAP (goat anti-TRAP), anti-
under the same standard conditions of illuminations (12-hr light/dark RANKL (goat anti-RANKL), and anti-OPG (goat anti-OPG). The sections
cycle), controlled temperature of 22 ± 1 °C, and food and water ad were incubated with a biotinylated secondary antibody for 2 h and
libitum during all the experimental period. The animals were subjected subsequently treated with a streptavidin–horseradish peroxidase con-
to a period of 7 days of acclimatization to the environment and then jugate (Universal Dako Labeled HRP Streptavidin–Biotin Kit®, Dako
randomly allocated into the following groups (n = 30) according to the Laboratories, CA, USA) for 1 h. The reaction was developed using the
following protocol: 1) scaling root planing - SRP (control) subjected to chromogen 3,30-diaminobenzidine (DAB chromogen Kit®, Dako La-
experimental periodontitis (EP) induction, SRP and irrigation with 1 mL boratories, CA, USA) and counterstained with Harris hematoxylin. All
of 0.9 % saline solution; 2) SRP + 0.12 % CHX, subjected to EP in- samples were accompanied by a negative control, i.e. specimens sub-
duction, SRP and irrigation with 1 mL 0.12 % chlorhexidine solution jected to the aforementioned procedures but without the primary an-
(Maquira Produtos Odontológicos, Maringa, Paraná, Brazil); and 3) tibodies.
SRP + 0.2 % CHX, SRP after EP induction and irrigation with 1 mL 0.2
% chlorhexidine solution (Maquira Produtos Odontológicos, Maringa, 2.5. Histological and histometric analysis
Paraná, Brazil).
Sections dyed by H&E were analyzed under light microscopy to
2.2. Acute experimental periodontitis induction (EP) establish the bone loss and characteristics of periodontal ligament in
the furcation region of first molars. The area of bone loss in the fur-
Animals were anesthetized by intramuscular injection with a mix- cation region was histometrically determined using an image analysis
ture of ketamine§ (70 mg/kg) and xylazine‖ (6 mg/kg). A cotton thread system (ImageJ Tool, San Antonio, TX, USA) as previously described
(Corrente Algodão no. 24, Coats Corrente, São Paulo, São Paulo, Brazil) (De Almeida et al., 2015). After excluding the first and last sections
was tied around the mandibular left first molar of each rat to induce where the furcation region was evident, 5 equidistant sections of each

2
N.R. Prietto, et al. Archives of Oral Biology 110 (2020) 104600

specimen block were selected and captured by a digital camera coupled Table 1
to a light microscope. For determination of changes in the area of al- Bone loss in mm2 (mean ± SD) in the furcation area according to groups and
veolar bone between groups (primary outcome), bone loss (mm2) in the experimental periods.
furcation region was determined histometrically (De Almeida et al., 7 days 15 days 30 days
2015). One blinded, trained examiner selected the sections for the
histometric and histological analysis. Another masked, calibrated ex- SRP only (control) 4.58 ± 2.51* 4.21 ± 1.25* 3.49 ± 1.48*
SRP + CHX 0.12% 1.86 ± 1.11 0.79 ± 0.27 0.34 ± 0.14
aminer conducted the histometric analysis.
SRP + CHX 0.2% 1.14 ± 0.51 0.98 ± 0.40 0.41 ± 0.21

2.6. Immunohistochemical analysis * Significantly lower than groups CHX 0.12 % and CHX 0.2 %. No differences
observed between both chlorhexidine concentrations. SRP, Scaling and root
An examiner who was blinded to the groups and treatments (CSS) planing; CHX, chlorhexidine.
conducted the immunohistochemical analyses. The values for each
section were measured three times by the same examiner on different
days to reduce variations in the data. Immunolabeling for TRAP,
RANKL and OPG was analyzed in the entire furcation region of the
mandibular first molar under ×400 magnification. To determine po-
tential differences in the pattern of immunolabeling between groups
(secondary outcome), a semi-quantitative analysis was performed.
Three histological sections from each group were used, and the im-
munolabeling criteria as follows: 0, total absence of immunoreactive
(IR) cells; 1, low immunolabeling (approximately one quarter of IR
cells); 2, moderate immunolabeling (approximately one half of IR cells);
and 3, high immunolabeling (approximately three quarters of IR cells)
(De Almeida et al., 2015).

2.7. Statistical analyses

Histometric data were analyzed using a statistics software (StataSE

14.1, StataCorp, College Station, TX, USA). Intra-examiner reproduci-
bility was checked with paired t-test and Pearson’s correlation coeffi-
cient before and during the histometric analysis. Seven sections from Fig. 1. Photomicrograph illustrating the bone septum in the furcation region of
each group were selected at random and analyzed twice with a 1-week ligature-induced periodontitis in the mandibular first molar after 7 days of
interval between analyses. Data normality was analyzed using the treatment. A - Group SRP (×12.5; HE); B - Group CHX 0.12 % (×12.5; HE); C -
Shapiro-Wilk test. Groups were compared via Kruskal-Wallis one-way Group CHX 0.2 % (×12.5; HE); D - Group SRP (×40; HE); E - Group CHX 0.12
analysis of variance (ANOVA). Multiple comparisons (post hoc) were % (×40; HE). The inflammatory infiltrate is delimitated by arrows in A, B and
C. Arrows in E highlight active osteoclasts.
performed using Dunn’s test, and P values < .05 indicated statistical
significance.
inflammatory infiltrate was moderate to discrete at 7 days after SRP
3. Results plus subgingival irrigation (Fig. 1B and 1C). It consisted of a mix of
acute and chronic inflammatory cells in an advanced under organiza-
3.1. Histometric analyses of lose bone in the furcation of teeth (PBF) tion connective tissue. The bone septum presented a regular outline
with some osteoclasts (Fig. 1E). Cementum resorption areas were ob-
Change in alveolar bone loss between groups was set as the primary served in most specimens.
outcome variable. Paired t-test statistics was calculated, and no differ- At 15 days after SRP alone, the acute inflammatory infiltrate re-
ences were observed in the mean values for comparison (p > 0.05). In duced but was still more evident than chronic inflammatory cells, e.g.
addition, the Pearson’s correlation coefficient revealed high correlation macrophages and lymphocytes. Connective tissue was disorganized
(0.89) between the two measurements for the histometric analyses. In with small number of fibroblasts and high number of neutrophils in
the intergroup analysis, the animals in the SRP group showed a lower degeneration. At 30 days after SRP, connective tissue occupied a large
area of bone than the animals in both chlorhexidine groups for all ex- portion of the furcation area. Inflammatory cells were located closer to
perimental periods (P < 0.05). The animals in the SRP + 0.12 % CHX the septum bone, which showed more regular outline (Fig. 2A). The
and in the SRP + 0.2 % CHX groups showed similar bone area at 7, 15 cementum still showed small areas of resorption with most of them
and 30 days (P > 0.05). In the intragroup analysis, the animals in the inactive (Fig. 2D). Cellular cement was deposited in some areas closer
SRP group showed a similar area of bone at 7, 15 and 30 days to the remaining septum.
(P > 0.05). The animals in the SRP + 0.12 % CHX and SRP + 0.2 % At 15 and 30 days after treatments the inflammatory infiltrate was
CHX groups showed a higher area of bone in each period, with a sig- absent in most of the specimens or very few inflammatory cells were
nificant difference (P > 0.05) specially when the 7 days’ period was observed in a completely organized connective tissue (Fig. 2B and C).
compared with the 30 days’ period. Results are summarized in Table 1. Interradicular septum was regular and covered with osteoblasts, os-
teocytes were abundant and osteoclasts rarely observed. Periodontal
3.2. Descriptive histologic analyses ligament was rich in vessels and collagen fibers. All cementum re-
sorption areas were inactive and undergoing repair or had already been
In the SRP group (control), a large number of neutrophils occupied replaced by cellular cement (Fig. 2E). No visual differences were ob-
the connective tissue at the furcation area at 7 days after treatment served between both concentrations of chlorhexidine.
(Fig. 1A). The acute inflammatory infiltrate spread all over the furca-
tion space reaching the area around the bone septum (Fig. 1D). The 3.3. Immunohistochemical analysis
bone consisted of thin and irregular trabeculae with resorption lacunae
and active osteoclasts. In both SRP plus chlorhexidine groups, the acute Change in the pattern of immunolabeling between groups was set as

3
N.R. Prietto, et al. Archives of Oral Biology 110 (2020) 104600

4. Discussion

Destructive forms of periodontal inflammation might lead to

breakdown of connective tissue extracellular matrix. The major de-
struction observed in this process is host immune system-mediated but
usually initiated by bacteria and their toxic byproducts and other sys-
temic causes, such as smoking and diabetes. Commercially, chlorhex-
idine is found at high doses (0.12–2.0 %) to obtain bactericidal effects
(Matthews, 2011). However, low and non-toxic concentrations of
chlorhexidine up to 0.05 % might modulate the immune response by
acting on neutrophils activity (Goultschin & Levy, 1986; Montecucco,
Bertolotto, & Ottonello, 2009).
Even though neutrophils are recruited to periodontal tissues to have
a protective role, they possess a high potential to damage the tissue.
After reaching the site, enzymes and reactive oxygen species (ROS) are
released by neutrophils in an attempt to deactivate inflammatory trig-
gers (Montecucco et al., 2009). However, neutrophils release proteases,
cytokines and myeloperoxidase (MPO), increasing oxidant release and
Fig. 2. Photomicrograph illustrating the bone septum in the furcation region of inactivating protective antiproteases (Montecucco et al., 2009; Test &
ligature-induced periodontitis in the mandibular first molar after 30 days of Weiss, 1986). The superoxide anions are quickly converted to hydrogen
treatment. A - Group SRP (×12.5; HE); B - Group CHX 0.12 % (×12.5; HE); C - peroxide, which is transformed into hypochlorous acid (HOCl) by MPO
Group CHX 0.2 % (×12.5; HE); D - Group SRP (×40; HE); E - Group CHX 0.2 % (Test & Weiss, 1986). Thus, the observed damage to periodontal tissues
(×40; HE). The remaining inflammatory infiltrate is delimitated by arrows in is mainly caused by HOCl by increasing cell oxidative stress and by
A. Arrows in D highlight areas of active cement resorption and in E areas of
increase of elastase-mediated breakdown of connective tissue compo-
cement formation.
nents, e.g. elastin, collagen, fibronectin and laminin. Control of neu-
trophil-mediated proteolysis is one of the key points in pharmacology to
Table 2 reduce inflammatory tissue injury. In this study, CHX seemed to reduce
OPG, RANKL and TRAP immunoexpression in the furcation area according to the number of inflammatory cells to the furcation area and promote a
groups and experimental periods. more balanced, organized and quicker repair of bone septum and per-
Period Groups OPG RANKL TRAP iodontal ligament. We suppose that the higher percentage of bone in
both chlorhexidine groups was due to the reaction of CHX with HOCl
7 days SRP only (control) ++ ++ ++
faster than HOCl-induced inactivation of inhibitors of neutrophil elas-
CHX 0.12% + ++ +++
CHX 0.2 % ++ + +
tases as previously described (Montecucco et al., 2009).
15 days SRP only (control) ++ ++ ++ The potential to neutralize the inflammation by CHX seems to be
CHX 0.12% + ++ +++ dependent on its cation-chelating properties (Gendron et al., 1999).
CHX 0.2 % ++ ++ + Also, CHX quickly deactivate large and small portions of lipopoly-
30 days SRP only (control) ++ ++ ++
saccharide (LPS) and lipoteichoic acid (LTA) from oral bacteria, pre-
CHX 0.12% +++ ++ +++
CHX 0.2 % ++ + +++ venting further perpetuation of inflammatory cell activation (Marinho,
Martinho, Leite, Nascimento, & Gomes, 2015). The capability of CHX to
+ low immunolabeling; ++ moderate immunolabeling; +++ high im- neutralize LPS and LTA before the stimulation of the innate immune
munolabeling. response is important since biofilm disruption by mechanical period-
ontal treatment causes the release of high levels of immunostimulatory
secondary outcome. Table 2 show the immunohistochemical results for bacterial cell wall components, which might exacerbate and/or perpe-
OPG, RANKL and TRAP immunolabeling, using a symbols system. Fig. 3 tuate periodontal inflammation and destruction (Leite, Aquino,
illustrate TRAP, OPG and RANKL immunolabeling in the furcation re- Guimarães, Cirelli, & Junior, 2014; Leite, de Aquino, & Guimarães,
gion. 2015). CHX inhibits the cell activation by LPS and LTA in a con-
centration-dependent manner with almost complete inhibition at a
3.3.1. OPG concentration of 0.0005 % and 0.0001 %, respectively. These data
Group SRP at 7, 15 and 30 days postoperative presented moderate might explain the lack of differences in tissue repair between the
immunolabeling. Group CHX 0.12 % presented low immunolabeling at SRP + CHX 0.12 % and the SRP + CHX 0.2 % groups. In addition, re-
7 and 15 days postoperative. Group CHX 0.12 % presented high im- specting differences in study designs, a previous systematic review has
munolabeling at 30 days postoperative. Group CHX 0.2 % presented demonstrated that there is no evidence that one concentration of
moderate immunolabeling at 7, 15 and 30 days postoperative. chlorhexidine rinse is more effective than another in reducing gingivitis
levels (James et al., 2017).
3.3.2. RANKL According to previous studies (Goultschin & Levy, 1986;
Groups SRP and CHX 0.12 % presented moderate immunolabeling Montecucco et al., 2009), CHX concentrations above 0.05 % might not
at 7, 15 and 30 days postoperative. Group CHX 0.2 % presented low lead to an extra modulation of the immune response. In this study,
immunolabeling at 7 and 30 days and moderate immunolabeling at 15 groups treated with chlorhexidine presented higher number of TRAP-
days postoperative. immunolabeled cells than SRP group. The opposite result should be
expected, since TRAP labelling is an indicator of osteoclast differ-
3.3.3. TRAP entiation. Two hypotheses can explain these results. One of them is that
Group SRP presented moderate immunolabeling at 7, 15 and 30 the osteoclasts present in the furcation region were not active, as well as
days postoperative. Group CHX 0.12 % presented high immunolabeling TRAP, which was accumulated into the developing medullary bone
at 7, 15 and 30 days postoperative. Group CHX 0.2 % presented low (Yamamoto, Yamagata, & Nagai, 1995). Other possible justification is
immunolabeling at 7 and 15 days and high immunolabeling at 30 days that groups treated with chlorhexidine were in a more advanced phase
postoperative. of the bone remodeling process after treatment and, therefore, a higher

4
N.R. Prietto, et al. Archives of Oral Biology 110 (2020) 104600

Fig. 3. Photomicrograph illustrating TRAP,

OPG and RANKL immunolabeling in the fur-
cation region of ligature-induced periodontitis
in the mandibular first molar.
Photomicrographs showing the im-
munolabeling pattern of TRAP (arrows) in the
SRP (A), CHX 0.12 % (B), and CHX 0.2 % (C)
groups. Photomicrographs showing the im-
munolabeling pattern of OPG (arrows) in the
SRP (D), CHX 0.12 % (E), and CHX 0.2 % (F)
groups. Photomicrographs showing the im-
munolabeling pattern of RANKL (arrows) in
the SRP (G), CHX 0.12 % (H), and CHX 0.2 %
(I) groups. Original magnification ×400; he-
matoxylin counterstaining. ab, alveolar bone;
chx; chlorhexidine; d, dentin; srp, scaling and
root planing (For interpretation of the refer-
ences to colour in this figure legend, the reader
is referred to the web version of this article).

number of TRAP-immunolabeled cells was observed. periodontal disease induction using ligature is extensively used because
Local administration of drugs in the periodontal pocket is an ef- the alveolar bone loss develops predictably after 7 days (Kuhr, Popa-
fective treatment for mechanical debridement. Localized therapies have Wagner, Schmoll, Schwahn, & Kocher, 2004). The presence of bacteria
received significant attention because of the pattern of destruction of is mandatory since germ-free rats do not develop significant alveolar
site-specific periodontal infections and the potential collateral effects of bone destruction after ligature placement. However, a limitation of the
systemic antimicrobials and anti-inflammatory agents (Tonetti & model is that the combination of bacteria with mechanical trauma
Cortellini, 2015). Another important reason for the development of during ligature placement may accelerate the periodontal tissue
effective local application of drugs to periodontal pockets stems from breakdown and bone remodeling (de Molon et al., 2014). The 7-day
the finding that systemic administration of many drugs (and antibiotics ligature model creates an acute and transitory inflammation resulting
in particular) results in poorly effective local concentrations of free from the combination of mechanical trauma and initial shift of equili-
active principle in the periodontal pouch and in the surrounding tissues brium from bacterial endotoxins to host response (Garcia de Aquino
(Tonetti & Cortellini, 2015). However, it is necessary to consider that et al., 2009; Lee, Lin, Fong, Ryder, & Ho, 2013). A high osteoclastic
recolonization is an important phenomenon that should be avoided by a activity is usually not sustained after ligature removal or tissue reces-
clinical strategy: optimal supragingival hygiene, disinfection of the sion, since the mechanical trauma is removed or the concentration of
whole mouth and / or use of antiseptic oral antiseptic (Tonetti & endotoxins reduces (Lee et al., 2013). Therefore, a remission of bone
Cortellini, 2015). resorption following the acute phase of inflammation is expected.
Macrophages can also be related to the inflammatory response on Nevertheless, the comparison of the effects of CHX with the control
periodontitis and gingivitis (Garaicoa-Pazmino et al., 2019). The ration group where only SRP was used may not be totally undermined with
between macrophages type M1 and M2 have been suggested to play a this model. We recommend anyway the results to be carefully inter-
role in the transition from healthy, gingivitis, and periodontitis preted considering the acute characteristic of the model and the dif-
(Garaicoa-Pazmino et al., 2019). Periodontitis samples displayed lower ferences in tissue metabolism between rodents and humans.
levels of macrophages dispersed in the stromal tissues compared with Subgingival administration of drugs may present several benefits in
gingivitis samples; however, it remained higher than in healthy tissues. comparison to systemic administration of a drug, such as guarantee of
The polarization of macrophages appears to be reduced in periodontitis delivery of adequate concentration of an agent, reduction of the
and showed similar levels to those observed in healthy tissues chances of adverse reactions, more precise control of the amount of the
(Garaicoa-Pazmino et al., 2019). Even though the authors have not drug being delivered at a site, immediate suspension of drug delivery if
found differences in macrophage polarization along periodontitis an adverse effect is observed, reduction in the chances of development
chronification, the macrophage role on periodontal healing after using of resistance to a drug, among others (Goodman & Robinson, 1990).
subgingival irrigation with CHX combined with SRP can be further Conversely, disadvantages with subgingival irrigation comprise the
evaluated. small reservoir for the drug and the high exponential kinetics dictated
Overall, the best results observed in CHX groups might be due to the by the crevicular fluid that may shorten the effective period of a drug
inhibition of inflammatory cells to release cytotoxic contents and en- (Tonetti & Cortellini, 2015). Therefore, it is imperative to use drugs
zymes causing periodontal tissue breakdown and by CHX ability to with high substantivity, i.e., increased capacity to attach to surfaces for
remove remaining of LPS and LTA, which could sustain the excessive a longer period, such as the case of CHX that can bind and be slowly
inflammatory process observed in the SRP only group. Inflammation released from periodontal structures for 12 h after application (Lang
resolution can also be explained in part considering the characteristics et al., 1982). Regardless, subgingival irrigation may not deliver the
of the inflammation induced with ligature insertion. Experimental drug to the deepest areas of a pocket especially due to anatomic

5
N.R. Prietto, et al. Archives of Oral Biology 110 (2020) 104600

features, which is circumvented with systemic delivery of a drug superoxide generation by induced human neutrophils. Journal of Periodontal Research,
(Goodman & Robinson, 1990). 22, 445–450.
Garaicoa-Pazmino, C., Fretwurst, T., Squarize, C. H., Berglundh, T., Giannobile, W. V.,
Larsson, L., et al. (2019). Characterization of macrophage polarization in periodontal
5. Conclusions disease. Journal of Clinical Periodontology, 46(8), 830–839.
Garcia de Aquino, S., Manzolli Leite, F. R., Stach-Machado, D. R., Francisco da Silva, J. A.,
Spolidorio, L. C., & Rossa, C., Jr (2009). Signaling pathways associated with the
Subgingival irrigation with chlorhexidine accelerated the healing expression of inflammatory mediators activated during the course of two models of
process after SRP. In addition, CHX seems to present an anti-infl- experimental periodontitis. Life Sciences, 84(21–22), 745–754.
ammatory property with histoprotective characteristics. Randomized Gendron, R., Grenier, D., Sorsa, T., & Mayrand, D. (1999). Inhibition of the activities of
matrix metalloproteinases 2, 8, and 9 by chlorhexidine. Clinical and Diagnostic
clinical trials are recommended to analyze the long-term effects of CHX Laboratory Immunology, 6, 437–439.
as an adjunct treatment to modulate the inflammatory process after Gokhale, S. R., & Padhye, A. M. (2013). Future prospects of systemic host modulatory
SRP. agents in periodontal therapy. Brazilian Dental Journal, 214, 467–471.
Golub, L. M., Sorsa, T., Lee, H. M., et al. (1995). Doxycycline inhibits neutrophil (PMN)-
type matrix metalloproteinases in human adult periodontitis gingiva. Journal of
Author declaration Clinical Periodontology, 22, 100–109.
Goodman, C. H., & Robinson, P. J. (1990). Periodontal therapy: Reviewing subgingival
We wish to confirm that there are no known conflicts of interest irrigations and future considerations. The Journal of the American Dental Association,
121(4), 541–543.
associated with this publication and there has been no significant fi- Goultschin, J., & Levy, H. (1986). Inhibition of superoxide generation by human poly-
nancial support for this work that could have influenced its outcome. morphonuclear leukocytes with chlorhexidine. Its possible relation to periodontal
We confirm that the manuscript has been read and approved by all disease. Journal of Periodontology, 57, 422–425.
Grenier, D. (1993). Reduction of proteolytic degradation by chlorhexidine. Journal of
named authors and that there are no other persons who satisfied the Dental Research, 72, 630–633.
criteria for authorship but are not listed. We further confirm that the James, P., Worthington, H. V., Parnell, C., Harding, M., Lamont, T., Cheung, A., et al.
order of authors listed in the manuscript has been approved by all of us. (2017). Chlorhexidine mouthrinse as an adjunctive treatment for gingival health. The
Cochrane Database of Systematic Reviews, 3, CD008676.
We confirm that we have given due consideration to the protection Kornman, K. S., Page, R. C., & Tonetti, M. S. (1997). The host response to the microbial
of intellectual property associated with this work and that there are no challenge in periodontitis: Assembling the players. Periodontology, 2000(14),
impediments to publication, including the timing of publication, with 112–143.
Kuhr, A., Popa-Wagner, A., Schmoll, H., Schwahn, C., & Kocher, T. (2004). Observations
respect to intellectual property. In so doing we confirm that we have on experimental marginal periodontitis in rats. Journal of Periodontal Research, 39(2),
followed the regulations of our institutions concerning intellectual 101–106.
property. Lang, N. P., Hotz, P., Graf, H., Geering, A. H., Saxer, U. P., Sturzenberger, O. P., et al.
(1982). Effects of supervised chlorhexidine mouthrinses in children. A longitudinal
We further confirm that any aspect of the work covered in this
clinical trial. Journal of Periodontal Research, 17(1), 101–111.
manuscript that has involved either experimental animals or human Lee, J. H., Lin, J. D., Fong, J. I., Ryder, M. I., & Ho, S. P. (2013). The adaptive nature of the
patients has been conducted with the ethical approval of all relevant bone-periodontal ligament-cementum complex in a ligature-induced periodontitis rat
bodies and that such approvals are acknowledged within the manu- model. BioMed Research International, 2013, 876316.
Leite, F. R. M., Aquino, S. G. D., Guimarães, M. R., Cirelli, J. A., & Junior, C. R. (2014).
script. RANKL expression is differentially modulated by TLR2 and TLR4 signaling in fibro-
We understand that the Corresponding Author is the sole contact for blasts and osteoblasts. Immunology Innovation, 2, 1–9.
the Editorial process (including Editorial Manager and direct commu- Leite, F. R. M., de Aquino, S. G., Guimarães, M. R., et al. (2015). Relevance of the myeloid
differentiation factor 88 (MyD88) on RANKL, OPG, and nod expressions induced by
nications with the office). He/she is responsible for communicating TLR and IL-1R signaling in bone marrow stromal cells. Inflammation, 38, 1–8.
with the other authors about progress, submissions of revisions and Marinho, A. C., Martinho, F. C., Leite, F. R., Nascimento, G. G., & Gomes, B. P. (2015).
final approval of proofs. We confirm that we have provided a current, Proinflammatory activity of primarily infected endodontic content against macro-
phages after different phases of the root canal therapy. Journal of Endodontics, 41,
correct email address which is accessible by the Corresponding Author 817–823.
and which has been configured to accept email from carolsantinoni@ Matthews, D. (2011). No difference between 0.12 % and 0.2 % chlorhexidine mouthrinse
msn.com. on reduction of gingivitis. Evidence Based Dentistry, 12, 8–9.
Messora, M. R., Pereira, L. J., Foureaux, R., Oliveira, L. F. F., Sordi, C. G., Alves, A. J. N.,
et al. (2016). Favourable effects of Bacillus subtilis and Bacillus licheniformis on
Acknowledgment experimental periodontitis in rats. Archives of Oral Biology, 66, 108–119.
Montecucco, F., Bertolotto, M., Ottonello, L., et al. (2009). Chlorhexidine prevents hy-
pochlorous acid-induced inactivation of alpha1-antitrypsin. Clinical and Experimental
The study was supported by the FAPERGS (the Research Support
Pharmacology & Physiology, 36, e72–77.
Foundation of Rio Grande do Sul) grant call 02/2013 ARD. Nicu, E. A., & Loos, B. G. (2016). Polymorphonuclear neutrophils in periodontitis and
their possible modulation as a therapeutic approach. Periodontology, 2000(71),
References 140–163.
Teronen, O., Konttinen, Y. T., Lindqvist, C., et al. (1997). Inhibition of matrix metallo-
proteinase-1 by dichloromethylene bisphosphonate (clodronate). Calcified Tissue
da Costa, L., Amaral, C., Barbirato, D. D. S., Leao, A. T. T., & Fogacci, M. F. (2017). International, 61, 59–61.
Chlorhexidine mouthwash as an adjunct to mechanical therapy in chronic period- Test, S. T., & Weiss, S. J. (1986). The generation of utilization of chlorinated oxidants by
ontitis: A meta-analysis. The Journal of the American Dental Association, 148(5), human neutrophils. Free Radical Biology and Medicine, 2, 91–116.
308–318. Tonetti, M. S., & Cortellini, P. (2015). Local drug administration for periodontal treat-
De Almeida, J., Ervolino, E., Bonfietti, L. H., et al. (2015). Adjuvant therapy with sodium ment. In N. P. Lang, & J. Lindhe (Eds.). Clinical periodontology and implant dentistry
alendronate for the treatment of experimental periodontitis in rats. Journal of (6th edition). New York: Wiley.
Periodontology, 86, 1166–1175. Tüter, G., Kurtis, B., & Serdar, M. (2002). Effects of phase I periodontal treatment on
de Molon, R. S., de Avila, E. D., Boas Nogueira, A. V., Chaves de Souza, J. A., Avila- gingival crevicular fluid levels of matrix metalloproteinase-1 and tissue inhibitor of
Campos, M. J., de Andrade, C. R., et al. (2014). Evaluation of the host response in metalloproteinase-1. Journal of Periodontology, 73, 487–493.
various models of induced periodontal disease in mice. Journal of Periodontology, Weiss, S. J. (1989). Tissue destruction by neutrophils. The New England Journal of
85(3), 465–477. Medicine, 320, 365–376.
Fernandes, L. A., de Almeida, J. M., Theodoro, L. H., Bosco, A. F., Nagata, M. J., Martins, Weiss, S. J., Peppin, G., Ortiz, X., Ragsdale, C., & Test, S. T. (1985). Oxidative auto-
T. M., et al. (2009). Treatment of experimental periodontal disease by photodynamic activation of latent collagenase by human neutrophils. Science, 227, 747–749.
therapy in immunosuppressed rats. Journal of Clinical Periodontology, 36(3), 219–228. Yamamoto, T., Yamagata, A., & Nagai, H. (1995). Histochemical reactivity of tartrate-
Firatli, E., Unal, T., Onan, U., & Sandalli, P. (1994). Antioxidative activities of some resistant acid phosphatase in the matrix of developing medullary bone. Acta
chemotherapeutics. A possible mechanism in reducing gingival inflammation. Journal Histochemica et Cytochemica, 28, 319–322.
of Clinical Periodontology, 21, 680–683.
Gabler, W. L., Bullock, W. W., & Creamer, H. R. (1987). The influence of chlorhexidine on