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Journal of Periodontal & Implant Science logoLink to Journal of Periodontal & Implant Science
. 2023 May 9;54(1):13–24. doi: 10.5051/jpis.2203580179

Adjunctive effect of 470-nm and 630-nm light-emitting diode irradiation in experimental periodontitis treatment: a preclinical study

Dongseob Lee 1,2, Jungwon Lee 1,3,✉, Sun-Hee Ahn 4, Woosub Song 4, Ling Li 1, Yang-Jo Seol 1,2, Yong-Moo Lee 1,2, Ki-Tae Koo 1,2,✉
PMCID: PMC10901685  PMID: 37336520

Abstract

Purpose

This study investigated the adjunctive effect of light-emitting diodes (LEDs) in the treatment of experimental periodontitis.

Methods

Experimental periodontitis was induced by placing ligatures around the mandibular second, third, and fourth premolars of 6 beagles for 3 months. After ligature removal, periodontitis progressed spontaneously for 2 months. The animals’ hemimandibles were allocated among the following 3 groups: 1) no treatment (control), 2) scaling and root planing (SRP), and 3) SRP with LED irradiation at 470-nm and 630-nm wavelengths (SRP/LED). The probing pocket depth (PPD) and gingival recession (GR) were measured at baseline, 6 weeks, and 12 weeks. The clinical attachment level (CAL) was calculated. After 12 weeks, histological and histomorphometric assessments were performed. The distances from the gingival margin to the apical extent of the junctional epithelium (E) and to the connective tissue (CT) attachment were measured, as was the total length of soft tissue (ST).

Results

PPD and CAL increased at 12 weeks compared with baseline in the control group (6.31±0.43 mm to 6.93±0.50 mm, and 6.46±0.60 mm to 7.61±0.78 mm, respectively). PPD and CAL decreased at 12 weeks compared with baseline in the SRP group (6.01±0.59 to 4.81±0.65 mm, and 6.51±0.98 to 5.39±0.93 mm, respectively). PPD and CAL decreased at 12 weeks compared with baseline in the SRP/LED group (6.03±0.39 to 4.46±0.47 mm, and 6.11±0.47 to 4.78±0.57 mm, respectively). The E/ST and CT/ST ratios significantly differed among the 3 groups (P<0.05). The clinical parameters and histologic findings demonstrated that 470-nm and 630-nm wavelength LED irradiation accompanying SRP could improve treatment results.

Conclusions

Within the study limitations, 470 nm and 630 nm wavelength LED irradiation might provide additional benefits for periodontitis treatment.

Keywords: Nonsurgical periodontal debridement, Periodontitis, Photobiomodulation therapy

Graphical Abstract

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INTRODUCTION

Periodontitis is a chronic inflammatory disease typified by periodontal tissue destruction resulting from the interactions among microbial products, cell populations, and mediators [1]. Dysbiosis of the periodontal microbiota means the perturbation of ecologically balanced biofilms related to periodontal tissue homeostasis [2]. The periodontal microbiota produces endotoxins that cause inflammatory cytokines and extracellular polymeric substances to form dental plaque biofilms [3]. In this context, bacterial reduction is the key target for periodontitis treatment in clinical situations [4].

Mechanical debridement of local factors on the tooth surface using scaling and root planing (SRP) plays a pivotal role in treating chronic periodontitis [5]. However, the anatomic complexity of teeth, including furcations, deep grooves, and concavities, impairs the proper mechanical removal of local factors due to access limitations [6,7]. Various therapeutic strategies have been suggested to improve treatment outcomes, such as antibiotics, photodynamic therapy, and laser therapy [8,9]. In recent studies light-emitting diode (LED) irradiation at 425 and 525 nm showed bactericidal effects [10].

Periodontal tissue healing is another issue relevant to treatment. Numerous studies have reported that light or laser irradiation can enhance wound healing [11,12,13,14]. LED irradiation has been shown to promote the proliferation and osteogenic differentiation of periodontal ligament stem cells [15,16]. The promotion of the healing process using LED irradiation may improve periodontal responses following SRP [17].

Phototherapy has recently been introduced to enhance the outcomes of periodontitis treatment based on bactericidal and photobiomodulatory effects [10,12,18,19]. LEDs have been suggested as an energy source for phototherapy [17,20]. Adjunctive phototherapy can be anticipated using LED irradiation at wavelengths exhibiting bactericidal and photobiomodulatory effects in periodontitis treatment. In our previous study, LED irradiation at 470 nm showed bactericidal effects, and LED irradiation at 630 nm after lipopolysaccharide (LPS) treatment reduced nitric oxide in RAW 264.7 macrophages, demonstrating treatment effects on LPS-induced RAW 264.7 inflammation in vitro [21]. To date, there is little in vivo evidence that LED irradiation with a combination of wavelengths might improve periodontal treatment results when combined with mechanical SRP.

The aim of this study was to investigate the adjunctive effect of LED irradiation in the treatment of experimental periodontitis in vivo.

MATERIALS AND METHODS

Ethical statement

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Seoul National University (No. SNU-210317-6) and followed the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines (version 2.0) [22].

Study design

The timeline of this study is presented in Figure 1A. Six 12-month-old beagle dogs were used to investigate the adjunctive effects of LED irradiation in the treatment of experimental periodontitis. The dogs were subjected to no treatment (control group), SRP only (SRP group), or SRP with LED irradiation (SRP/LED group). The right and left mandibles of each subject were separately allocated to randomly assigned intervention groups. The randomization was conducted using a computer-generated protocol.

Figure 1. (A) The timeline of this study. Spontaneous progression of periodontitis for 2 months was observed in this study following 3 months of experimental periodontitis induction for mandibular P2, P3, and P4. Clinical parameters, including probing pocket depth, gingival recession, and clinical attachment level, were measured at baseline, 6 weeks, and 12 weeks of observation. (B) Customized LED irradiation device (1.2 cm × 1.6 cm, width × height). (C) The application of a customized LED irradiation device (wavelength: 470 nm) for the SRP/LED group. (D) The application of a customized LED irradiation device (wavelength: 630 nm) for the SRP/LED group.

Figure 1

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SRP: scaling and root planing, LED: light-emitting diode.

Experimental device

A customized LED irradiation device was manufactured for this experiment (Figure 1B-D). The width and height were gradually increased from the anterior to posterior region of the device to 1.2 cm in width and 1.6 cm in height at the anterior region and 2 cm in width and 2 cm in height at the posterior region. Two LED irradiation devices were made of different wavelength sources, which were 470 nm (blue, 13 V, 10 mA) and 630 nm (red, 8.5 V, 20 mA), and both devices provided identical energy density (energy per unit area) of 10 J/cm2 when applied for 20 minutes without any photosensitizer.

Experimental procedures

Experimental periodontitis induction phase

Experimental periodontitis was induced at the mandibular second, third, and fourth premolars (P2, P3, and P4). General anesthesia was induced by an intravenous injection of a combination of tiletamine/zolazepam as an anesthetic (5 mg/kg), xylazine hydrochloride as a sedative (2.33 mg/kg), and atropine sulfate hydrate as an antispasmodic (0.05 mg/kg). Local anesthesia was conducted using 2% lidocaine HCl containing 1:100,000 epinephrine before the surgical procedure. Sulcular incisions were made with a 15C blade, and mucoperiosteal flaps were subsequently raised. A high-speed #330 bur was used to prepare a notch at the level of the cementoenamel junction, which was made with a #330 bur to tie the ligature wire and silk for experimental periodontitis induction. The surgical area was sutured with 5-0 Monosyn (Figure 2). After the experiment, the animals were treated with intravenous injections of antibiotics (cefazoline; 20 mg/kg) and analgesics (tramadol HCl; 5 mg/kg).

Figure 2. Clinical photos and periapical radiographs in the periodontitis induction and progression stages. The periodontal tissue showed inflammatory signs, resulting in progressive alveolar bone loss.

Figure 2

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The animals were regularly examined at 4-week intervals to maintain the ligatures during the experimental periodontitis induction period. Periapical radiographs and clinical photographs were taken at check-ups every 4 weeks. The ligatures were removed after 2 months of experimental periodontitis induction (Figure 2).

Spontaneous progression of periodontitis phase

After ligature removal, spontaneous progression of periodontitis was allowed for 2 months (Figure 2). After 2 months of spontaneous progression of periodontitis (baseline), clinical parameters, including the probing pocket depth (PPD), gingival recession (GR) (each buccal and lingual side), and width of keratinized gingiva, were recorded.

Clinical observations

After 2 months of spontaneous progression of periodontitis, the hemimandibles of the animals were allocated to 3 groups: control, SRP, and SRP/LED (Figure 3). In the control group, no intervention was given to the experimental teeth. In the SRP group, SRP was performed with an ultrasonic scaler. In the SRP/LED group, SRP was performed and then LED irradiation at wavelengths of 470 nm (blue, 13 V, 10 mA) and 630 nm (red, 8.5 V, 20 mA) was applied for 20 minutes, respectively, providing the same energy density (10 J/cm2) (Figure 1C and D). Every 2 weeks, plaque control using chlorhexidine-soaked cotton and a toothbrush was performed. Clinical parameters, including PPD and GR (each buccal and lingual side) were measured using a UNC-15 probe, and the clinical attachment level (CAL) was calculated at 6 weeks and 12 weeks after the interventions.

Figure 3. Clinical photographs and periapical radiographs of the control, SRP, and SRP/LED groups. At baseline, all groups demonstrated inflammatory signs in periodontal tissue. (A) Persistent inflammation was observed in the control group. (B) Periodontal inflammation was resolved after 6 weeks in the SRP group. (C) The resolution of periodontal inflammation was observed after 6 weeks in the SRP/LED group.

Figure 3

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SRP: scaling and root planing, LED: light-emitting diode.

Histologic processing

All animals were euthanized 12 weeks after baseline under general anesthesia with a combination of intravenous tiletamine/zolazepam and xylazine hydrochloride and then injected intravenously with potassium chloride (0.75 g/kg). The mandibles of the animals were dissected and subsequently immersed in 10% neutral formalin for 10 days. After decalcification with 10% EDTA solution for 10 weeks, each block was dehydrated in a graded series of ethanol solutions and then embedded in paraffin. Serial sections with 5-µm thickness were obtained in the buccolingual plane of the teeth. The centermost sections of each root (mesial and distal root) were subjected to hematoxylin and eosin staining. All histological slides were scanned on a high-throughput panoramic scan (Panoramic 250 Flash III; 3DHISTECH, Budapest, Hungary), and each section image was visualized and analyzed using software (CaseViewer ver. 2.1; 3DHISTECH).

Histomorphometric analysis

Thirty-six teeth (72 roots) from 6 animals were used for the analysis of 3 groups (control, SRP, and SRP/LED). Twelve teeth (24 roots) were assigned to each group. The length from the gingival margin to the apical extent of the junctional epithelium (E) and connective tissue (CT) attachment were measured. The total length of the soft tissue (ST) dimension was measured by summing E and CT. The E/ST and CT/ST ratios (expressed as percentages) in each group were calculated and compared.

Statistical analysis

The Shapiro-Wilk test was used to check whether variables followed a normal distribution. Repeated-measures analysis of variance (ANOVA) was performed to compare repeated measurements of each clinical parameter, including PPD, GR, and CAL at each time point. The differences in each clinical parameter between baseline and 6 weeks, between 6 weeks and 12 weeks, and between baseline and 12 weeks in the 3 groups were compared with 1-way ANOVA. One-way ANOVA was also performed to compare E/ST and CT/ST among the 3 groups. Post hoc multiple comparisons were performed using the Tukey test. All statistical analyses were performed using GraphPad Prism version 9.3.1 for Windows (GraphPad Software, San Diego, CA, USA). Statistical significance was set at 5% (P<0.05).

RESULTS

Clinical findings

At the ligature-induced periodontitis sites, signs of inflammation were observed, including swelling and redness. There was no tooth mobility or severe bone loss at the time of ligature removal (baseline). At 6 weeks and 12 weeks post-intervention, persistent inflammation signs were observed in the control group. Inflammatory signs were not observed in the SRP and SRP/LED groups 6 weeks and 12 weeks after the intervention.

The values of the PPD, GR, and CAL changed at each time point, as described in Table 1. For the PPD, there were significant differences between baseline and 6 weeks, between 6 weeks and 12 weeks, and between baseline and 12 weeks in all groups except between baseline and 6 weeks in the control group (P<0.05). Regarding GR, statistically significant differences were observed between 6 weeks and 12 weeks and between baseline and 12 weeks only in the control group (P<0.05). The CAL showed statistically significant differences between baseline and 6 weeks, between 6 weeks and 12 weeks, and between baseline and 12 weeks in all groups (P<0.05).

Table 1. Clinical parameter changes at baseline, 6 weeks, and 12 weeks in the 3 groups.

Group Time PPD (mm) GR (mm) CAL (mm)
Control Baseline 6.31±0.43 0.15±0.31 6.46±0.60
6 wk 6.53±0.58 0.60±0.50 7.13±0.91c)
12 wk 6.93±0.50a)b) 0.68±0.56a)b) 7.61±0.78a)b)
SRP Baseline 6.01±0.59 0.50±0.51 6.51±0.98
6 wk 5.24±0.68c) 0.58±0.54 5.82±0.95c)
12 wk 4.81±0.65a)b) 0.58±0.58 5.39±0.93a)b)
SRP/LED Baseline 6.03±0.39 0.08±0.15 6.11±0.47
6 wk 5.18±0.45c) 0.31±0.44 5.49±0.54c)
12 wk 4.46±0.47a)b) 0.32±0.50 4.78±0.57a)b)
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Values are presented as mean ± standard deviation.

PPD: probing pocket depth, GR: gingival recession, CAL: clinical attachment level, SRP: scaling and root planing, LED: light-emitting diode.

a)Statistically significant difference between baseline and 12 weeks (P<0.05).

b)Statistically significant difference between 6 weeks and 12 weeks (P<0.05).

c)Statistically significant difference between baseline and 6 weeks (P<0.05).

The SRP/LED group did not show differences in changes in the clinical parameters (PPD, GR, and CAL) from baseline to 12 weeks compared with the SRP-only group (Figure 4).

Figure 4. Clinical parameters including probing pocket depth, gingival recession, and clinical attachment. (A) Differences in probing pocket depth between baseline and 12 weeks, 6 weeks and 12 weeks, and baseline and 6 weeks. (B) Differences in gingival recession between baseline and 12 weeks, 6 weeks and 12 weeks, and baseline and 6 weeks. (C) Differences in the clinical attachment level between baseline and 12 weeks, 6 weeks and 12 weeks and baseline and 6 weeks.

Figure 4

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SRP: scaling and root planing, LED, light-emitting diode.

a)Indicates statistical significance compared to the control group (P<0.05).

b)Indicates statistical significance compared to the SRP group (P<0.05).

Histological analysis

Representative histologic views of the 3 groups are shown in Figure 5. In the control group, severe inflammatory cell infiltration was observed in the E and CT area compared to the SRP and SRP/LED groups (Figure 5B, C, F, G, J, and K). Migration of the junctional E was observed, indicating the onset of periodontal disease. Irregular and extended E was observed in most coronal ST in the control group; however, rete pegs (epithelial extensions that project into the underlying CT) could be observed, unlike the SRP and SRP/LED groups (Figure 5B, F, and J). Fewer blood vessels were not observed in most coronal areas in the control group than in the SRP or SRP/LED groups (Figure 5B, F, and J). The distribution of blood vessels at the apical area was reduced in the SRP/LED group compared with the control or SRP group (Figure 5D, H, and L). The direction of the collagen fibers in the CT was more irregular in the control group than in the SRP or SRP/LED group (Figure 5D, H, and L).

Figure 5. Representative histological views of the 3 groups. Hematoxylin and eosin staining. (A, E and I) Histological views of the control, SRP, and SRP/LED groups. The region of interest was chosen and analyzed for its vertical position in the junctional epithelium (black, blue, and red boxes). (B, F, and J) Higher magnification and the most coronal part of the epithelium in each group. Increased blood vessels, and loss of rete pegs were observed in the control group compared to the other groups. (C, G, and K) Higher magnification and the middle part of the epithelium in each group. More inflammatory cell infiltration into the epithelium and connective tissue was observed in the control group than in the other groups. (D, H, and L) Higher magnification and the most apical part of the epithelium in each group. Regular collagen fiber arrangement was shown in the SRP and SRP/LED groups. A blue arrowhead indicates a rete peg, where epithelial extensions projected into the underlying connective tissue.

Figure 5

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SRP: scaling and root planing, LED, light-emitting diode.

Histomorphometric analysis

The E/ST values in the control, SRP, and SRP/LED groups were 73.90%±6.03%, 67.28%±3.91%, and 62.20%±4.75%, respectively (Figure 6A). Statistically significant differences were found between the control and SRP groups, between the control and SRP/LED groups, and between the SRP and SRP/LED groups (P<0.0001, P<0.0001, and P=0.0026, respectively). The CT/ST values in the control, SRP, and SRP/LED groups were 26.10%±6.03%, 32.72%±3.91%, and 37.80%d ± 4.75%, respectively (Figure 6B). Once again, significant differences were identified between the control and SRP groups, between the control and SRP/LED groups, and between the SRP and SRP/LED groups (P<0.0001, P<0.0001, and P=0.0026, respectively).

Figure 6. The ratio of epithelial and connective tissue to the total length of soft tissue in each group (expressed as a percentage). (A) The E/ST ratio in the 3 groups. A statistically significant difference was demonstrated between the SRP/LED group and the other groups. (B) The percentage of connective tissue in soft tissue in the 3 groups. There was a significant difference between the SRP/LED group and the other groups.

Figure 6

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SRP: scaling and root planing, LED, light-emitting diode.

a)P=0.0026, b)P<0.0001.

DISCUSSION

Various adjunctive therapies accompanying SRP have been proposed as ways to obtain better outcomes. Antibiotics are widely used for adjunctive therapy and provide an additional effect compared to SRP alone in PDD and bleeding on probing reduction. However, systemic side effects and reinfection are noted as disadvantages of antibiotics [8]. Laser therapy has been used as an adjunctive periodontal treatment. Many ranges of wavelength lasers have been used for promoting wound healing (biostimulation), root modification (biomodification), and the photodynamic effect. A low-energy red laser was reported to promote growth of human fibroblasts and enhance blood flow, lowering inflammatory cytokine levels [23]. In addition, it was reported that LED irradiation might also promote proliferation and osteogenic differentiation in periodontal ligament stem cells [15,16]. In previous studies, Er:YAG lasers (47 and 83 mJ) after SRP improved irregular surfaces, such as the smear layer [24], and more collagen fibers adhered to Er:YAG laser-treated root fragments [25]. Photodynamic therapy (antimicrobial) was suggested to overcome the limitations of SRP. In a meta-analysis, photodynamic laser therapy after SRP was revealed to reduce PPD and increase CAL [26]. Aggregatibacter actinomycetemcomitans was significantly reduced by photodynamic therapy. Based on a previous study, serial applications of lasers at specific wavelengths (405, 635, and 810 nm) after SRP could additionally reduce the PPD and CAL at 1 year (P<0.001) [27].

However, LED devices have simpler and smaller structures than laser devices, providing an adjunctive effect similar to those of Er:YAG, and CO2 lasers. From previous studies, specific-wavelength LED devices have been introduced as a photodynamic therapy because of their antimicrobial and anti-inflammatory effects [28,29]. We recently found that 470-nm and 630-nm wavelengths of LED irradiation showed antibacterial and anti-inflammatory effects in vitro [21]. In this context, we investigated the adjunctive effects of 470-nm and 630-nm wavelengths of LED irradiation in the treatment of experimental periodontitis in vivo. To assess the adjunctive effects of LED in periodontal treatment, a ligature-induced periodontitis model was generated in dogs. In this study, the induction and spontaneous progression of periodontitis were confirmed by several clinical parameters. Significant differences were shown over time in the control group, demonstrating an increasing PPD, GR, and CAL (P<0.05) (Table 1). In a previous study, nonsurgical periodontal treatment (SRP) demonstrated improved clinical parameters, resulting in the resolution of periodontitis [5]. Similarly, in our study, the PPD and CAL were significantly different among baseline, 6 weeks, and 12 weeks in the SRP and SRP/LED groups (P<0.05) (Table 1).

Although LED irradiation followed by SRP did not show an adjunctive effect on clinical parameters compared with SRP treatment only, histomorphometric improvements were observed. These results may be affected by the adjunctive effect of LED irradiation. In the histomorphometric analysis, the SRP/LED group demonstrated greater improvements in periodontal tissue healing than the control or SRP group. A significantly lower E/ST ratio was observed in the SRP/LED group than in the other 2 groups (Figure 6A). More CT attachment was also observed in the SRP/LED group than in the other groups, with a significant difference (Figure 6B). Collagen production and proliferation in skin could be promoted by LED irradiation devices exerting photo biostimulation and modification effects [30,31]. In periodontology, after nonsurgical treatment for periodontitis, periodontal tissue mostly heals via the formation of a long junctional E within 2 weeks [32,33]. In general, healthy junctional E contributes to defense mechanisms against micro-organisms and their products [34,35]. Junctional E and CT that were tightly connected to the tooth apparatus were considered healthy periodontal tissue. However, when periodontitis occurs, presenting inflammation signs, the PPD could increase [36]. This may create a subgingival niche for anaerobes to proliferate. As a result, more CT attachment relative to less epithelial attachment may help maintain healthy periodontal tissue.

The histological findings were in accordance with the improved clinical parameters in the SRP and SRP/LED groups. In particular, dense and well-aligned collagen fibers were more abundant in the SRP/LED group than in the control and SRP groups (Figure 5D, H, and L). This phenomenon could be supported by the photobiomodulation effect. However, the changes in clinical parameters from baseline to 12 weeks were not significantly different between the SRP and SRP/LED groups, although different histologic findings were observed between the 2 groups (Figure 4A and C). Despite different histologic findings, a short follow-up period may be insufficient to identify effects on clinical parameters. In previous studies comparing a post-SRP photodynamic therapy group with an SRP treatment-alone group, the PPD and CAL showed significant differences after at least 3 months (P<0.05) [37,38].

Within the limits of this study, 470-nm and 630-nm wavelength LED irradiation accompanying SRP could improve treatment results based on changes in histologic and clinical parameters. However, it must be emphasized that a drawback of the present study was the lack of a healthy periodontal group as a control to compare the 3 groups included in this study. Clinical studies with large samples are needed to evaluate the adjunctive effects of LED irradiation in the future.

Footnotes

Funding: This work was supported by the Development of Advanced Technology for Electronic Systems Program – Optical Convergence for Human Care Technology Development Project (20010763, Development of oral care device with LED standard light source for oral health improvement) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).

Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Author Contributions:
  • Conceptualization: Jungwon Lee, Ki-Tae Koo.
  • Formal analysis: Yang-Jo Seol, Yong-Moo Lee.
  • Investigation: Ling Li, Sun-Hee Ahn, Woosub Song.
  • Methodology: Jungwon Lee, Dongseob Lee.
  • Project administration: Ki-Tae Koo.
  • Writing - original draft: Dongseob Lee, Jungwon Lee.
  • Writing - review & editing: Sun-Hee Ahn, Woosub Song, Ling Li, Yang-Jo Seol, Yong-Moo Lee, Ki-Tae Koo.

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