INTRODUCTION:

Today, cosmetic and facial aesthetics were deemed important aspects of a human being. Orthodontic treatment is the treatment that aims to achieve good aesthetic facial profile and functional conditions.^1,2

The treatment duration is one of the patient’s considerations before undertaking orthodontic treatment. The average length of fixed orthodontic treatment takes about two to three years. This is sometimes difficult for patients to accept, especially adult patients. The long duration of orthodontic treatment can have negative effects, such as enamel demineralization, root resorption, gingival inflammation, caries, and periodontitis.^3,4

Various methods were developed to reduce the orthodontic treatment time by increasing the rate of tooth movement, thereby making it easier for both the patient and the orthodontist.⁵ Methods of accelerating orthodontic tooth movement are classified into two categories: (1) invasive surgical methods such as corticotomy and (2) non-surgical invasive methods such as pharmacology, application of electric currents, resonant vibrations, electromagnetic fields, and photobiomodulation.^6,7 The photobiomodulation method offers several advantages, such as being non-invasive, easy to use, inexpensive, not requiring expensive special machines, and exerting no local or systemic negative effects, over other methods.⁶

Photobiomodulation is a therapy using low-level laser exposure or light-emitting diode (LED) light, which has been proven to produce various beneficial biological effects. The photobiomodulation mechanism involves photobiological reactions, which is the absorption of light with a certain wavelength by photoreceptor molecules. Photobiological responses can be categorized into primary and secondary reactions. The primary reaction comes from the synergy-interpolated photons and photoreceptors that can be observed within a few seconds or minutes after light radiation.⁸ Light waves are absorbed by cytochrome C oxidase that is a photoreceptor in mitochondria causing an increased cellular respiration as well as the production of ATP, nitric oxide, and very low levels of reactive oxygen species.⁷ Secondary reaction is an effect that occurs in response to a primary reaction occurring within hours or days. Effects that arise from primary responses are augmented and transferred to other parts of the cell, further producing physiological outcomes such as alterations in cell membrane permeability, adjustments in intracellular calcium levels, enhanced cellular metabolism, and syntheses of RNA and DNA.⁸

Orthodontic tooth movement (OTM) results from a biological response that facilitates tooth movement within the alveolus. OTM depends on remodeling the periodontal ligament and alveolar bone. A pressured periodontal ligament will trigger bone resorption, whereas a tensioned periodontal ligament will trigger bone formation. Bone resorption is required to allow OTM in the direction of orthodontic force to be applied; hence, the amount of tooth movement will be promptly associated with the rate of bone resorption.^9,10 New bone formation is performed by osteoblasts on the tension side, whereas bone resorption is regulated by osteoclasts on the pressure side.^11,12

Angiogenesis contributes to the bone remodeling process.¹³ Angiogenesis is the development of new blood vessels from existing blood vessels.¹⁴ The increased activity of blood vessels will contribute to the accelerated bone turnover or remodeling. The accelerated remodeling process will result in a shorter orthodontic treatment time. Angiogenesis plays an important role in increasing bone remodeling to shorten the orthodontic treatment time.¹⁵ The objective of this study is to investigate the influence of LED blue-light exposure on the amount of blood vessels on the pressure and tension sides during OTM.

MATERIALS AND METHODS:

This study was categorized as laboratory experimental research. This research has been accepted by the Research Ethics Commission of the Faculty of Dentistry, Universitas Gadjah Mada, with the amount number: 00458/KKEP/FKG-UGM/EC/2020. This study used 48 male Wistar rats (Rattus norvegicus) aged 2 months having a weight of 200–250 grams that were obtained from LPPT unit IV. The research subjects were randomly selected and divided into four groups: the control group (C), 25-second exposure of blue LED light (T1), 30-second exposure of blue LED light (T2), and 35-second exposure of blue LED light (T3) (n = 12 Wistar rats). Wistar rats were generally anesthetized using ketamine and xylazine intramuscularly in the right upper thigh muscle before receiving treatment. A light orthodontic force (35 grams force) was applied to both mandibular incisors of Wistar rats using a NiTi open coil spring having a size of 0.010"× 0.030" (American Orthodontics®, USA). This force was measured using tension gauge (Dentaurum®, Germany). The bracket (American Orthodontics®, USA) was placed 3 mm from the incisal ends of the 2 mandibular incisors (Figure 1). In the control group, Wistar rats were not exposed to the blue LED light, whereas the treatment groups were exposed to the blue LED light for 25 seconds (T1), 30 seconds (T2), and 35 seconds (T3). At a wavelength of 490 nm and a power density of 1000 mW/cm², the blue LED light came from Light Cure LED D (Guilin Woodpecker Medical Instrument®). The blue LED light exposure was carried out at the center of resistance, namely on the labial gingiva, 5 mm from the gingival edge between the mandibular incisors with an optical fiber distance of 1 mm to the gingiva once a day for the treatment duration (Figure 2).

Wistar rats were euthanized using an overdose of ketamine and xylazine. Observations were made on days 0, 3, 7, and 14 after treatment. Hematoxylin–eosin staining was performed to histologically examine the amount of blood vessels on the periodontal tissue ligament. The amount of blood vessels on the tension and pressure sides is calculated by counting the visible amount of blood vessels during an observation under a binocular light microscope with 400× magnification. Observations were made in five visual fields randomly selected as the regions of interest, extending from the incisal to the apical directions of the periodontal ligament on both the pressure and tension sides.

Data from the observation of the amount of blood vessels were tested for normality using Shapiro–Wilk test and analyzed for homogeneity with Levene’s test. The data were analyzed using the two-way ANOVA test with a confidence level of 95% (P < 0.05). Then, the post-hoc LSD test was performed.

Figure 1. Schematic of the experimental Wistar rat OTM model. Installation of an open coil spring on the mandibular incisors to moved the teeth distally

Figure 2. The exposure to blue LED light in the Wistar rat

RESULT:

The results showed that the average amount of blood vessels visible on the pressure side was the highest in the 35-second blue LED light exposure group as compared to the control group, 25-second blue LED light exposure group, and 30-second blue LED light exposure group. The average amount of blood vessels on the tension side was mostly found in the 35-second blue LED light exposure group followed by the 30-second blue LED light exposure group, 25-second blue LED light exposure group, and control group. The results of the Shapiro–Wilk normality test and Levene’s homogeneity test showed that the data on the amount of blood vessels on the pressure and tension sides were normally distributed and homogeneous (P < 0.05).

Two-way ANOVA test results showed that the exposure of blue LED light increased the amount of blood vessels on the pressure and tension sides (P <0.05) (Tables 1 and 2). Post-hoc LSD test showed significant differences in the increased amount of blood vessels on the pressure side occurred in the 35-second (T3) blue LED light exposure treatment group (P < 0.05), whereas the 30-second (T2) and 25-second (T1) blue LED light exposure treatment groups showed no improvement as compared to the control group (P > 0.05). The increase in the average amount of blood vessels occurred from day 0 to day 3 (P < 0.05).

Post-hoc LSD test of the amount of blood vessels on the tension side showed significant differences of treatment groups as compared to the control group (P < 0.05). The increase in the amount of blood vessels was mostly found in the 35-second (T3) blue LED light exposure treatment group followed by the 30-second (T2) and 25-second (T1) blue LED light exposure treatment groups (P < 0.05). The increase in the amount of blood vessels occurred on day 0 to day 14 (P < 0.05).

Table 1. The average and standard deviation (SD) of the amount of blood vessels in the control and treatment groups (T) on days 0, 3, 7, and 14 on the pressure side

Day	Amount of blood vessels				P-value
Day	Control	T1	T2	T3
0	1.27 ± 0.12	1.20 ± 0.20	1.40 ± 0.53	1.40 ± 0.53	0.001^*
3	1.33 ± 0.12	1.93 ± 0.12	2.00 ± 0.40	2.67 ± 0.31
7	2.07 ± 0.31	2.00 ± 0.20	2.27 ± 0.12	3.07 ± 1.75
14	2.00 ± 0.40	2.20 ± 0.20	1.87 ± 0.95	2.20 ± 0.35

Notes :

Control : Not exposed to blue LED light

T1 : 25-second LED blue-light exposure group

T2 : 30-second LED blue-light exposure group

T3 : 35-second LED blue-light exposure group

*by two-way ANOVA followed by posthoc LSD test, significant differences between groups (p < 0.05).

Table 2. The average and standard deviation (SD) of the amount of blood vessels in the control and treatment groups (T) on days 0, 3, 7, and 14 on the tension side

Day	Amount of blood vessels				P-value
Day	Control	T1	T2	T3
0	1.33 ± 0.31	1.25 ± 0.22	0.87 ± 0.42	1.07 ± 0.42	0.001^*
3	1.47 ± 0.27	1.80 ± 0.28	2.40 ± 0.53	2.47 ± 0.13
7	1.67 ± 0.50	2.67 ± 0.20	2.48 ± 0.23	3.33 ± 0.12
14	2.67 ± 0.42	2.73 ± 0.12	2.73 ± 0.31	3.13 ± 0.51

Notes :

Control : Not exposed to blue LED light

T1 : 25-second LED blue-light exposure group

T2 : 30-second LED blue-light exposure group

T3 : 35-second LED blue-light exposure group

*by two-way ANOVA followed by posthoc LSD test, significant differences between groups (p < 0.05).

A histological examination with 400× magnification was performed to determine the amount of blood vessels in the periodontal ligament on the pressure and tension sides. Figure 3 illustrates the amount of vessels on the pressure side periodontal ligament. Moreover, Figure 4 illustrates the amount of blood vessels in the periodontal ligament on the tension side.

Figure 3. Histological view of the periodontal ligament on the pressure side at 400× magnification. The red arrow shows the blood vessels. Control = Not exposed to blue LED light; T1 = 25-second LED blue-light exposure; T2 = 30-second LED blue-light exposure; T3 = 35-second LED blue-light exposure.

Figure 4. Histological view of the periodontal ligament on the tension side at 400× magnification. The red arrows show the blood vessels. Control = Not exposed to blue LED light; T1 = 25-second LED blue-light exposure; T2 = 30-second LED blue-light exposure; T3 = 35-second LED blue-light exposure.

DISCUSSION:

The average amount of blood vessels on the pressure side increased after 35 seconds of blue LED light exposure. An increase in the amount of blood vessels in rats that were exposed to blue LED light for 35 seconds is explained by the fact that the exposure to blue LED light may have triggered the formation of new blood vessels by increasing the expression of vascular endothelial growth factor (VEGF). LED exposure can affect cellular metabolism by triggering the intracellular photo biochemical reactions to produce various effects; one of them is the stimulation of angiogenesis.⁷ LED light can increase the proliferation and migration of endothelial cells, which are the constituent cells of blood vessels.¹⁶ The mechanism involves the absorption of LED light waves by cytochrome c oxidase, which is a photoreceptor in the mitochondria that further increases the amount of nitric oxide (NO).⁷ NO will trigger the formation of new blood vessels by increasing VEGF production. VEGF is one of the most important mitogens that induce angiogenesis.¹⁷The average amount of blood vessels on the pressure side increased between day 0 and day 3. This increase in the amount of blood vessels was attributed to the high VEGF expression on day 3, possibly because of the narrowing of the blood vessels due to orthodontic forces that naturally reduced blood supply, resulting in hypoxia on the pressure side. Hypoxia induces the formation of the active transcription factor hypoxia-inducible factor 1 (HIF-1) and activates the production of VEGF that induces the formation of blood vessels.¹⁸ The exposure to red LED and blue LED light for 10 minutes in 5 consecutive days increases angiogenesis; hence, it can accelerate the healing of ischemic wounds.¹⁷

The amount of blood vessels on the pressure side did not change significantly between day 3 and day 14, possibly because it was a lag phase. The movement of orthodontic teeth is divided into three stages: the initial phase, the lag phase, and the post lag phase. The initial phase happens shortly after the application of orthodontic force and occurs between 24 hours and 2 days. The next stage is the lag phase in which the tooth movement is minimal or sometimes there is no movement at all due to the hyalinization of the periodontal ligament on the pressure side. Tooth movement holds 20–30 days until all the necrotic tissue is excluded along with resorption of the surrounding bone.¹⁹

An increase in the amount of blood vessels on the tension side after exposure to blue LED light for 25 seconds, 30 seconds, and 35 seconds was attributed to the fact that the exposure to blue LED light triggered angiogenesis that increased the amount of blood vessels. The average amount of blood vessels on the tension side increased significantly from day 0 to days 3, 7, and 14. Twenty-four hours after applying orthodontic force, the blood vessels around the root on the tension side will expand.¹⁸ An increase in the amount of blood vessels occurred continuously on the tension side on days 3, 7 and 14 because VEGF expression on those 3 days increased along with VEGF expression by osteoblasts.²⁰ An increase in VEGF was found in fibroblasts and osteoblasts on the tension side of the periodontal ligament one day after the application of synthetic orthodontic force, and LED light could increase the production of DNA and RNA by cells.^8,21 VEGF is the main mediator of angiogenesis and increases vascular permeability. VEGF plays a major role in bone remodeling because it is involved in bone resorption and formation.²¹

The difference in the exposure time to blue LED light between groups influenced the amount of blood vessels on the pressure and tension sides. There was a significant difference in the amount of blood vessels on the pressure and tension sides among rats that were exposed to blue LED light for 25 seconds, 30 seconds, and 35 seconds. The average amount of blood vessels on the pressure and tension sides of the blue LED exposure for 35 seconds was the highest among the other groups. The increase in the average amount of blood vessels was attributed to the different doses received by each group. The Arndt–Schulz law states that activating the cellular response requires a precise set of parameters called fluence or dose. The size of the dose will be proportional to the time of exposure and intensity.²² The increase in exposure time was related to an increase in the amount of blood vessels on the pressure or tension side. The three treatment groups were exposed to blue LED light with the same intensity, that is 1000 mW/cm², but with different exposure times resulting in different doses. A 25-second exposure to blue LED light produced a dose of 25 J/cm²; 30 seconds of exposure to blue LED light produced a dose of 30 J/cm²; and 35 seconds of exposure to blue LED light produced a dose of 35 J/cm².

An increase of exposure time will increase the dose that influenced the effectiveness of the blue LED light while increasing the amount of blood vessels on the pressure and tension sides. The highest effectiveness was indicated by the exposure to blue LED light for 35 seconds in proportion to the highest administered dose. These results were consistent with the previous research stating that the exposure to LED light at a dose of 24 J/cm² could increase OTM and miniscrew stabilization.⁶

CONCLUSION:

Based on this study, it can be concluded that an exposure to blue LED light increases the amount of blood vessels that plays an important role in the orthodontic tooth movement on the pressure and tension sides. The exposure to blue LED light for 25 seconds has been able to increase the amount of blood vessels on the tension side, whereas the pressure side requires the exposure to blue LED light for 35 seconds.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 18.05.2021 Modified on 27.06.2021

Research J. Pharm.and Tech 2022; 15(3):1196-1200.

DOI: 10.52711/0974-360X.2022.00200