Introduction

Chronic periodontitis is an infectious disease caused by periodontal pathogens resulting in inflammation, attachment loss, bone resorption, and characterized by pocket formation and/or gingival recession [4, 21]. The goal of periodontal therapy is eliminate the supra- and subgingival deposits from the root surfaces in order to prevent disease initiation and progression [14]. Mechanical treatment for removal deposits involves supra- and subgingival scaling and root planing (SRP) with substantial clinical efficacy [7, 35, 39, 46]. Mechanical therapy alone, however, may not always be predictable and may fail to eradicate the pathogenic bacterial species because of their location within the periodontal tissues or in other areas inaccessible by periodontal instruments during close debridement, which warrants for surgical alternatives [1, 41]. These concerns provide the limiting factors for the long-term stability of the treatment outcomes of nonsurgical techniques and are the basis for adjunctive therapies with antibiotics, antiseptics as well as nonchemical modalities [42, 55, 61, 70]. Yet, research shows conflicting results about the efficacy of these methods without any consensus on the best method for enhancing the outcomes of the mechanical treatment. The complexity of the etiology of periodontal diseases may contribute to the disagreement. Microbial population around the periodontium consists of more than 600 species of bacteria [50], nonbacterial species [60], and their virulence factors such as lipopolysaccharide, fimbria, and capsule [31]. Microbial factors collectively provoke host responses [20, 47, 51, 56], which are also determined by genetic and environmental factors [37]. Perpetuation of the host response by a persistent bacterial challenge disrupts homeostatic mechanisms and results in release of biologic mediators such as cytokines (e.g., interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α [TNF-α]), proteases (e.g., matrix metalloproteinases (MMPs)) and prostanoids (e.g., prostaglandin E2 (PGE2)). These mediators of inflammation promote extracellular matrix destruction in periodontium and stimulate bone resorption [25, 48, 63]. Successful treatment of the periodontal diseases and the stability of the outcomes have to take these issues into account while new approaches and modalities should be considered. To this end, lasers could provide a good option.

Use of lasers in dentistry has been gaining popularity over the past few years. Dental lasers have been classified based on the difference in type of gain medium, wavelength, delivery system, emission modes, tissue absorption, and clinical applications. A wide range of dental lasers including argon and helium lasers, diode lasers, neodymium yttrium aluminum garnet (Nd:YAG) lasers, holmium:YAG, and erbium family lasers (Er:YAG, Er,Cr:YSGG) and CO2 lasers are available for clinical use each with specific advantages [3, 32]. The diode laser is a semiconductor laser that generally includes a combination of gallium (Ga), arsenide (Ar), and other elements such as aluminum (Al) and indium (In) to convert electrical energy into light energy. The wavelength range is about 800–980 nm. The diode laser does not interact with dental hard tissues making it convenient for soft tissue operations; cutting and coagulating gingiva and oral mucosa, soft tissue curettage, or sulcular debridement [3]. Moritz et al. [44] have demonstrated significant bacterial decrease and reduction of inflammation when using a diode laser of 805 nm wavelength combined with SRP. Other authors have also reported favorable results with the use of diode laser in treatment of periodontitis or perimplantitis [6, 19, 38] while few have not found additional benefit in the use of gallium arsenide laser adjunct to SRP [16]. It is still not fully clear how the healing is enhanced in response to adjunctive use of lasers. The data from elsewhere in the human body suggests that lasers can be effective tools in pain reduction, anti-inflammation, and acceleration of wound healing [10, 11, 57]. Diode laser (780 nm) has been shown to inhibit gene expression of the pro-inflammatory interleukin (IL)-1β from aortic smooth muscle cells, modulate MMP activity [23] and reduce monocyte chemotactic protein-1, IL-1α, IL-10, interleukin-1β (IL-1β), and IL-6 in lipopolysaccharide-stimulated macrophages [24]. Acute lung inflammation can be attenuated, IL-10 production can be increased and TNF-α can be reduced [15]. Research further demonstrated that irradiation with diode laser (685 and 830 nm) can significantly inhibit edema formation, vascular permeability, and hyperalgesia in zymosan-induced arthritis in rats [17].

Lasers can be effective on oral microbial species and “disinfect” the periodontal environment [43, 44]. Lasers may also modulate the oral inflammatory response [53]. Limited evidence suggests that diode laser could reduce the periodontal inflammation and matrix metalloproteinase-8 (MMP-8) levels with no significant reduction in gingival crevicular fluid (GCF) IL-1β levels [52]. IL-1β and MMP-8 levels can also be significantly reduced by Nd:YAG laser [53] implying that the type of laser could play role on the outcomes of the therapy. In contrast, a recent study showed that while significant improvements in clinical parameters by diode laser can be attained, the levels of MMP-1 and tissue inhibitor matrix metalloproteinase-1 (TIMP-1) in GCF did not change [5]. Meanwhile, other forms of laser treatment such as the low-level laser therapy have been shown to achieve reductions in the percentage of sites with bleeding on probing and in mean probing depths and in GCF IL-1β compared with the SRP alone [40]. Clearly, there is no consensus on the treatment outcomes with lasers and long-term results are still unknown. Within these lines, it should be noted that the choice of the outcome inflammatory parameters may be important on the study results since the healing is regulated by different molecular mechanism at different stages. For example, IL-1 is a potent pro-inflammatory cytokine which enables the recruitment of cells towards infection sites, promotes bone resorption, and stimulates PGE2 release by monocytes and fibroblasts and the release of MMPs that degrade extracellular matrix proteins [18]. IL-6, on the other hand, is a pleiotropic cytokine with a broad range of humoral and cellular immune effects, relating to inflammation, host defense and tissue injury [49] promoting the osteoclast differentiation from progenitor cells [30]. IL-8 is the best-known chemokine for recruitment and activation of human neutrophils [8] with both inflammation-retarding effects during gingival inflammation and inflammation-enhancing effects during periodontal destruction resulting from increased release of lysosomal enzymes and MMPs from neutrophils [65, 66]. MMPs are proteolytic enzymes that play essential role in degradation and remodeling of extracellular matrix proteins [2, 9, 71]. The proteolytic activity of MMPs is strictly regulated by their TIMPs [59]. In this study, we hypothesized that tissue response to laser treatment can be marked by various inflammatory mediators. Thus, the aim of this study was to test the hypothesis that the diode laser will enhance the treatment outcomes of the SRP in periodontitis and this will be accompanied by the changes in the levels of mediators of inflammation and tissue turnover.

Materials and methods

Participants and study design

This study was a randomized and controlled 6-month clinical trial using a parallel design. Thirty patients (18 men and 12 women) with chronic periodontitis [4], who were referred for periodontal treatment at the Department of Periodontology at the Faculty of Dentistry of Selcuk University, for periodontal treatment between January 2010 and July 2010 were included. Patients who were diagnosed with chronic periodontitis in the Oral Diagnosis and Radiology Department were randomly assigned in two groups to receive SRP alone (control group, n = 15) or SRP followed by diode laser (test group, n = 15) by an experienced investigator (SSH) who did not collect data or perform the procedures. Written informed consent was obtained from all subjects. The study protocol was approved by the Ethics Commission of Selcuk University for human subjects (2009/79). Exclusion criteria were periodontal treatment received for the last 1 year; systemic diseases that could influence the outcome of the therapy, pregnancy, smoking, immunosuppressive chemotherapy; and use of antibiotics and anti-inflammatory drugs for the last 6 months. Patients were included if they had at least 14 teeth with at least two teeth with ≥5 mm probing depth at each quadrant.

Clinical procedure

All patients received oral hygiene instructions and supragingival scaling in a single appointment 1 week apart before treatment. Supragingival scaling was performed for each patient in all groups using hand instruments (Gracey Curettes, Hu-Friedy, Chicago, IL, USA) and ultrasonic devices (Satelec, Merignac, France). Full-mouth subgingival scaling and root planing under local anesthesia was performed in a single appointment for each patient in all groups using an ultrasonic scaler and hand instruments.

SRP and diode laser therapy was performed in the same visit. All treatments were performed under local anesthesia. Laser treatment was performed by using a 940 nm indium–gallium–aluminum–phosphate diode laser (Ezlase, Biolase, USA). The periodontal pocket was set at 1.5 W with a pulse interval of 20 ms and pulse length of 20 ms delivering 20 s/cm2 and 15 J/cm2 of energy. Irradiation was accomplished with a 300 μm fiber optic delivery system. The fiber was inserted into the periodontal pocket base in parallel alignment with the root surface, the device was activated, and the fiber was slowly moved from apical to coronal in a sweeping motion during the laser light emission. This was done mesially to distally at the buccal aspect for 10 s and distally to mesially at the lingual aspect for 10 s reaching a total of 20 s for each tooth. The periodontal pocket was irrigated with saline solution after each session of irradiation. In order to control for the same conditions, pockets were also rinsed with saline after SRP in the control group. Both patients and the operator wore protective glasses during laser application.

The clinical evaluation of the patients was determined by plaque index (PI; Silness & Löe 1964), gingival index (GI; Löe 1967), probing pocket depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP). Clinical parameters were performed at six sites per tooth (mesio-buccal, mid-buccal, disto-buccal, mesio-palatal, mid-palatal, and disto-palatal). The maxillary anterior region (maxillary incisors, canine, and premolar teeth) was used as the test site for the evaluation of site-specific clinical parameters and GCF sampling. GCF sampling and clinical index scores were recorded at baseline and after 1, 3, and 6 months after the treatment. All clinical measurements and treatments were performed by the same experienced investigator (MS). Patients did not know which group they were assigned to until interventions were performed. Only statistician was masked during the whole study period.

GCF sampling and analysis

The deepest three pockets with probing depth of ≥5 mm were chosen for GCF sampling. GCF was collected using filter paper strips (Periopaper, Oraflow Inc., Plainview, NY, USA), from non-adjacent pocket sites in the anterior region. GCF sampling sites were selected according to the baseline measurements. Baseline GCF collection was done before oral hygiene instructions and supragingival scaling. The area was isolated to prevent samples from being contaminated by saliva. The sample site was gently air-dried and all supragingival plaque was removed. The paper strips were inserted into the crevice until mild resistance was felt and left in place for 30 s. Strips contaminated with blood were discarded. GCF volume was measured with a calibrated Periotron™ 8000 m (Oraflow Inc.). The three samples from the deepest pockets sides were pooled in an Eppendorf tube together and diluted in phosphate buffer saline up to 650 μl. The strips were stored at −80 °C until assayed.

GCF samples were analyzed for IL-1β (Invitrogen™ Corporation 542 Flynn Road, Camarillo, CA, USA), IL-6 and IL-8 (DIAsource ImmunoAssays S.A.-Rue de l’Industrie, 8-B-1400, Nivelles, Belgium), MMP-1 and MMP-8 (RayBio®, Norcross, GA, USA), and TIMP-1 (Calbiochem®, Merck KGaA, Darmstadt, Germany) using commercially available kits in accordance with the manufacturer’s instructions.

Sample size calculation and statistical analysis

CAL was set as the primary outcome and used to estimate the sample size. The secondary outcome was BOP (percent) difference between methods. If a CAL difference in the change between methods of 1 mm was to be detected at α = 0.05 with an 82 % power, the appropriate number of participants per group was 15 patients. The statistical analysis was performed using commercially available software (SPSS v.20.0, IBM, Chicago, IL, USA). The Shapiro–Wilks normality test was used to verify the normality of the data. If the data were normally distributed, parametric tests were used for intragroup (repeated measurements ANOVA/Tukey’s test) and between groups (independent samples T test) testing (whole-mouth clinical parameters/site-specific clinical parameters). If the data were not normally distributed, nonparametric tests was used for intragroup (Friedman/Wilcoxon test with Bonferroni’s correction) and between groups (Mann–Whitney U test; all biochemical parameters) significant.

Results

All subjects completed the entire study. Healing was uneventful in all cases. No adverse effects, such as discomfort, burning sensation, dentin hypersensitivity, or pain related to the laser irradiation were reported by any of the subjects. The baseline demographic data of the male and female volunteers are given per group in Table 1.

Table 1 Baseline demographic data
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Clinical assessments

The results of the whole-mouth clinical measurements (mean ± SD) between baseline and time points in test and control groups are displayed in Figs. 1 and 2. In both groups, all clinical parameters showed statistically significant reductions at all time points compared to baseline (p < 0.05). The mean PD at baseline was 3.6 ± 0.3 in the laser group and 3.5 ± 0.5 in the control group. After treatment, these values became 1.8 ± 0.2 and 2.8 ± 0.2 at 1 month, 1.7 ± 0.2 and 2.7 ± 0.2 at 3 months, and 1.7 ± 0.2 and 2.7 ± 0.2 at 6 months, respectively. The mean CAL at baseline was 2.7 ± 0.4 in the laser group and 2.8 ± 0.6 in the control group. After treatment, these values decreased to 1.8 ± 0.2 and 2.1 ± 0.4 at 1 month, 1.7 ± 0.2 and 2.0 ± 0.4 at 3 months, and 1.7 ± 0.2 and 1.9 ± 0.4 at 6 months, respectively. The reduction in PD and CAL was significantly higher for the test group (p < 0.05). The mean PI at baseline was 1.9 ± 0.1 in the laser group and 2.0 ± 0.2 in the control group. After treatment, these values were 1.2 ± 0.1 and 1.4 ± 0.2 at 1 month, 1.1 ± 0.1 and 1.2 ± 0.2 at 3 months, and 1.3 ± 0.2 and 1.4 ± 0.2 at 6 months, respectively. The mean GI at baseline was 1.8 ± 0.1 in the laser group and 1.9 ± 0.2 in the control group. After treatment, these values were reduced to 1.2 ± 0.1 and 1.3 ± 0.1 at 1 month, 1.0 ± 0.1 and 1.1 ± 0.1 at 3 months, and 1.2 ± 0.1 and 1.3 ± 0.1 at 6 months, respectively. The mean BOP percentage at baseline was 81 ± 7 in the laser group and 83 ± 10 in the control group. After treatment, these percentages changed into 16 ± 4 and 28 ± 6 at 1 month, 10 ± 5 and 16 ± 5 at 3 months, and 19 ± 9 and 31 ± 13 at 6 months, respectively. The change in PI and GI after 1 and 6 months and change in BOP at 1 and 3 months were also significantly higher for the test group (p < 0.05). GCF volumes were significantly reduced in both groups after treatment compared to baseline (p < 0.001; Fig. 1). The mean GCF value at baseline was 0.50 ± 0.15 in the laser group and 0.41 ± 0.12 in the control group. After treatment, these values were 0.15 ± 0.05 and 0.19 ± 0.08 at 1 month, 0.11 ± 0.03 and 0.15 ± 0.06 at 3 months, and 0.18 ± 0.07 and 0.13 ± 0.07 at 6 months, respectively. No statistically significant differences were observed in GCF volumes between two groups (p > 0.05).

Fig. 1
figure 1

Whole-mouth PI, GI, BOP (percent) values and GCF volume. *p < 0.05, significant difference between two treatment groups

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Fig. 2
figure 2

Whole-mouth PD and CAL values. *p < 0.05, significant difference between two treatment groups

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The site-specific PD and CAL values were significantly reduced in both groups at all time points compared to baseline (p ≤ 0.001; Fig. 3). The mean site-specific PD at baseline was 6.2 ± 0.5 in the laser group and 5.8 ± 0.8 in the control group. After treatment, these values were 4.1 ± 0.6 and 3.7 ± 0.8 at 1 month, 2.9 ± 0.3 and 3.0 ± 0.5 at 3 months, and 2.8 ± 0.2 and 3.1 ± 0.6 at 6 months, respectively. The mean site-specific CAL at baseline was 5.3 ± 0.6 in the laser group and 5.2 ± 1.1 in the control group. After treatment, these values were 3.2 ± 0.7 and 3.2 ± 1.1 at 1 month, 2.0 ± 0.4 and 2.5 ± 0.9 at 3 months, and 1.9 ± 0.4 and 2.5 ± 0.9 at 6 months, respectively. No significant differences were observed between groups (p ≥ 0.05).

Fig. 3
figure 3

Site-specific PD and CAL values. *p < 0.05, significant difference between two treatment groups

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Changes in cytokine levels in GCF

IL-1β, IL-6, and IL-8 were assessed in GCF as markers of cytokine-mediated inflammatory response (Fig. 4). At baseline, no significant differences were found between test and control groups. The mean IL-1β level at baseline was 22.42 ± 13.94 pg/30 s in the laser group and 25.82 ± 14.41 pg/30 s in the control group. After treatment, these values were 9.84 ± 6.55 and 11.95 ± 15.34 pg/30 s at 1 month, 7.02 ± 4.09 and 5.57 ± 4.50 pg/30 s at 3 months, and 4.70 ± 3.30 and 5.75 ± 4.79 pg/30 s at 6 months, respectively. The mean IL-6 level at baseline was 21.38 ± 12.29 pg/30 s in the laser group and 15.43 ± 7.04 pg/30 s in the control group. After treatment, these values were 8.97 ± 8.76 and 6.80 ± 6.26 pg/30 s at 1 month, 3.53 ± 2.31 and 5.41 ± 5.31 pg/30 s at 3 months, and 3.62 ± 2.53 and 2.47 ± 1.47 pg/30 s at 6 months, respectively. After treatment, levels of IL-1β and IL-6 significantly reduced at 1 month (p < 0.05), stayed low at 3 months (p < 0.05) and 6 months (p < 0.05). There were no statistically significant differences between groups. IL-8 levels significantly increased in both groups after treatment and stayed high for 6 months (p < 0.05), respectively. The mean IL-8 level at baseline was 231.23 ± 49.27 pg/30 s in the laser group and 248.95 ± 69.19 pg/30 s in the control group. After treatment, these values were 323.10 ± 47.84 and 327.50 ± 48.12 pg/30 s at 1 month, 295.61 ± 71.90 and 312.91 ± 21.86 pg/30 s at 3 months, 301.03 ± 58.84 and 334.01 ± 49.53 pg/30 s at 6 months, respectively. The intergroup difference was not significant for IL-8 levels in GCF (p > 0.05).

Fig. 4
figure 4

Total amounts of IL-1β, IL-6, and IL-8 in GCF samples. *p < 0.05, significant difference between two treatment groups

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Changes in MMP and TIMP1 levels in GCF

MMP1, MMP-8, and TIMP-1 were measured in GCF as markers of the tissue turnover (Fig. 5). At baseline, no significant differences were found between test and control groups. MMP-1 levels significantly reduced in both groups after treatment and stayed low for 3 months (p < 0.05) while there was an increase at 6 months (p > 0.05). The mean MMP-1 level at baseline was 14.44 ± 6.33 pg/30 s in the laser group and 11.62 ± 6.87 pg/30 s in the control group. After treatment, these values were 10.24 ± 4.31 and 5.43 ± 4.47 pg/30 s at 1 month, 6.40 ± 4.70 and 5.17 ± 5.56 pg/30 s at 3 months, and 9.46 ± 2.76 and 8.87 ± 5.10 pg/30 s at 6 months, respectively. MMP-8 levels significantly reduced in both groups and stayed low (p < 0.05). The mean MMP-8 level at baseline was 616.16 ± 244.66 pg/30 s in the laser group and 632.35 ± 288.95 pg/30 s in the control group. After treatment, these values were 231.40 ± 125.17 and 453.15 ± 201.88 pg/30 s at 1 month, 127.15 ± 126.83 and 288.36 ± 248.39 pg/30 s at 3 months, and 59.26 ± 40.53 and 85.53 ± 109.20 pg/30 s at 6 months, respectively. The GCF level of MMP-8 was lower in the test group at 1 month compared to the control group (p < 0.05). TIMP-1 levels significantly reduced at 1 and 3 months in response to both treatment types compared to baseline (p < 0.05) while there was an increase in the control group at 6 months returning to baseline levels. The mean TIMP-1 level at baseline was 8.54 ± 7.92 pg/30 s in the laser group and 11.97 ± 6.58 pg/30 s in the control group. After treatment, these values were 1.84 ± 0.52 and 3.87 ± 3.99 pg/30 s at 1 month, 1.68 ± 0.35 and 3.21 ± 1.37 pg/30 s at 3 months, 1.56 ± 0.23 and 11.14 ± 10.59 pg/30 s at 6 months, respectively. The intergroup difference was not significant at 1 month (p > 0.05). The GCF level of TIMP-1 was lower in test group at 3 and 6 months (p < 0.05).

Fig. 5
figure 5

Total amounts of MMP-1, MMP-8, and TIMP-1 in GCF samples. *p < 0.05, significant difference between two treatment groups

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Clinical and biochemical changes were also analyzed for initially moderate (4–6 mm) and deep (≥7 mm) pockets to investigate the effect of additional laser application on pockets with different depths. No significant differences were observed (data not shown).

Discussion

The use of lasers in dentistry field has been the subject of numerous studies. Postoperative healing was uneventful in all cases and no complications such as abscesses, infections, or dentin hypersensitivity were observed. We found that both treatment modalities resulted in significant improvements in all clinical parameters after periodontal treatment. The whole-mouth clinical reductions were greater in the test group compared to the control group. In general, these changes were not accompanied with differences between groups suggesting that other mechanisms (e.g., bactericidal) in addition to the inflammation may regulate the wound healing process in response to laser therapy. One possibility is a localized impact on the gingival/crevicular epithelium. This was first suggested by Romanos et al. in a pig model [54]. In their work, instrumentation of the soft periodontal tissues with a diode laser (980 nm) led to a complete epithelial removal in comparison to conventional treatment methods with hand instruments in pigs. In another study, Kreisler et al. [38] suggested that higher reduction in PD was probably related to the de-epithelization of the periodontal pockets leading to an enhanced connective tissue attachment. These studies confirm previous work where laser application provided a de-epithelization and resulted in a reduced epithelial migration as well as an increased connective tissue formation [12, 33]. Our results support this notion and suggest that the penetration of the laser may not evoke a substantial inflammatory difference compared to the mechanical treatment alone while resulting in an enhanced clinical healing.

Previous work by our group reported that the use of diode laser at the “periodontal pocket setting (decontamination mode)” used in this study enhanced the mRNA expression of insulin growth factor, vascular endothelial growth factor, and transforming growth factor-β in human gingival fibroblasts demonstrating a potent impact on the connective tissue metabolism [29]. Taken together with these in vitro observations, our results suggest that while there was no significant difference in the inflammatory parameters, MMP-1 and TIMP-1 were differentially regulated by the diode laser treatment and connective tissue metabolism may be affected by the laser. In the absence of a profound inflammation-mediated regulation, this change most probably could be attributed to the epithelial changes where epithelium modulates the connective tissue turnover during wound healing. Recent evidence by Sume et al. [62] and Kantarci et al. [36] support this notion and demonstrate that epithelial connective tissue cross-talk is critical for the homeostasis of the periodontal structures. Further research is needed regarding the penetration capacity of and extent of the tissue response to laser therapy in order to identify the epithelium-mediated changes.

Our clinical results are in agreement with those obtained by Kreisler et al. [38] who demonstrated differences between the groups for PD and CAL. In addition, our data also support findings by Qadri et al. [52] who observed differences for laser group in PD, PI, and GI compared to conventional treatment. Likewise, Moritz et al. [44] and Lui et al. [40] who both demonstrated superior results for laser group in the terms of PD and BOP. Contrary to our findings, Yilmaz et al. [72] and De Micheli et al. [16] suggested that diode laser did not result in any additional clinical benefit when compared with conventional treatment. These controversial reports might be the result of different wavelengths, application power densities (685 nm at 30 mW), and application time. In a recent study, Gokhale et al. [26] reported that diode laser application (980 nm, 2.5 W) as an adjunct to periodontal flap surgery did not improve clinical parameters but its bactericidal effect was clearly evident by greater reduction of colony forming units of obligate anaerobes.

In this study, while GCF IL-1β and IL-6 levels decreased, IL-8 levels increased significantly in both groups after treatment compared to baseline. IL-1β, IL-6, and IL-8 level changes in GCF did not show any significant difference between groups. Lui et al. [40], who used combined photodynamic and low-laser therapies as an adjunct to nonsurgical treatment, reported that GCF IL-1β levels reduced after treatment but did not show any significant differences between LASER + SRP group and only SRP group 1 month after treatment. Contrary to our findings, Qadri et al. suggested that diode laser did not affect the inhibition of IL-1β levels in GCF after treatment [52]. This observation might be the result of their application energy density being low (4.5–8.75 J/cm2) and having limited treatment sites. Most studies reported that GCF IL-8 levels decreased after nonsurgical periodontal therapy [22, 34, 64, 67]. In our study, GCF IL-8 level increased in both groups after treatment. Chung et al. [13] suggested that an increase in IL-8 level is associated with an increase in β-glucuronidase activity. Sfakianakis et al. [58] indicated that IL-8 is involved in cell proliferation and angiogenesis in wound healing. Thus, the increase in IL-8 levels could be associated with wound healing including cellular sources such as epithelial cells.

MMPs are responsible for the degradation of various extracellular molecules, including collagen, elastin, proteoglycans, and laminins [25]. MMP-1 and MMP-8 are two major collagenases involved in breakdown of collagen fibers in periodontitis [69]. Aykol et al. [5] reported that diode laser did not promote additional effects to the conventional periodontal treatment in MMP-1 reduction in 1, 3, and 6 months after therapy. Qadri et al. [52] also did not observe statistically significant differences between diode laser and control group in GCF MMP-8 levels after treatment. In our study, the long-term response on MMP-8 is not significantly different between groups. However, as shown in Fig. 5, 1-month data demonstrates that there is a clear and significant difference between the test and control groups. The differences between these studies may be due to laser wavelength (635–830 versus 940 nm in our study), energy density (4.5–8.75 versus 15 J/cm2 in our study) and study design (quadrants instead of the whole mouth). In our study, GCF TIMP-1 levels decreased in both groups after treatment. Aykol et al. demonstrated that there was no statistically significant difference in TIMP-1 levels between the groups at any time points [5]. However, we have observed significant differences in TIMP-1 levels between the groups at 3 and 6 months after treatment. The decrease of TIMP-1 levels after treatment could indicate that inflammatory process has subsided and the gingival tissues have healed [28]. In addition, previous work by Haerian and others suggest that TIMP-1 may be regulated by MMP-1 activity where in healthy tissues TIMP-1 may not be upregulated [27, 45]. This brings up the issue that in our study there is no upregulation of TIMP-1 in the test group while there is an increase in MMP-1 at 6 months. The data may be explained in two ways: (1) TIMP-1 may not be only regulated by MMP-1, which is a known fact that other MMPs are associated with TIMP-1 [68]. (2) As pointed out by Haerian, the assay only detects the total enzyme where there could be difference between active and latent forms [28]. Within the limits of this study (e.g., no blinding for investigator and participants, number of volunteers), taken together, the results of this study suggest that the use of the diode laser as an adjunct to scaling and root planning produces significant improvement in the whole-mouth clinical parameters compared to conventional treatment. When biochemical parameters were compared between groups, laser group has been shown to be more effective than the mechanical treatment in reducing the GCF MMP-8 levels suggesting the epithelial involvement in healing in response to laser treatment.