Treatment of experimental periodontitis with chlorhexidine as adjuvant to scaling and root planing

Journal Pre-proof Treatment of experimental periodontitis with chlorhexidine as adjuvant to scaling and root planing Nubia Rosa Prietto, Thiago Marchi Martins, Carolina dos Santos ´ Marcumini Pola, Edilson Ervolino, Amalia ´ Santinoni, Natalia ´ Machado Bielemann, Fabio Renato Manzolli Leite

PII:

S0003-9969(19)30804-0

DOI:

https://doi.org/10.1016/j.archoralbio.2019.104600

Reference:

AOB 104600

To appear in:

Archives of Oral Biology

Received Date:

9 August 2019

Revised Date:

3 October 2019

Accepted Date:

3 November 2019

Please cite this article as: Prietto NR, Martins TM, dos Santos Santinoni C, Pola NM, Ervolino E, Bielemann AM, Leite FRM, Treatment of experimental periodontitis with chlorhexidine as adjuvant to scaling and root planing, Archives of Oral Biology (2019), doi: https://doi.org/10.1016/j.archoralbio.2019.104600

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Title: Treatment of experimental periodontitis with chlorhexidine as adjuvant to scaling and root planing

Nubia Rosa Priettoa, MS, Thiago Marchi Martinsa, PhD, Carolina dos Santos Santinoni*b, PhD, Natália Marcumini Polaa, PhD, Edilson Ervolinoc, PhD; Amália Machado Bielemann, PhDa; Fábio Renato Manzolli Leited, PhD

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Graduate Program in Dentistry, School of Dentistry, Federal University of Pelotas, Pelotas, Brazil

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University of Western Sao Paulo, Presidente Prudente, Brazil c

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Dental School of Presidente Prudente, Graduate Program in Dentistry (GPD - Master's Degree),

Department of Basic Sciences, Dental School of Araçatuba, São Paulo State University, São Paulo,

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Brazil

Section of Periodontology, Department of Dentistry and Oral Health, Aarhus University, Aarhus,

*Corresponding Author

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Carolina dos Santos Santinoni

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Denmark

Universidade do Oeste Paulista - UNOESTE José Bongiovani, 700 – Cidade Universitária Presidente Prudente, SP, Brasil

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19050-920

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Telephone: +55 (18) 3229-1259 E-mail: [email protected]

2 HIGHLIGHTS

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Chlorhexidine 0.12 % or 0.2 % presented the same effect on periodontitis healing

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Chlorhexidine should be considered an antimicrobial and anti-inflammatory agent

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Chlorhexidine seems to modulate the bone remodeling process

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ABSTRACT

Objectives: To assess whether subgingival irrigation with 0.12% or 0.2% chlorhexidine (CHX) immediately after scaling and root planing (SRP) enhances periodontal tissue repair compared to

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irrigation with saline solution (control). Materials and Methods: Periodontitis was ligature-induced in rat molars for 7 days. Animals were distributed into three groups: 1) SRP group, SRP and irrigation

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with 0.9% saline (n = 30); 2) SRP + 0.12% CHX group, SRP and 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,

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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

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(RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP). Results: Both chlorhexidine groups presented less inflammation and improved tissue repair along the entire

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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

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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.

Key words: Periodontitis; periodontal diseases; rats; wound healing; dental scaling; chlorhexidine

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4 INTRODUCTION Periodontitis is an inflammatory condition, which gradually leads to destruction of the supportive tissues of teeth. The host inflammatory response triggered by bacteria compounds or other causes of periodontal diseases, e.g. diabetes, smoking, among others, is the major responsible for the tissue destruction [1]. Extracellular matrix components, e.g. collagen, fibronectin, and proteoglycans, maintain the structural integrity of the anchoring apparatus. Extracellular matrix components degradation causes irreversible loss of connective tissue and alveolar bone [2]. Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, are strategic regulators of the healing

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process [3] and the ratio between their activity and inhibition influences the healing outcome [3]. Different approaches to modulate host inflammatory response have been suggested to induce a more balanced periodontal healing. The idea is to inhibit inflammatory pathways leading to tissue breakdown

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and to trigger those associated with cell proliferation and differentiation [4]. Therefore, MMP inhibition would be appreciated after periodontal treatment. Tetracyclines, bisphosphonates and chlorhexidine

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have been shown to inhibit the activity of several MMP family members [5-7]. Chlorhexidine (CHX) is an antimicrobial agent with broad spectrum antimicrobial activity. This

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cationic bis-biguanide reduces dental biofilm proliferation, therefore its use in subgingival irrigation. CHX presents proteolytic activity of certain periodontal pathogens [8] and antioxidative capacity [9]

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through inhibition of superoxide anion generation in red and white blood cells [10,11]. Chlorhexidine scavenge long-lived nitrogen-chlorine oxidants capable of degrading protein sulfhydryl groups linked

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to endogenous autoactivation of neutrophil collagenase [12,13]. Due to the role of neutrophils in the protection of periodontal tissues against bacteria invasion but also in the degradation of the extracellular

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matrix, a substance that controls bacteria proliferation and collagenase activity would be interesting. Recently, a systematic review compared treatment of patients with periodontitis who underwent mechanical periodontal therapy (scaling and root planing - SRP) combined or not with CHX as an adjunct [14]. The authors considered clinical parameters such as probing depth (PD) and clinical attachment level (CAL) as primary outcomes. Groups treated with CHX plus SRP had slightly better results than did those treated only with SRP [14].

5 Pondering lack of studies that evaluate the effects of chlorhexidine on periodontitis healing, the purpose of this study was to compare the efficacy chlorhexidine plus conventional mechanical scaling root planing (SRP) on alveolar bone loss in a model of acute experimental periodontitis in rats.

MATERIALS AND METHODS

Experimental model The experimental protocol was approved by the Ethics Committee on Animal Experimentation

Experiments (ARRIVE) guidelines were used for reporting.

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(protocol 1587) of the Federal University of Pelotas. The Animal Research: Reporting of in vivo

The sample size was determined to recognize a significant difference of 20% among groups in

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a scenario with 10% of standard deviation, power set at 80% power and 95% confidence interval. Data from changes in the area of alveolar bone (our primary outcome) after ligature-induced periodontitis

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from a previous study were used [15]. A sample size of eight animals per group was required. Considering potential attrition rates due to potential tooth loss, ligature loss, anesthesia complications,

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among others, 10 animals per experimental time were used.

Ninety 2-month-old rats (Rattus norvegicus albinus, Wistar) weighing 130 to 160 g were

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obtained from the Central Animal Facility of the Federal University of Pelotas. The rats were kept in individual cages under the same standard conditions of illuminations (12-hr light/dark cycle), controlled

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temperature of 22 ± 1°C, and food and water ad libitum during all the experimental period. The animals were subjected to a period of 7 days of acclimatization to the environment and then randomly allocated

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into the following groups (n = 30) according to the following protocol: 1) scaling root planing - SRP (control) subjected to experimental periodontitis (EP) induction, SRP and irrigation with 1 mL of 0.9% saline solution; 2) SRP + 0.12% CHX, subjected to EP induction, SRP and irrigation with 1 mL 0.12% chlorhexidine solution (Maquira Produtos Odontológicos, Maringa, Paraná, Brazil); and 3) SRP + 0.2% CHX, SRP after EP induction and irrigation with 1 mL 0.2% chlorhexidine solution (Maquira Produtos Odontológicos, Maringa, Paraná, Brazil).

6 Acute experimental periodontitis induction (EP) Animals were anesthetized by intramuscular injection with a mixture of ketamine§ (70 mg/kg) and xylazine‖ (6 mg/kg). A cotton thread (Corrente Algodão no. 24, Coats Corrente, São Paulo, São Paulo, Brazil) was tied around the mandibular left first molar of each rat to induce biofilm accumulation and consequent periodontitis [16]. The ligature was maintained for 7 days and SRP was performed immediately after ligature removal by the same trained operator using 10 distal–medial traction movements of a curette (-2 Mini Five curette, Hu-Friedy, Chicago, IL) over the buccal and lingual surfaces of the treated area [16]. The interproximal and furcation areas were scraped using cervical–

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occlusal traction movements of the same curette.

Subgingival irrigation

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Irrigation was performed just once, immediately after SRP. Group control received irrigation with 1 mL of 0.9% saline solution. Groups treated with chlorhexidine received 1 mL of 0.12%

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chlorhexidine solution (Maquira Produtos Odontológicos, Maringa, Paraná, Brazil) or 1 mL of 0.2% chlorhexidine solution (Maquira Produtos Odontológicos, Maringa, Paraná, Brazil). All the solutions

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Laboratorial Processing

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were inserted slowly into the periodontal pocket, using a 1 ml syringe and insulin needle without bevel.

At 7, 15, or 30 days after treatment, the animals were euthanized with an overdose of thiopental

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(150 mg/kg) (Cristália, Itapira, São Paulo, Brazil). Mandibles were dissected and fixed in 4% formaldehyde for 48h. Specimens were demineralized in 10% EDTA, washed, dehydrated in graded

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alcohol concentrations, cleared in xylene, and embedded in paraffin. The paraffin blocks were serially cut in a mesio-distal direction along the long axis of the teeth to generate 5 μm-thick longitudinal sections. After excluding the first and the last sections where the furcation region was evident, five equidistant sections of each specimen block were selected and stained with hematoxylin and eosin (HE) for histologic and histometric analysis [17]. Other histologic sections were subjected to the indirect immunoperoxidase method. The histological sections were deparaffinized and rehydrated through a

7 graded series of ethanol. For antigen retrieval, the slides were incubated in phosphate buffer solution (0.1 M, pH 7.4) at 95 ºC for 10 min. At the end of each step of the immunohistochemical reaction, the histological slides were washed with a phosphate buffer solution (0.1 M, pH 7.4). Subsequently, the slides were immersed in 3% hydrogen peroxide for 1 h and then 1% albumin bovine sérum for 12 h to block the endogenous peroxidase and nonspecific sites, respectively. The slides containing samples of each of the experimental groups were divided into three batches and each batch was incubated with one of the following primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA): anti-TRAP (goat anti-TRAP), anti-RANKL (goat anti-RANKL), and anti-OPG (goat anti-OPG). The sections were

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incubated with a biotinylated secondary antibody for 2 h and subsequently treated with a streptavidin– horseradish peroxidase conjugate (Universal Dako Labeled HRP Streptavidin–Biotin Kit®, Dako Laboratories, CA, USA) for 1 h. The reaction was developed using the chromogen 3,30-

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diaminobenzidine (DAB chromogen Kit®, Dako Laboratories, CA, USA) and counterstained with Harris hematoxylin. All samples were accompanied by a negative control, i.e. specimens subjected to

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Histological and Histometric Analysis

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the aforementioned procedures but without the primary antibodies.

Sections dyed by H&E were analyzed under light microscopy to establish the bone loss and

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characteristics of periodontal ligament in the furcation region of first molars. The area of bone loss in the furcation region was histometrically determined using an image analysis system (ImageJ Tool, San

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Antonio, TX, USA) as previously described [16]. After excluding the first and last sections where the furcation region was evident, 5 equidistant sections of each specimen block were selected and captured

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by a digital camera coupled to a light microscope. For determination of changes in the area of alveolar bone between groups (primary outcome), bone loss (mm2) in the furcation region was determined histometrically [16]. One blinded, trained examiner selected the sections for the histometric and histological analysis. Another masked, calibrated examiner conducted the histometric analysis.

Immunohistochemical analysis

8 An examiner who was blinded to the groups and treatments (CSS) 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 potential 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 immunolabeling criteria as follows: 0, total absence of immunoreactive (IR) cells; 1, low immunolabeling (approximately one quarter of IR cells); 2, moderate immunolabeling

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(approximately one half of IR cells); and 3, high immunolabeling (approximately three quarters of IR cells) [16].

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Statistical Analyses

Histometric data were analyzed using a statistics software (StataSE 14.1, StataCorp, College

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Station, TX, USA). Intra-examiner reproducibility was checked with paired t-test and Pearson’s correlation coefficient before and during the histometric analysis. Seven sections from each group were

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selected at random and analyzed twice with a 1-week interval between analyses. Data normality was analyzed using the Shapiro-Wilk test. Groups were compared via Kruskal-Wallis one-way analysis of

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variance (ANOVA). Multiple comparisons (post hoc) were performed using Dunn’s test, and P values

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<.05 indicated statistical significance.

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RESULTS

Histometric Analyses of Lose Bone in the Furcation of Teeth (PBF) Change in alveolar bone loss between groups was set as the primary outcome variable. Paired t-test statistics was calculated, and no differences were observed in the mean values for comparison (p > 0.05). In addition, the Pearson’s correlation coefficient revealed high correlation (0.89) between the two measurements for the histometric analyses. In the intergroup analysis, the animals in the SRP group

9 showed a lower area of bone than the animals in both chlorhexidine groups for all experimental periods (P < 0.05). The animals in the SRP + 0.12% CHX and in the SRP + 0.2% CHX groups showed similar bone area at 7, 15 and 30 days (P > 0.05). In the intragroup analysis, the animals in the SRP group showed a similar area of bone at 7, 15 and 30 days (P > 0.05). The animals in the SRP + 0.12% CHX and SRP + 0.2% CHX groups showed a higher area of bone in each period, with a significant difference (P > 0.05) specially when the 7 days’ period was compared with the 30 days’ period. Results are summarized in Table 1.

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Descriptive histologic analyses

In the SRP group (control), a large number of neutrophils occupied the connective tissue at the furcation area at 7 days after treatment (Fig. 1A). The acute inflammatory infiltrate spread all over the

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furcation space reaching the area around the bone septum (Fig. 1D). The bone consisted of thin and irregular trabeculae with resorption lacunae and active osteoclasts. In both SRP plus chlorhexidine

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groups, the acute inflammatory infiltrate was moderate to discrete at 7 days after SRP plus subgingival irrigation (Fig. 1B and 1C). It consisted of a mix of acute and chronic inflammatory cells in an advanced

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under organization connective tissue. The bone septum presented a regular outline with some osteoclasts (Fig. 1E). Cementum resorption areas were observed in most specimens.

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At 15 days after SRP alone, the acute inflammatory infiltrate reduced but was still more evident than chronic inflammatory cells, e.g. macrophages and lymphocytes. Connective tissue was

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disorganized with small number of fibroblasts and high number of neutrophils in degeneration. At 30 days after SRP, connective tissue occupied a large portion of the furcation area. Inflammatory cells

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were located closer to the septum bone, which showed more regular outline (Fig. 2A). The cementum still showed small areas of resorption with most of them inactive (Fig. 2D). Cellular cement was deposited in some areas closer to the remaining septum. At 15 and 30 days after treatments the inflammatory infiltrate was absent in most of the specimens or very few inflammatory cells were observed in a completely organized connective tissue (Fig. 2B and 2C). Interradicular septum was regular and covered with osteoblasts, osteocytes were

10 abundant and osteoclasts rarely observed. Periodontal ligament was rich in vessels and collagen fibers. All cementum resorption areas were inactive and undergoing repair or had already been replaced by cellular cement (Fig. 2E). No visual differences were observed between both concentrations of chlorhexidine.

Immunohistochemical analysis Change in the pattern of immunolabeling between groups was set as secondary outcome. Table

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2 show the immunohistochemical results for OPG, RANKL and TRAP immunolabeling, using a symbols system. Fig. 3 illustrate TRAP, OPG and RANKL immunolabeling in the furcation region.

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OPG

Group SRP at 7, 15 and 30 days postoperative presented moderate immunolabeling. Group

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CHX 0.12% presented low immunolabeling at 7 and 15 days postoperative. Group CHX 0.12% presented high immunolabeling at 30 days postoperative. Group CHX 0.2% presented moderate

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immunolabeling at 7, 15 and 30 days postoperative.

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RANKL

Groups SRP and CHX 0.12% presented moderate immunolabeling at 7, 15 and 30 days

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postoperative. Group CHX 0.2% presented low immunolabeling at 7 and 30 days and moderate

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immunolabeling at 15 days postoperative.

TRAP

Group SRP presented moderate immunolabeling at 7, 15 and 30 days postoperative. Group

CHX 0.12% presented high immunolabeling at 7, 15 and 30 days postoperative. Group CHX 0.2% presented low immunolabeling at 7 and 15 days and high immunolabeling at 30 days postoperative.

11 DISCUSSION Destructive forms of periodontal inflammation might lead to breakdown of connective tissue extracellular matrix. The major destruction observed in this process is host immune system-mediated but usually initiated by bacteria and their toxic byproducts and other systemic causes, such as smoking and diabetes. Commercially, chlorhexidine is found at high doses (0.12–2.0%) to obtain bactericidal effects [18]. However, low and non-toxic concentrations of chlorhexidine up to 0.05% might modulate the immune response by acting on neutrophils activity [10,19]. Even though neutrophils are recruited to periodontal tissues to have a protective role, they

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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 triggers [19]. However, neutrophils release proteases, cytokines and myeloperoxidase (MPO), increasing oxidant

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release and inactivating protective antiproteases [19,20]. The superoxide anions are quickly converted to hydrogen peroxide, which is transformed into hypochlorous acid (HOCl) by MPO [20]. Thus, the

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observed damage to periodontal tissues is mainly caused by HOCl by increasing cell oxidative stress and by increase of elastase-mediated breakdown of connective tissue components, e.g. elastin, collagen,

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fibronectin and laminin. Control of neutrophil-mediated proteolysis is one of the key points in pharmacology to reduce inflammatory tissue injury. In this study, CHX seemed to reduce the number

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of inflammatory cells to the furcation area and promote a more balanced, organized and quicker repair of bone septum and periodontal ligament. We suppose that the higher percentage of bone in both

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chlorhexidine groups was due to the reaction of CHX with HOCl faster than HOCl-induced inactivation of inhibitors of neutrophil elastases as previously described [19].

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The potential to neutralize the inflammation by CHX seems to be dependent on its cationchelating properties [7]. Also, CHX quickly deactivate large and small portions of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from oral bacteria, preventing further perpetuation of inflammatory cell activation [21]. The capability of CHX to neutralize LPS and LTA before the stimulation of the innate immune response is important since biofilm disruption by mechanical periodontal treatment causes the release of high levels of immunostimulatory bacterial cell wall components, which might exacerbate and/or perpetuate periodontal inflammation and destruction [22,23]. CHX inhibits the cell

12 activation by LPS and LTA in a concentration-dependent manner with almost complete inhibition at a concentration of 0.0005% and 0.0001%, respectively. These data might explain the lack of differences in tissue repair between the SRP + CHX 0.12% and the SRP + CHX 0.2% groups. In addition, respecting differences in study designs, a previous systematic review has demonstrated that there is no evidence that one concentration of chlorhexidine rinse is more effective than another in reducing gingivitis levels [24]. According to previous studies [10,19], CHX concentrations above 0.05% might not lead to an extra modulation of the immune response. In this study, groups treated with chlorhexidine presented

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higher number of TRAP-immunolabeled cells than SRP group. The opposite result should be expected, since TRAP labelling is an indicator of osteoclast differentiation. Two hypotheses can explain these results. One of them is that the osteoclasts present in the furcation region were not active, as well as

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TRAP, which was accumulated into the developing medullary bone [25]. Other possible justification is that groups treated with chlorhexidine were in a more advanced phase of the bone remodeling process

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after treatment and, therefore, a higher number of TRAP-immunolabeled cells was observed. Local administration of drugs in the periodontal pocket is an effective treatment for mechanical

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debridement. Localized therapies have received significant attention because of the pattern of destruction of site-specific periodontal infections and the potential collateral effects of systemic

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antimicrobials and anti-inflammatory agents [26]. Another important reason for the development of effective local application of drugs to periodontal pockets stems from the finding that systemic

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administration of many drugs (and antibiotics in particular) results in poorly effective local concentrations of free active principle in the periodontal pouch and in the surrounding tissues [26].

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However, it is necessary to consider that recolonization is an important phenomenon that should be avoided by a clinical strategy: optimal supragingival hygiene, disinfection of the whole mouth and / or use of antiseptic oral antiseptic [26]. Macrophages can also be related to the inflammatory response on periodontitis and gingivitis [27]. The ration between macrophages type M1 and M2 have been suggested to play a role in the transition from healthy, gingivitis, and periodontitis [27]. Periodontitis samples displayed lower levels of macrophages dispersed in the stromal tissues compared with gingivitis samples; however, it remained

13 higher than in healthy tissues. The polarization of macrophages appears to be reduced in periodontitis and showed similar levels to those observed in healthy tissues [27]. Even though the authors have not found differences in macrophage polarization along periodontitis chronification, the macrophage role on periodontal healing after using subgingival irrigation with CHX combined with SRP can be further evaluated. Overall, the best results observed in CHX groups might be due to the inhibition of inflammatory cells to release cytotoxic contents and enzymes causing periodontal tissue breakdown and by CHX ability to remove remaining of LPS and LTA, which could sustain the excessive inflammatory process

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observed in the SRP only group. Inflammation resolution can also be explained in part considering the characteristics of the inflammation induced with ligature insertion. Experimental periodontal disease induction using ligature is extensively used because the alveolar bone loss develops predictably after 7

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days [28]. The presence of bacteria is mandatory since germ-free rats do not develop significant alveolar bone destruction after ligature placement. However, a limitation of the model is that the combination of

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bacteria with mechanical trauma during ligature placement may accelerate the periodontal tissue breakdown and bone remodeling [29]. The 7-day ligature model creates an acute and transitory

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inflammation resulting from the combination of mechanical trauma and initial shift of equilibrium from bacterial endotoxins to host response [30,31]. A high osteoclastic activity is usually not sustained after

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ligature removal or tissue recession, since the mechanical trauma is removed or the concentration of endotoxins reduces [31]. Therefore, a remission of bone resorption following the acute phase of

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inflammation is expected. Nevertheless, the comparison of the effects of CHX with the control group where only SRP was used may not be totally undermined with this model. We recommend anyway the

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results to be carefully interpreted considering the acute characteristic of the model and the differences in tissue metabolism between rodents and humans. Subgingival administration of drugs may present several benefits in comparison to systemic

administration of a drug, such as guarantee of delivery of adequate concentration of an agent, reduction of the chances of adverse reactions, more precise control of the amount of the drug being delivered at a site, immediate suspension of drug delivery if an adverse effect is observed, reduction in the chances of development of resistance to a drug, among others [32]. Conversely, disadvantages with subgingival

14 irrigation comprise the small reservoir for the drug and the high exponential kinetics dictated by the crevicular fluid that may shorten the effective period of a drug [26]. Therefore, it is imperative to use drugs with high substantivity, i.e., increased capacity to attach to surfaces for a longer period, such as the case of CHX that can bind and be slowly released from periodontal structures for 12 hours after application [33]. Regardless, subgingival irrigation may not deliver the drug to the deepest areas of a pocket especially due to anatomic features, which is circumvented with systemic delivery of a drug [32].

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CONCLUSIONS

Subgingival irrigation with chlorhexidine accelerated the healing process after SRP. In addition, CHX seems to present an anti-inflammatory property with histoprotective characteristics. Randomized

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clinical trials are recommended to analyze the long-term effects of CHX as an adjunct treatment to

AUTHOR DECLARATION

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modulate the inflammatory process after SRP.

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We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

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We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

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We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We further confirm that any aspect of the work covered in this manuscript that has involved either experimental animals or human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible

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for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected].

ACKNOWLEDGMENTS The study was supported by the FAPERGS (the Research Support Foundation of Rio Grande

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do Sul) grant call 02/2013 ARD.

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17. Fernandes, L. A., de Almeida, J. M., Theodoro, L. H., Bosco, A. F., Nagata, M. J., Martins, T.

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M., Garcia, V. G. (2009). Treatment of experimental periodontal disease by photodynamic therapy in immunosuppressed rats. Journal of clinical periodontology, 36(3), 219-228.

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18. Matthews, D. (2011). No difference between 0.12% and 0.2% chlorhexidine mouthrinse on reduction of gingivitis. Evidence Based Dentistry, 12, 8-9.

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19. Montecucco, F., Bertolotto, M., Ottonello, L., et al. (2009). Chlorhexidine prevents hypochlorous acid-induced inactivation of alpha1-antitrypsin. Clinical and Experimental Pharmacology & Physiology, 36, e72-77.

20. Test, S.T., Weiss, S.J. (1986). The generation of utilization of chlorinated oxidants by human neutrophils. Free Radical Biology and Medicine, 2, 91-116.

18 21. Marinho, A.C., Martinho, F.C., Leite, F.R., Nascimento, G.G., Gomes, B.P. (2015). Proinflammatory Activity of Primarily Infected Endodontic Content against Macrophages after Different Phases of the Root Canal Therapy. Journal of Endodontics, 41, 817-823. 22. Leite, F.R.M., Aquino, S.G.D., Guimarães, M.R., Cirelli, J.A., Junior, C.R. (2014). RANKL expression is differentially modulated by TLR2 and TLR4 signaling in fibroblasts and osteoblasts. Immunology Innovation, 2, 1-9. 23. 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 TLR

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and IL-1R Signaling in Bone Marrow Stromal Cells. Inflammation, 38, 1-8.

24. James, P., Worthington, H. V., Parnell, C., Harding, M., Lamont, T., Cheung, A., Riley, P. (2017). Chlorhexidine mouthrinse as an adjunctive treatment for gingival health. The Cochrane

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database of systematic reviews, 3, CD008676.

25. Yamamoto, T., Yamagata, A., Nagai, H. (1995). Histochemical reactivity of tartrate-resistant

Cytochemica, 28, 319-322.

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acid phosphatase in the matrix of developing medullary bone. Acta Histochemica et

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26. Tonetti MS; Cortellini P. Local drug administration for periodontal treatment. In: LANG, N.P., LINDHE, J. Clinical Periodontology and Implant Dentistry. 2015. 6th Edition. New York:

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Wiley.

27. Garaicoa-Pazmino, C., Fretwurst, T., Squarize, C. H., Berglundh, T., Giannobile, W. V.,

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Larsson, L., & Castilho, R. M. (2019). Characterization of macrophage polarization in periodontal disease. Journal of clinical periodontology, 46(8), 830-839.

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28. Kuhr, A., Popa-Wagner, A., Schmoll, H., Schwahn, C., & Kocher, T. (2004). Observations on experimental marginal periodontitis in rats. Journal of periodontal research, 39(2), 101-106.

29. de Molon, R. S., de Avila, E. D., Boas Nogueira, A. V., Chaves de Souza, J. A., Avila-Campos, M. J., de Andrade, C. R., & Cirelli, J. A. (2014). Evaluation of the host response in various models of induced periodontal disease in mice. Journal of periodontology, 85(3), 465-477.

19 30. 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 expression of inflammatory mediators activated during the course of two models of experimental periodontitis. Life sciences, 84(21-22), 745-754. 31. Lee, J. H., Lin, J. D., Fong, J. I., Ryder, M. I., & Ho, S. P. (2013). The adaptive nature

of the bone-periodontal ligament-cementum complex in a ligature-induced periodontitis rat model. Biomed Res Int, 2013, 876316.

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32. Goodman, C. H., & Robinson, P. J. (1990). Periodontal therapy: reviewing subgingival

irrigations and future considerations. Journal of the American Dental Association, 121(4), 541-543.

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33. Lang, N. P., Hotz, P., Graf, H., Geering, A. H., Saxer, U. P., Sturzenberger, O. P., &

Meckel, A. H. (1982). Effects of supervised chlorhexidine mouthrinses in children. A

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longitudinal clinical trial. Journal of periodontal research, 17(1), 101-111.

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FIGURE LEGENDS

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Fig. 1. Photomicrograph illustrating the bone septum in the furcation region of ligature-induced periodontitis in the mandibular first molar after 7 days of treatment. A - Group SRP (12.5; HE); B -

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Group CHX 0.12% (12.5; HE); C - Group CHX 0.2% (12.5; HE); D - Group SRP (40; HE); E Group CHX 0.12% (40; HE). The inflammatory infiltrate is delimitated by arrows in A, B and C.

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Arrows in E highlight active osteoclasts.

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Fig. 2. Photomicrograph illustrating the bone septum in the furcation region of ligature-induced

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periodontitis in the mandibular first molar after 30 days of treatment. A - Group SRP (12.5; HE); B Group CHX 0.12% (12.5; HE); C - Group CHX 0.2% (12.5; HE); D - Group SRP (40; HE); E -

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Group CHX 0.2% (40; HE). The remaining inflammatory infiltrate is delimitated by arrows in A.

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Arrows in D highlight areas of active cement resorption and in E areas of cement formation.

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Fig. 3. Photomicrograph illustrating TRAP, OPG and RANKL immunolabeling in the furcation region

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of ligature-induced periodontitis in the mandibular first molar. Photomicrographs showing the immunolabeling pattern of TRAP (black arrows) in the SRP (A), CHX 0.12% (B), and CHX 0.2% (C)

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groups. Photomicrographs showing the immunolabeling pattern of OPG (red arrows) in the SRP (D), CHX 0.12% (E), and CHX 0.2% (F) groups. Photomicrographs showing the immunolabeling pattern

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of RANKL (blue arrows) in the SRP (G), CHX 0.12% (H), and CHX 0.2% (I) groups. Original magnification 400; hematoxylin counterstaining. ab, alveolar bone; chx; chlorhexidine; d, dentin; srp,

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scaling and root planing.

23 Table 1. Bone loss in mm2 (mean ± SD) in the furcation area according to groups and experimental periods. 7 days

15 days

30 days

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

SRP + CHX 0.2%

1.14 ± 0.51

0.98 ± 0.40

0.41 ± 0.21

*

Significantly lower than groups CHX 0.12% and CHX 0.2%. No differences observed between both

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chlorhexidine concentrations. SRP, Scaling and root planing; CHX, chlorhexidine.

24 Table 2. OPG, RANKL and TRAP immunoexpression in the furcation area according to groups and experimental periods.

7 days

SRP only (control) CHX 0.12% CHX 0.2 %

15 days

SRP only (control) CHX 0.12% CHX 0.2 %

RANKL

TRAP

++

++

++

+

++

+++

++

+

+

++

++

++

+

++

+++

++

SRP only (control)

30 days

OPG

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Groups

++

CHX 0.12%

+++

CHX 0.2 %

++

++

+

++

++

++

+++

+

+++

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Period

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+ low immunolabeling; ++ moderate immunolabeling; +++ high immunolabeling.