Increased expression of genes after periodontal treatment with photodynamic therapy

Photodiagnosis and Photodynamic Therapy (2014) 11, 41—47

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Increased expression of genes after periodontal treatment with photodynamic therapy Eric Jacomino Franco MSC, MD a,∗, Robert Edward Pogue b, Luis Henrique Toshihiro Sakamoto c, Larissa Lemos Mendanha Cavalcante b,c, Daniel Rey de Carvalho a, Rosângela Vieira de Andrade b a

Department of Periodontology, School of Dentistry, Catholic University of Brasília, Brasília-DF, CEP 71966-700, Brazil b Laboratory of Genomic Sciences and Molecular Biotechnology, Catholic University of Brasília, Brasília-DF, CEP 70790-160, Brazil c Children’s Hospital of Brasília-José Alencar-Brasília-DF, CEP 70.071-900, Brazil Received 9 June 2013; received in revised form 7 October 2013; accepted 11 October 2013 Available online 30 October 2013

KEYWORDS Photodynamic therapy; Periodontal treatment; Gene expression

Summary Background: The current study was devised with the objective of using a split-mouth, controlled clinical trial to compare conventional mechanical debridement (scaling and root planing) treatment (T1) with conventional mechanical treatment followed by photodynamic therapy (PDT) (T2) in patients with severe periodontitis. Methods: Four PDT sessions were completed, and clinical parameters such as bleeding upon probing (BOP positive), plaque index (PI), probing pocket depth (PPD) and clinical attachment loss (CAL) were evaluated before and after the treatment series. In addition, gingival biopsies were collected at the start and finish of treatment, and were used for qPCR gene expression analysis of TNFA, IL1B, IL8, IL10, IL17, MMP13, FGF2, RANK, RANKL and OPG. Results: The clinical results showed a significant improvement in BOP with treatment T2 (p = 0.03). The molecular data showed an up-regulation of FGF2, RANK and OPG gene expression after T2. The expression levels of the other genes were not significantly different between T1 and T2. PDT increased the expression of RANK and OPG, which could indicate a reduction in osteoclastogenesis. Furthermore, the use of PDT in conjunction with conventional treatment significantly increased the expression of FGF2, which has an important role in the periodontal repair process.

∗ Corresponding author at: Department of Periodontology, School of Dentistry, Catholic University of Brasília, Campus I, QS 07 Lote 01 EPCT, Bloco S, Sala 213, Águas Claras, DF, CEP 71966-700, Brazil. Tel.: +55 61 81214785; fax: +55 61 33569612. E-mail address: [email protected] (E.J. Franco).

1572-1000/$ — see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.pdpdt.2013.10.002

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E.J. Franco et al. Conclusions: PDT technology could be a means to improve conventional periodontitis treatment. Our results suggest that PDT acts in part by controlling bone resorption and increasing the expression of genes important for tissue repair. © 2013 Elsevier B.V. All rights reserved.

Introduction Periodontal disease is an inflammatory disease of multifactorial etiology that develops in response to the presence of specific bacteria. It has the potential to compromise protective structures and the retention of teeth, resulting in attachment loss, irreversible bone loss, and eventually, tooth loss [1,2]. The prevalence of periodontal disease is high; it is considered one of the most common infections observed in all populations and represents a significant cause for concern in public health worldwide [3—8]. Conventional periodontal treatments typically involve radicular mechanical treatments that aim to remove the causative disease agents through the use of manual and ultrasonic techniques. More recently, photodynamic therapy (PDT) has begun to be incorporated as a periodontal treatment with specific antimicrobial characteristics. However, neither the effectiveness nor the mode of action of PDT at human periodontal sites is as yet completely clear, especially with regard to the possible molecular alterations resulting from this treatment. Considering the etiopathogenesis of periodontal disease and the importance of the host as a main factor [9—14], the genes selected for this study are related to immunoinflammatory processes (TNFA, IL1B, IL8, IL10, IL17 and MMP13), bone loss and periodontitis (RANK, RANKL and OPG), and to the repair process (FGF2). The expression levels of these genes were tested in the gingival tissues from periodontitis patients before and after scaling treatment followed by PDT. Furthermore, clinical periodontal parameters such as bleeding upon probing (BOP positive), plaque index (PI), probing pocket depth (PPD) and clinical attachment loss (CAL) were evaluated before and after treatment in order to observe the clinical efficiency of PDT in the treatment of severe periodontitis.

Materials and methods Patient selection For patient selection, a database of 628 patients from the dental clinic at the Catholic University of Brasília (UCB) was used. Fifteen patients were selected based on the following inclusion criteria: aged 35—44 years, diagnosis of severe chronic periodontitis, presence of at least 20 teeth with at least one posterior tooth in each quadrant, and periodontal pockets ≥5 mm on at least seven teeth. Patients subjected to periodontal treatment within the previous six months, patients with systemic diseases that could influence the therapy, smokers, and patients taking antibiotics, corticoids, immunosuppressants or anti-inflammatories within the previous six months were excluded. All patients received

orientation regarding the research and signed an informed consent form. This research was approved by the UCB ethics committee (#52/2010).

Clinical evaluation This study was designed using the split mouth system in which the two quadrants of one side of the mouth were treated with conventional mechanical debridement (deep scaling and root planing) (treatment 1/T1), and the other two quadrants were treated with deep scaling and root planing followed by PDT (treatment 2/T2). All patients received orientation during the first treatment session regarding oral hygiene techniques according to individual necessity. This included information about the use of interdental brushes, soft brushes, and dental floss. In addition, motivational techniques were employed throughout the treatment. It is important to note that all patients were treated by the same therapist. Side 1 (T1) Scaling and root planing was performed using Graceytype (Hu-Friedy® -Chicago, IL, USA) periodontal curettes (5/6; 7/8, 11/12 and 13/14), which were new and properly sharpened. Subgingival scaling was performed with 2% lidocaine anesthetic, with an adrenaline vasoconstrictor (1:100,000). Side 2 (T2) Scaling and root planing was performed with Gracey-type periodontal curettes (Hu-Friedy® -Chicago, IL, USA) (5/6; 7/8, 11/12 and 13/14), which were new and properly sharpened. Sub-gingival scaling was performed with 2% lidocaine anesthetic, with an adrenaline vasoconstrictor (1:100,000). After conventional scraping, PDT was performed using a laser diode (MMoptics-São Carlos-SP, Brazil) at a wavelength of 660 nm, potency of 60 mW/cm2 and energy density of 5.4 J/cm2 . Methylene blue (0.01%) was applied to the subgingival sites using a sterile disposable syringe as a photosensitizing agent, and was left in place for 5 min (the pre-irradiation period). Periodontal pockets were then exposed to laser diode light using a 0.4 mm optic fiber (MMoptics-São Carlos-SP, Brazil). PDT was applied to six sites per tooth (mesiobuccal; buccal; distobuccal; mesiolingual; lingual and distolingual) for 15 s each, for a total of 90 s per tooth. Four sessions were performed, with intervals of seven days between the sessions. All clinical parameters were registered on periograms containing the following clinical data: bleeding upon probing (BOP positive), plaque index (PI), probing pocket depth (PPD) and clinical attachment loss (CAL). These parameters were recorded before treatment and 90 days after treatment. For the measurement of the clinical parameters,

Increased expression of genes a periodontal probe with Williams markings (Hu-Friedy® Chicago, IL, USA) was used.

Collection of samples for gene expression analysis For each patient, four gingival tissue samples were collected from each side of the mouth, each measuring 1 mm2 . Two samples were collected before treatment and the other two samples were collected 30 days after treatment. The gingival sample removal was performed according to the methods described by Dong et al. [15]. Samples were stored in RNA later (Invitrogen, Grand Island, NY, USA) at −80 ◦ C.

43 Table 1

Patient selection and exclusion criteria.

n

(%)

Condition

628 239 197

100.0 38.1 31.4

177

28.2

91

14.5

18

2.9

All patients Smokers Patients with systemic diseases Patients who used any medication in the last 6 months Periodontal treatment in the last 6 months Patients with all research criteria

RNA extraction and cDNA synthesis RNA extraction was performed using the Rneasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Genomic DNA was removed using DNase I (Qiagen Inc., Valencia, CA, USA). The RNA was quantified using the Qubit fluorometer (Invitrogen) and the quality was determined using a Bioanalyser (Agilent Technologies Inc., USA). Reverse transcriptase reactions were performed using a high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s protocol.

the normal gingival tissue of ten healthy volunteers. The scaled values of NRQ were used to perform all comparisons.

Statistical analysis Statistical analysis was performed using SPSS software (version 17.0; SPSS Inc.). Fisher’s exact test was performed to determine differences between the categorical variables. An independent sample t-test was used to determine the differences between the treatment groups and time points. p-Values lower than 0.05 were considered significant.

Real-time quantitative PCR (qPCR)

Results

Quantitative PCR reactions were performed using the steponeplus real-time PCR System (Applied Biosystems, Carlsbad, CA, USA) and the TaqMan Universal PCR MasterMix and TaqMan gene expression assays — FAM-MGB, according to the manufacturer’s instructions. The Assay IDs used were: RANK-HS00921369 M1*, RANKL-HS00243519 M1, OPG-HS00171068 M1, TNFAHS99999043 M1, IL1B-HS01555410 M1, IL8-HS99999034 M1, IL10-HS00961619 M1, IL17-HS00174383 M1*, MMP13HS00233992 M1*, FGF2-HS00266645 M1* and constitutive control genes GAPDH-HS02758991-G1* and 18SrRNAHS99999901-s1. The assays were performed in triplicate using 2 ␮L (500 ng cDNA/␮L) of each sample, and the templates were added to a 10 ␮L final reaction volume. The amplification conditions were as follows: 2 min at 50 ◦ C and 10 min at 95 ◦ C for the initial holding stage, followed by 40 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. The amplicons varied from 63 to 150 base pairs (bp) in length.

Clinical results

qPCR analysis The Cq (quantification cycle) value of each sample was calculated using the StepOne software. The mean of the Cq values of the triplicates for each sample was compiled and transferred to the QBasePlus v2.1 (Biogazelle) specialized software. The qBase method was used for relative mRNA expression analysis, using the GAPDH and 18s rRNA genes as the reference targets for input normalization. The values of NRQ (Normalized Relative Quantification) were then scaled by a sample consisting of a total mRNA pool extracted from

The rigorous inclusion and exclusion criteria used in this study were essential in order to construct a homogeneous sample group. Fifteen patients were selected from a 628patient database, according to the criteria stated previously (Table 1). The clinical results indicated a significant reduction in the percentage of periodontal sites with bleeding upon probing (BOP positive) after conventional treatment associated with PDT (T2) when compared with conventional treatment alone (T1). These data are relevant as they demonstrate the efficiency of PDT in reducing periodontitis (Fig. 1). The other clinical parameters analyzed (plaque index [PI], probing pocket depth [PPD] and clinical attachment loss [CAL]) did not present statistically significant differences between treatments T1 and T2. However, for all parameters there was a significant improvement when the pre-treatment T2 group was compared to the posttreatment T2 group. (Table 2)

qPCR analysis of target genes The integrity and quality of the total RNA obtained was tested by Bioanalyser (Agilent Technologies Inc., USA). The RIN (RNA Integrity Number) values ranged from 9.2 to 10.0, and the ratio ranged from 1.8 to 2.0. This indicated that intact RNA, free of genomic DNA, was successfully isolated. The expression levels of genes related to immunoinflammatory processes (TNFA, IL-1B, IL-8, IL-10, IL-17 and

44

E.J. Franco et al.

Figure 1 Bleeding upon probing (BOP) of periodontal sites before and after treatment T1 and T2. A significant reduction of bleeding was observed after treatment T2 (*p = 0.03).

MMP13), genes known to be involved in bone loss in periodontitis (RANK, RANKL and OPG), and one gene involved in the repair process (FGF2) were determined before and after T1 and T2. The gene expression assays were 100% efficient, with a slope value of −3.32 and an r-value > 0.99. When the expression levels were compared with those of healthy gingival tissue, only T2 showed an up-regulation of the RANK, OPG and FGF2 genes. RANK showed a two-fold up-regulation after treatment compared with before treatment (T2). OPG showed nearly a 2.5 fold greater expression level after T2, and FGF2 showed nearly a three times greater expression level after T2 (Fig. 2). The molecular data showed significant differences in the expression levels of RANK and FGF2 after T1 and T2, with both genes being up-regulated in the T1 and T2 treated sides of mouths when compared with the pre-treatment biopsies. RANK showed a 2.5-fold up-regulation in the patient samples that had undergone T2 compared with those that had instead undergone T1. Likewise, FGF2 showed a two-fold up-regulation for T2 compared with T1 (Fig. 3). There were no statistically significant differences for the others genes tested.

Discussion Photodynamic therapy (PDT) is considered an adjuvant treatment for conventional mechanical periodontal therapy.

Table 2

A principle advantage of this therapy is its specific and localized anti-microbial action in periodontal pockets, which reduces the signs of inflammation without unwanted sideeffects on the adjacent periodontal tissues. In addition it has the potential to improve scarring at periodontal sites [16—22]. Nevertheless, little is known about the molecular mechanisms involved in the response to this treatment. Data generated from molecular findings can be of great utility for the advancement of this area of dentistry. This could also lead to a better understanding of the etiopathogenesis of oral diseases and the development of more sensitive diagnostic methods using molecular biomarkers [23,24]. The present study therefore focused principally on gene expression before and after conventional mechanical periodontal treatment compared to conventional treatment with PDT. Clinical results were also correlated with the molecular data. Although periodontitis is one of the most prevalent human pathologies associated with bone loss, the mechanism of osteoclast formation and bone resorption related to this disease has not been completely elucidated. It is known that some inflammatory cytokines, including IL-1␤, TNF-␣, PGE2, INF-␥ and IL-6, stimulate bone resorption and are present in periodontal tissues. The role of the key markers of osteoclastic activity (RANK, RANKL and OPG) has been wellstudied in healthy and inflamed periodontal tissues [25—27]. RANKL and OPG are known to be important positive and

Clinical parameters before treatment and after 90 days (T1 and T2 treatment).

Clinical parameter

Before T1

PI PPD ≤3 mm PPD 4-5 mm PDD ≥6 mm CAL 1-2 mm CAL 3—4 mm CAL ≥5 mm

75.55 68.75 25.44 5.81 20.22 3.19 7.61

± ± ± ± ± ± ±

5.1 4.6 3.9 1.4 2.9 0.6 2.3

After 90 d. T1

p Value

Beforeb T2

± ± ± ± ± ± ±

<0.005 <0.005 0.07 0.01 <0.005 0.8 0.8

74.78 68.02 24.8 7.18 19.87 5.56 5.81

23.49 76.96 19.04 3.93 15.20 3.59 7.17

1.9 4.1 3.2 1.2 2.1 0.6 1.7

PI, plaque index; PPD, probing pocket depth; CAL, clinical attachment loss.

± ± ± ± ± ± ±

5.2 4.5 3.7 1.9 3.1 1.0 2.1

After 90 d. T2

p Value

± ± ± ± ± ± ±

<0.005 <0.005 <0.005 0.005 0.01 0.01 0.005

16.81 82.74 14.92 2.32 11.70 2.37 4.1

1.7 3.0 2.4 0.8 2.0 0.4 1.8

Increased expression of genes

45

Figure 2 Gene expression before and after periodontal treatment with PDT (T2). qPCR was performed to identify the expression profile of RANK, OPG and FGF2 before and after periodontal treatment with PDT (T2). The data are presented as a Relative Quantitation (RQ), and the qBase method was performed for the relative mRNA expression analysis. GAPDH and 18s rRNA genes were used as the reference targets for input normalization. RANK (p = 0.04); OPG (p = 0.02); FGF2 (p = 0.01).

negative regulators, respectively, of osteoclastogenesis and bone resorption [27,28]. The genes analyzed here were chosen mainly due to their known relationship to the etiopathogenesis of periodontal disease, emphasizing the role of the host as a principal component in periodontal disease. Genes related to inflammation (TNFA, IL1B, IL8, IL10, IL17 and MMP13), bone loss in periodontitis (RANK, RANKL and OPG) and the repair process (FGF2) were therefore selected for expression analysis. The present study observed statistically significant differences in the expression of RANK and OPG before and after PDT (T2). This was not observed after conventional periodontal therapy (T1). It is known that both RANK and OPG, and also RANKL are involved in the bone resorption process. It is important to stress that the main characteristic of bone loss mediated by inflammation in periodontitis is an increase in osteoclastic

activity without a corresponding increase in bone formation. It is well established that inflammation and the immune system are directly involved in the development of periodontitis. More recently, the role of the immune system in bone metabolism has been recognized. A better understanding of the role of the immune system in periodontal disease may be critical for the development of new methods for the prevention and treatment of pathological bone loss in diseases like periodontitis [25]. The increased expression of RANK, and particularly in OPG indicates that there was a reduction in the osteoclastogenic activity. This finding is extremely relevant from a clinical perspective since evidence that PDT may be efficient in interrupting localized bone loss processes has been suggested by others using in vivo studies [12,25]. It is known that the RANK protein, along with its ligand (RANKL) and OPG, is involved in the bone resorption process

Figure 3 Gene expression after periodontal treatment (T1 and T2). qPCR was performed to identify the expression level of RANK and FGF2 in gingival tissues after periodontal treatment (T1 and T2). The data are presented as a Relative Quantitation (RQ), where the qBase method was performed for relative mRNA expression analysis. GAPDH and 18s rRNA genes were used as the reference targets for input normalization. RANK (p = 0.01); FGF2 (p = 0.04).

46 [12]. The expression of RANKL activates a signaling pathway in osteclastic precursors, which induces differentiation into the mature form. OPG regulates bone loss and acts as an antagonist for interaction between RANK and RANKL, thereby impeding osteoclastogenesis. In periodontal tissues, increased levels of RANKL are found in inflamed regions, and the balance between the expression of RANKL and OPG seems to directly influence bone resorption [12,25]. Additionally, it is known that when the concentration of OPG is relatively higher than that of RANKL, OPG attaches itself to RANKL and inhibits its interaction with RANK. In this way, OPG reduces the formation of osteoclasts and promotes apoptosis of preexisting osteoclasts [29]. The RANKL/OPG ratio in inflamed periodontal tissues can be elevated either by an increase in RANKL, a decrease in OPG, or a combination of the two [30,31]. Not only does the RANKL/OPG ratio increase at periodontal inflammation sites, but it is also related to disease severity [12,25,30]. Liu et al. [32] induced lesions in rats and performed treatment with Metformin. This treatment produced a reduction in the RANKL/OPG ratio. In other words, there was a relative downregulation of RANKL and a relative upregulation of OPG. Metformin reduced bone loss, promoted osteoblast differentiation and inhibited osteoclastogenesis. The increase in OPG expression observed in the present study suggests that PDT treatment may also have an important role in controlling bone loss. Yamashita et al. [33] suggested that increased expression of RANK reduces the threshold for RANKL, thus inducing osteoclastogenesis. In other words, osteclastic precursors express more RANK and therefore require less RANKL in order to differentiate into osteoclasts. However, this effect could be compensated by an upregulation of OPG. The present study demonstrates that the expression of OPG was significantly increased after T2, corroborating once again its importance in the control of osteoclastogenesis. Manfrin et al. [34] analyzed the expression of RANKL-RANK-OPG through immunohistochemistry after dental re-implantation in rats. OPG and RANK were simultaneously expressed, with the expression of RANKL being the most variable. Elevated OPG levels may also be linked to osteoblast maturation, and could potentially act as a decoy receptor for RANKL. If this were the case, it would be necessary for OPG to be co-expressed with RANK or the RANK-RANKL signaling pathway would be constitutively activated. The increased OPG expression level relative to RANK may indicate a down-regulation of resorption in order to permit the reconstitution of tissues. These data are in agreement with our findings, as OPG was found to be expressed at a level that was 2.5 times greater following T2. Similarly, the differential expression of the FGF2 gene was twice as high after T2 as after T1. FGF2 is known to be expressed in various tissues and to have effects on various cell types. The protein encoded by this gene promotes the proliferation of fibroblasts and osteoblasts and has a particularly strong induction effect on angiogenic activity [35,36]. These activities are directly associated with the regeneration of periodontal tissues [37]. Thus, the observed up-regulated expression of FGF2 after PDT treatment suggests that this form of therapy efficiently promotes periodontal repair, confirming previous findings in clinical studies [20,37]. It is worth noting that mRNA expression does not necessarily reflect the level or activity of the corresponding protein, although it permits us to understand

E.J. Franco et al. possible mechanisms of and/or the molecular responses to clinical interventions. No statistically significant differences were found for the other genes tested. This could be due to the existence of dental plaque after treatment, which could induce immune-inflammatory processes. It could also be because the expression effects of the treatment on the other genes analyzed did not persist in the gingival tissues for the full 30 days. Significant improvements for all of the other clinical parameters were observed after treatment. When comparing the parameters between T1 and T2, a significant reduction in the percentage of periodontal sites with bleeding was observed after PDT (T2) treatment compared to the T1 treatment (p = 0.03). This finding is important because it suggests that the disease activity at these periodontal sites was interrupted or reduced. These results support earlier in vivo studies showing a reduction in periodontal bone loss and clinical signs of inflammation, such as bleeding on probing after periodontal treatment with PDT [16,38—41]. Christodoulides et al., 2008 [40], clinically examined 24 patients who were given PDT as an adjunctive treatment to mechanical periodontal treatment and reported a significant improvement in the bleeding index after PDT treatment. Cappuyns et al. [41] also evaluated PDT for use in the treatment of residual periodontal pockets and noted a significant reduction in BOP, pocket depth, and level of periodontal pathogens after six months of treatment. These data demonstrate that PDT has a strong influence on the reduction of inflammatory activity in periodontitis when compared to conventional periodontal treatment, as it can alter the clinical manifestations of periodontitis. Based on the clinical and molecular findings of this study, we conclude that PDT used in conjunction with conventional dental scaling leads to an up-regulation of RANK and OPG expression, which could indicate a reduction in osteclastogenic activity. The addition of PDT to conventional treatment also significantly increases the expression of FGF2, which has an important role in the periodontal repair process. The significant improvement observed in the periodontal sites treated with PDT highlights the efficiency of the proposed treatment. This observation could be explained by the differential expression of genes related to osteoclastogenesis and periodontal repair. The combined use of PDT with conventional mechanical scaling and root planing therefore represents a promising new treatment modality for dealing with severe and chronic periodontitis.

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