Effects of Antimicrobial Photodynamic Therapy on Transforming Growth Factor- β 1 Levels in the Gingival Crevicular Fluid

Abstract

Background and objective: Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjunctive therapy to scaling and root planing (SRP). The transforming growth factor-β1 (TGF-β) has been considered as an anti-inflammatory cytokine, and its levels in the gingival crevicular fluid (GCF) could monitor the periodontal repair. This study evaluated the adjunct effect of aPDT compared with SRP, analyzing the TGF-β levels in GCF after nonsurgical and surgical therapy in chronic periodontitis patients. Methods: Fifteen patients, presenting bilaterally lower molars with class III furcation lesions, were selected. Each pair of teeth was randomly assigned to a control group (CG) or test group (TG). Initially, SRP was performed in the CG, and SRP + aPDT in the TG. Forty-five days later, flap surgery plus SRP, and flap surgery plus SRP + aPDT were performed in CG and TG, respectively. GCF was collected and an enzyme-linked immunosorbent assay (ELISA) test was conducted to determine the amount and concentration of TGF-β in the GCF at baseline, 45 days post-initial therapy, and 21 days after surgery. Results: Statistically significant differences between groups were found in relation to GCF volume 21 days after the surgical procedures (p=0.03) and TGF-β concentration in GCF 45 days post-initial therapy (p=0.04), favoring the TG. Conclusions: There was an additional effect of the aPDT protocol compared with SRP for the TGF-β concentration in GCF 45 days after nonsurgical therapy, and for the GCF volume 21 days after surgical therapy.

Introduction

C urrent concepts for periodontal treatment are based on mechanical scaling and root planing (SRP) to remove bacterial plaque and calculus. However, this removal can be impaired in sites with difficult access for adequate instrumentation (e.g., furcation lesions, severe periodontal pockets, and concavities). Multirooted teeth, for example, have special anatomical characteristics that make access difficult for adequate instrumentation in the furcation area, and this is one of the reasons for the more frequent loss of these teeth.^1
–3 Therefore, the assessment of adjunctive therapy to conventional procedures of SRP, such as the antimicrobial photodynamic therapy (aPDT), has been proposed.⁴

The biological principle of aPDT is based on inactivation of target cells, microorganisms, or molecules by the use of a photosensitizer (or photoactive dye) and a laser light source (low-power visible light with a suitable wavelength). In aPDT, the laser light source activates the photosensitizer that is linked to a target cell (bacteria, for example) and that is capable of absorbing light of a specific wavelength and transforming it into useful energy. The transfer of energy from the activated photosensitizer to the target cell's available oxygen molecules results in the formation of toxic oxygen species, such as singlet oxygen and free radicals. These chemical substances can damage proteins, lipids, nucleic acids, and other cellular components, such as mitochondria, lysosomes, and nuclei and cell membranes.^4,5 Previous studies have demonstrated the bactericidal effect of aPDT against periodontal pathogens and, in addition, the potential of some key virulence factors (lipopolysaccharide and proteases) were shown to be reduced by photosensitization.^6

–11

Recently, an in vitro study demonstrated that the aPDT has also the potential to inactivate host inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), through an independent mechanism of the bacterial lysis.⁶ As an adjunctive therapy, studies have demonstrated that aPDT can improve the clinical outcomes compared with SRP therapy alone in nonsurgical periodontal treatment.^12

–16 Furthermore, similar clinical results and effects on crevicular TNF-α and receptor activator of nuclear factor-κB ligand (RANKL) levels were found in patients with aggressive periodontitis after nonsurgical treatment with aPDT or SRP.^17,18 Hence, aPDT may be used as an adjunct to conventional surgical or nonsurgical periodontal therapy, especially for the cases in which these procedures have shown limited results.^12

–16 Therefore, it is possible to assume that the aPDT may contribute to restoring the periodontal homeostasis and to promoting a faster repair after the treatment.

The transforming growth factor-β1 (TGF-β) has been considered as an anti-inflammatory cytokine, a key mediator in the limitation and resolution of inflammation, and its levels in gingival crevicular fluid (GCF) can be useful in monitoring the progress of periodontal repair.^19,20 TGF-β is a pleiotropic cytokine that can regulate cell growth, differentiation, and matrix production. Therefore, to further understand the effect of aPDT on periodontal repair, the present randomized controlled clinical study aimed to assess the TGF-β levels in GCF samples collected from the furcation lesions in patients with chronic periodontitis during nonsurgical and surgical periodontal therapy, using aPDT associated with SRP, or SRP alone.

In the current study, we tested the hypothesis that aPDT as an adjunctive therapy to SRP may optimize periodontal repair through increase of TGF-β levels in GCF.

Material and Methods

Study population

Fifteen subjects (6 males and 9 females, ages 36–65), presenting bilaterally lower molars with class III furcation lesions and scheduled for extraction, were included in this study. Subjects were selected from the pool of patients of the School of Dentistry of Ribeirão Preto, University of São Paulo. The inclusion criteria for the study were: adult subjects with chronic periodontitis, presenting bilaterally lower molars with class III furcation lesions, scheduled for extraction, without endodontic treatment or presence of periapical or pulpal alterations, and caries or restorations close to the gingival margin. The exclusion criteria were: pregnant or lactating women; anti-inflammatory, antibiotic, or hormone use during the 6 months before the study; evidence of systemic modifiers of periodontal disease, such as osteoporosis, smoking, diabetes, or the use of drugs that influence periodontal tissues; compromised heart condition or any other systemic disorder that required antibiotic prophylaxis; and periodontal treatment within the previous 6 months.

Subjects were instructed as to the character and purpose of the study, and all signed an informed consent form. The study protocol was reviewed and approved by the Institution's Human Research Committee (number: 2007.1.1084.58.3).

Study design

The study used the split-mouth design and all patients were treated by the same experienced operator (P.F.A.). Each pair of teeth with class III furcation lesions was randomly assigned, through a computer-generated random number table, to control or test groups (CG and TG, respectively). The randomization was performed by a blinded investigator immediately following the end of the SRP. In the initial therapy, only SRP was performed in the CG, whereas in the TG, the SRP was associated with aPDT, using a phenothiazine photosensitizer and a laser source with wavelength of 660 nm. At 45 days post-initial therapy, flap surgery plus SRP and flap surgery plus SRP + aPDT were performed on the CG and TG, respectively. At 21 days post-surgery, the newly formed granulation tissues in the class III furcation defects were collected carefully for assessing gene expression by the quantitative polymerase chain reaction (PCRq) technique (data not yet reported), and the teeth were extracted. All patients were recalled for prophylaxis once a week during the period of the study.

Oral hygiene program

Fourteen days before treatment, all patients were instructed about the cause and consequences of periodontal disease, and received oral hygiene instructions, according to individual needs. Supragingival plaque retention factors were removed, cavities were filled, and supragingival professional tooth cleaning was performed 7 days before clinical examination.

Initial therapy

After clinical examination, radiographs, diagnosis, and treatment planning, the sites that were not included in the study received subgingival SRP, and extractions were made, if indicated. Furthermore, occlusal adjustment was performed when necessary.

In sequence, the teeth selected for the study were treated. Mechanical subgingival instrumentation was conducted in both groups, under local anesthesia (Alphacaíne^®, DFL, RJ, Brazil), using an ultrasonic device with a scaler tip (Swivel Ultrasonic Inserts, AF Swivel Direct Flow Straight 25K, Hu-Friedy Co., Chicago, IL) and hand instruments (Gracey curettes nos. 7/8, 11/12 and 13/14, and mini five Gracey curettes nos. 7/8, 11/12 and 13/14, Hu-Friedy Co., Chicago, IL), with subsequent rinsing with sterile saline. Instrumentation was performed until the operator felt that the root surfaces were adequately debrided and planed. Following, in the TG, a diode laser (Helbo Therapielaser, Helbo Photodynamic Systems GmbH & Co KG, Grieskirchen, Austria) with a wavelength of 660 nm and power density of 60 mW/cm² was employed together with a phenothiazine chloride photosensitizer (main component is toluidine-blue, Helbo Blue, Helbo Photodynamic Systems GmbH & Co KG, Grieskirchen, Austria) in a concentration of 10 mg/mL.^17,18 The photosensitizer was applied placing the applicator (blunt cannula) at the bottom of the periodontal pockets (in six sites per tooth: furcation area and proximal sites) and was continuously deposited in a coronal direction in order to achieve complete filling of the pockets and coating of the root surfaces. After 1 min, the pockets were irrigated with sterile saline to remove the photosensitizer in excess. In sequence, the pockets were exposed to the laser light, using a fiberoptic applicator (Helbo 3D Pocket Probe, Helbo Photodynamic Systems GmbH & Co KG, Grieskirchen, Austria) of 0.6 mm diameter, during 10 sec per site. The treatment was performed in six sites per tooth, totaling 1 min of treatment per tooth.

Surgical procedures

Forty-five days after initial therapy, the class III furcation lesions were accessed surgically in the same session. Thus, flap surgery plus SRP and flap surgery plus SRP + aPDT were performed in the CG and TG, respectively. Following local anesthesia (Alphacaíne^®, DFL, RJ, Brazil), intrasulcular incisions were made, and buccal and lingual mucoperiosteal flaps were raised. The granulation tissue was removed from the defect, and treatment was accomplished according to the group assignment, as in the initial therapy. In both groups, the flaps were coronally positioned to cover the furcation, and sutured. Analgesic medication was prescribed for 3 days, and chlorhexidine rinse (0.12%) was used twice daily was prescribed throughout the experimental period (21 days). Patients were instructed regarding the standard concerns in the postoperative period, such as no physical exercise, feeding advice, and plaque-control techniques. Sutures were removed after 7 days. At 21 days post-surgery, the newly formed granulation tissue in the class III furcation defects was collected, and the teeth were extracted.

Clinical evaluation

The clinical measurements of each subject was assessed at baseline and 45 days post-initial therapy. All measurements were performed by one experienced periodontal examiner, allowing an intra-experimental comparison of the values. The examiner underwent calibration training at the beginning of the study. The probing depths (PDs), plaque index (PI, developed by Silness & Loe, 1964) and bleeding on probing (BOP) were assessed. BOP was assessed simultaneously with the pocket measurements, using a manual periodontal probe (PCP 12, Hu-Friedy, Chicago, IL), and the presence or absence of bleeding up to 30 sec after probing was recorded.

Collection of gingival crevicular fluid (GCF) samples

GCF samples were collected by one blinded investigator from the furcation area of the experimental teeth at baseline, 45 days post-initial therapy (immediately before the flap surgery), and 21 days after the surgical procedures. Prior to GCF sampling, the supragingival plaque was removed from the selected sites, and these were gently dried by air syringe and isolated by cotton rolls to avoid saliva contamination. Sterile periopaper strips (PerioPaper, Oraflow, Amityville, NY) were carefully inserted into the periodontal pocket of furcation area (buccal surface) until mild resistance was felt, and were left in place for 30 sec. This procedure was repeated in the same site with 3 min intervals between sampling.²¹ Care was taken to avoid mechanical injury, and strips contaminated with blood, saliva, or debris were discarded. Samples were always taken from the same sites at the three visits. The absorbed GCF volume for each strip was determined by an electronic gingival fluid measuring device (Periotron 6000, Oraflow, Amityville, NY), which had been calibrated with known serial volumes of human serum. Subsequently, the strips were placed into sterile microtubes and kept at −70°C until laboratory analysis (the strips from each site were placed in the same microtube). The readings from the electronic instrument were converted to an actual volume (microliters) by reference to the standard curve.

For elution of the GCF components from the strips, 250 μL of phosphate-buffered saline (PBS, pH 7.2) were added to each microtube containing the strips. Subsequently, the microtubes were vortexed for 1 min and then centrifuged at 2000g for 15 min at 4°C. The strips were then discarded, and the supernatant was aliquoted into sterile tubes, which were stored at −70°C until enzyme-linked immunosorbent assay (ELISA) analysis was conducted to determine the amount and concentration of TGF-β in the GCF (primary outcome measure) at baseline, 45 days post-initial therapy (immediately before the flap surgeries), and 21 days after the surgeries.

ELISAs

The amount of transforming growth factor-β1 (TGF-β) in the GCF samples was determined by ELISA using aliquots of each sample and commercially available kits (R&D Systems, Minneapolis, MN) in accordance with the manufacturer's instructions, from duplicate measurements. The GCF samples were assayed at the dilution of 1:6, according to standardization performed in our laboratory. These results were expressed in pg/mL and converted to pg/μL, and then multiplied by the initial sample volume (250 μL buffer+total GCF volume) to obtain results as pg/sample.²² The calculation of the TGF-β concentration in each GCF sample was performed by dividing the total amount of TGF-β (pg/sample) by the total GCF volume of the sample. Thus, the results were also expressed as picograms of TGF-β per microliter of GCF.

Statistical analysis

The GCF volume and the total amount and concentration of TGF-β in GCF were recorded as mean and standard deviations (SD). In order to verify the normality of the data, the method of Kolmogorov and Smirnov was used. For the intergroup comparisons (control group X test group), the nonparametric test (Wilcoxon) was applied, as the data of one of the groups failed the normality test. For the intragroup comparisons (baseline X 45 days post-initial therapy X 21 days after the surgical procedures; in each group), the nonparametric test (Kruskal–Wallis) or parametric test (one-way analysis of variance [ANOVA]) was performed. The level of significance of 5% was used for all statistical comparisons.

The mean values and SD for the clinical variables were calculated for each group and the normality of the data distribution was checked. For the intergroup comparisons (control group X test group) or for the intragroup comparisons (baseline X 45 days post-initial therapy), the nonparametric test (Wilcoxon) was performed, if the data of one of the groups failed the normality test. Paired t test was used when the data were considered normal for the parameter analyzed. The level of significance of 5% was used for all statistical comparisons.

Results

Initially, fifteen patients (n=15 patients; 30 sites analyzed; TG: 15 sites; CG: 15 sites) were selected for the study to determine the amount and concentration of TGF-β in the GCF at baseline, 45 days post-initial therapy (immediately before the flap surgeries) and 21 days after the surgeries; however, one patient used anti-inflammatories and antibiotics after the surgeries, and, therefore, was excluded from the statistical analysis related to the 21 day postoperative period. At the end of the experimental period, that is, 21 days after the surgical procedures, the soft tissues healed completely and did not present clinical signs of inflammation.

There was a statistically significant difference between the TG and CG for the GCF volume at 21 days after the surgical procedures, favoring the TG. At baseline, the GCF volume was 2.75±2.22 μL in the TG, and was 3.11±2.37 μL in the CG (p=0.35); 45 days post-initial therapy, the GCF volume was 2.74±2.64 μL in the TG, and was 2.85±3.02 μL in the CG (p=0.35); however, 21 days after the surgical procedures, the GCF volume in the TG was significantly lower than in the CG (TG=2.34±1.81 μL; CG=3.04±2.00 μL; p=0.03). The intragroup analysis demonstrated no statistically significant differences for the GCF volume between the three periods of evaluation (Table 1 and Fig. 1).

FIG. 1.

Intragroup and intergroup comparisons of the mean values±SD for the gingival crevicular fluid (GCF) volume (μL). Periods of evaluation: baseline; 45 days post-initial therapy (immediately before the flap surgery – T1); 21 days after the surgical procedures (T2). *Statistically significant difference at p<0.05. No statistically significant changes over time in both groups (p=0.85 for test group [TG]; p=0.45 for control group [CG]).

Table 1.

Intragroup and Intergroup Comparisons of the Mean Values±SD for the GCF Volume (μL)

	TG	CG	p - Intergroup
Baseline	2.75±2.22	3.11±2.37	0.35
T1	2.74±2.64	2.85±3.02	0.35
T2	2.34±1.81	3.04±2.00	0.03^a
p - Intragroup	0.85	0.45

Periods of evaluation: baseline; 45 days post-initial therapy (immediately before the flap surgery – T1); 21 days after the surgical procedures (T2). Intragroup comparisons: ANOVA test for the TG and Kruskal-Wallis test for the CG. Intergroup comparisons: Wilcoxon test.

Statistically significant difference at p<0.05.

GCF, gingival crevicular fluid; TG, test group; CG, control group.

In relation to the amount of TGF-β, there was no statistically significant difference between the TG and CG at baseline, 45 days post-initial therapy, and 21 days after the surgical procedures. Furthermore, similar amounts of TGF-β were found at the different time points in each group, with no statistically significant differences (Table 2).

Table 2.

Intragroup and Intergroup Comparisons of the Mean Values±SD for the TGF-β Levels (pg) in the GCF

	TG	CG	p - Intergroup
Baseline	84.26±175.43	64.76±91.17	0.83
T1	49.34±58.89	39.75±104.59	0.16
T2	147.74±358.87	58.66±113.86	0.43
p - Intragroup	0.98	0.37

Periods of evaluation: baseline; 45 days post-initial therapy (immediately before the flap surgery – T1); 21 days after the surgical procedures (T2). Intragroup comparisons: Kruskal–Wallis test. Intergroup comparisons: Wilcoxon test.

Statistically significant difference at p<0.05.

TGF-β, transforming growth factor-β1; GCF, gingival crevicular fluid; TG, test group; CG, control group.

There was a statistically significant difference between TG and CG in the concentration of TGF-β in GCF at 45 days post-initial therapy, favoring the TG. At baseline, the TGF-β concentration in GCF was 45.59±100.22 pg/μL in the TG, whereas in the CG it was 25.64±50.35 pg/μL (p=1); 45 days post-initial therapy, the TGF-β concentration in GCF was significantly higher in the TG (TG=38.35±71.56 pg/μL; CG=5.92±11.14 pg/μL; p=0.04); in the 21-day postoperative period, there was no statistically significant difference between the TG and the CG in GCF TGF-β concentration (TG=170.04±534.38 pg/μL; CG=18.51±31.45 pg/μL; p=0.37). The concentrations of TGF-β in GCF showed no statistically significant changes over time in both groups (intra-group analysis: p=0.81 for TG; p=0.3 for CG) (Fig. 2).

FIG. 2.

Intragroup and intergroup comparisons of the mean values±SD for the TGF-β levels in gingival crevicular fluid (GCF) (pg/μL). Periods of evaluation: baseline; 45 days post-initial therapy (immediately before the flap surgery – T1); 21 days after the surgical procedures (T2). *Statistically significant difference at p<0.05. No statistically significant changes over time in both groups (p=0.81 for test group [TG]; p=0.3 for control group [CG]).

Both groups had ∼100% of visible plaque deposits at baseline on the surface of the analyzed teeth. Plaque scores were markedly reduced, and no significant differences were observed between plaque scores of surfaces treated by both therapies 45 days after initial therapy. BOP was reduced from 100% at baseline to 80% in the TG and 86% in the CG 45 days after nonsurgical periodontal therapy. There were no statistically significant differences between the two groups at 45 days after initial therapy with regard to BOP (p=0.81).

There were no statistically significant differences between the two groups at baseline (TG: 7.07±1.62 / CG: 6.80±1.61) and at 45 days (TG: 6.20±2.11/ CG: 6.27±1.71) after nonsurgical periodontal therapy in relation to PD. However, there was a statistically significant reduction in both groups between baseline and 45 days after initial therapy (TG: p=0.006 / CG: p=0.01).

Discussion

The evaluation of changes in GCF TGF-β levels after periodontal treatment provides an important information relative to the early wound healing process.¹⁹ Therefore, the present randomized, controlled, clinical study evaluated the adjunct effect of the aPDT to SRP during the nonsurgical and surgical periodontal therapy, through the evaluation of the TGF-β levels in GCF samples, using class III furcation lesions in patients with chronic periodontitis. The class III furcation lesions were selected for this study only in order to represent the periodontal sites with difficult access for an effective decontamination by classic mechanical methods, such as SRP.^1
–3 As the teeth with class III furcation lesions were scheduled for extraction, the newly formed tissues under the flap could be collected 21 days after the surgeries and assessed for the expression of a number of genes^23,24 (data not yet reported). The results of this study showed an additional effect of the aPDT protocol to SRP for the TGF-β concentration in GCF 45 days post-initial therapy (p=0.04) and for the GCF volume 21 days after the surgical procedures (p=0.03). However, although the PDs were significantly reduced in both groups, no statistically significant differences were observed between the TG and CG at 45 days post-initial therapy. Furthermore, there were no statistically significant differences between the groups at 45 days after initial therapy with regard to BOP and plaque scores. Hence, although, the study has not found clinical differences between the groups at 45 days after initial therapy, difference in relation to TGF-β concentration in GCF was observed.

To the authors' knowledge, this is the first study that analyzes TGF-β levels in the GCF and its volume in class III furcation lesions before and after periodontal treatment, and, therefore, comparisons with earlier studies are very difficult. Furthermore, studies about the application of aPDT in the treatment of periodontal disease have used different protocols (different types of photosensitizers and their concentration and duration of application; different types of light sources with different wavelengths, power densities, and energy, durations of light application, and frequencies of the aPDT application), also impairing comparisons between this study and others.^13

–18 The choice of an aPDT protocol was based on recent controlled clinical studies that showed similar clinical results and effects on crevicular TNF-α and RANKL levels between the SRP and an aPDT protocol alone, using the same protocol of the present study, in the nonsurgical treatment of aggressive periodontitis.^17,18

It is important to mention that the complexity of the anatomy of class III furcation defects may explain the high SD present in the results of this study. Furthermore, although bilateral defects are more reliable in discriminating between test and control treatments, because of less variability in environmental factors, in the present study, in many cases, within the same patients, the morphology of defects were considerably different. The morphology of the bony defect and amount of periodontium that remains apical and lateral to the defect (the class III furcation lesions can be associated with vertical or circumferencial defects, as observed in the present study) play a role in the healing capabilities of these sites.

It is important to report that as a split-mouth design was used and some studies have reported an antimicrobial effect even using photosensitizer alone, care was taken to avoid the dye accidentally contacting the tissues and teeth of the control group.^25
–27

The anatomy of the class III furcation defects in mandibular molars, because of its complexity, is responsible for the lack of predictability of the SRP procedures (nonsurgical or surgical periodontal therapy), and is a reason for the more frequent loss of these teeth.^{1
–3,28

–31} The aPDT is an easy handling technique, and, therefore, seems to be appropriate for sites with difficult access for instrumentation in the whole extension of the contaminated root. Therefore, in the current study, the hypothesis that the adjunct use of aPDT to SRP may result in an additional improvement for reducing the bacterial load in class III furcation lesions and, consequently, that significant changes in GCF volume and TGF-β levels in GCF may be detected after periodontal treatment, was tested.

The results of the SRP group corroborate those in clinical trials that have reported the ineffectiveness of SRP procedures in the nonsurgical or surgical periodontal treatment of class III furcation defects.^30,32 However, in the SRP + aPDT group, there were also no statistically significant differences after nonsurgical and surgical periodontal therapy for the GCF volume and total amount and concentration of the TGF-β in GCF. On the other hand, there was a statistically significant difference between the SRP group and the SRP + aPDT group in relation to TGF-β concentration in GCF 45 days post-initial therapy and for the GCF volume 21 days after the surgical procedures, favoring the TG.

Investigators have reported that the change in GCF volume is the most sensitive and least subjective indicator of alterations in periodontal inflammation.^33,34 Based on GCF volume, our data suggest that the surgical treatment with SRP + aPDT, when compared with the surgical treatment, using only SRP, contributed to decrease in inflammation after 21 days. This result is in accordance with those of a recent study, also evaluating the effects of adjunctive aPDT in chronic periodontitis.¹⁵ Assessing 20 patients, the authors found significantly lower values for the sulcus fluid flow rate (relative Periotron units) in the test group (SRP + aPDT) than in the control group (SRP) 3 months after treatment.¹⁵ Also, in controlled clinical studies in patients with chronic periodontitis, the adjunct use of aPDT to SRP, when compared with SRP alone, resulted in a statistically significant improvement of BOP scores, indicating also a higher reduction of periodontal inflammation.^13

–16 Another study, in patients with aggressive periodontitis, also showed a significant reduction in BOP scores after application of aPDT alone, which was similar to the SRP group.¹⁷ Therefore, based on the results of these studies, it may be concluded that the aPDT was able to contribute to the reduction of inflammation. On the other hand, it is important to consider that the beneficial effect on inflammation may be attributed to additional treatment with low-level laser, which has been used in aPDT.^35
–37

TGF-β is a key mediator in the limitation and resolution of inflammation, and its levels in GCF can be useful to monitor the progress of periodontal repair.^19,20 This growth factor has important effects on regulation of matrix metalloproteinases (MMPs): it suppresses collagenase production by fibroblasts and macrophages, while enhancing the expression of tissue inhibitors of MMPs (TIMPs), resulting, consequently, in a decrease of connective tissue matrix destruction.³⁸ Furthermore, TGF-β has effects on th cell proliferation and differentiation process, increases the synthesis of extracellular matrix molecules, and is considered as a chemotactic agent for bone cells.³⁸ Therefore, the higher the TGF-β concentration in GCF (the greater the amount of TGF-β in the smallest GCF volume) may suggest a better resolution of inflammation and repair processes 45 days after the nonsurgical periodontal treatment associated with aPDT, when compared with SRP alone. After surgical treatment, a greater amount (2.5 times higher than the control group) and concentration of TGF-β in GCF (∼10 times higher than the control group) were also found for aPDT associated with SRP; however, there were no statistically significant differences between the groups. As mentioned previously, these results may also be attributed only to additional application of low-level laser, as it is an effective tool to reduce inflammation and promote wound repair.^35,39,40

Our TGF- β concentration in GCF findings are in agreement with those from other studies, which detected lower TGF- β concentration in GCF at more inflamed sites.^41,42 Expression of GCF constituents as concentration could result in higher levels particularly at healthy sites, where GCF volume is very low. Moreover, it has been stated that a greater increase of GCF volume may lead to decreased cytokine concentration in GCF samples. Therefore, the higher TGF-β concentration in GCF in the TG 45 days after the nonsurgical periodontal treatment may suggest a better resolution of inflammation and repair process, when compared with SRP alone.

It is important to consider that the aPDT was applied in a single session, but according to the manufacturer's recommendation, repeated aPDT applications should be performed during the first weeks of healing in order to enhance the antimicrobial effect and to optimize the periodontal repair. A recently published meta-analysis has concluded that the combined approach (SRP with aPDT) provides potential clinical improvements,¹² and further studies are necessary to determine which is the best aPDT protocol to enhance antimicrobial action (if multiple aPDT applications may improve the therapy outcomes) and biostimulatory effect of low-level laser in the treatment of periodontal disease. Furthermore, future randomized, controlled, clinical studies should be conducted to compare SRP alone with SRP + aPDT, aPDT alone, SRP + low-intensity laser, low-intensity laser alone, SRP + photosensitizer, and photosensitizer alone in order to clarify the effects of each therapy and their association on periodontal disease.

Conclusions

This study concluded that there was an additional effect of the aPDT protocol to SRP for TGF-β concentration in GCF 45 days after nonsurgical therapy, and for GCF volume 21 days after surgical therapy.

Footnotes

Acknowledgments

This study was supported financially by The State of São Paulo Research Foundation São Paulo, SP, Brazil (FAPESP: protocol number 07/04916-9) and Coordination for the Development of Personnel in Higher Education (CAPES) Brasília, DF, Brazil.

Author Disclosure Statement

No competing financial interests exist.

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