Effects of a Low Level Laser on Periodontal Tissue in Hypofunctional Teeth

Malocclusions, such as an open bite and high canines, are often encountered in orthodontic practice. Teeth without occlusal stimuli are known as hypofunctional teeth, and numerous atrophic changes have been reported in the periodontal tissue, including reductions in blood vessels in the periodontal ligament (PDL), heavy root resorption, and reduced bone mineral density (BMD) in the alveolar bone. Low Level Laser (LLL) has been shown to have a positive effect on bone formation and the vasculature. Although the recovery of hypofunctional teeth remains unclear, LLL is expected to have a positive influence on periodontal tissue in occlusal hypofunction. The aim of the present study was to elucidate the relationship between LLL and periodontal tissue in occlusal hypofunction. Twenty-four male rats aged 5 weeks were randomly divided into control and hypofunctional groups. An anterior metal cap and bite plate were attached to the maxillary and mandibular incisors in the hypofunctional group to simulate occlusal hypofunction in the molars. LLL irradiation was applied to the maxillary first molar through the gingival sulcus in half of the rats. Rats were divided into four groups; control, control+LLL, hypofunctional, and hypofunctional+LLL. Exposure to LLL irradiation was performed for 3 minutes every other day for 2 weeks. Animals were examined by Micro-CT at 5 and 7 weeks and were subsequently sacrificed. Heads were resected and examined histologically and immunohistologically. The hypofunctional group had obvious stricture of the PDL. However, no significant differences were observed in the PDL and alveolar bone between the hypofunctional+LLL and the control groups. In addition, the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)-positive cells were higher in the hypofunctional + LLL group than in the hypofunctional group. These results indicated that LLL enhanced the production of bFGF and VEGF in the periodontal tissue of hypofunctional teeth.


Introduction
Malocclusions, such as an open bite, high canines, or underoccluded teeth, are often encountered in orthodontic practice. Teeth without occlusal stimuli are known as hypofunctional teeth, and numerous atrophic changes have been reported in the periodontal ligament (PDL) of these teeth [1,2]. The extent of root resorption was also shown to be significantly greater in hypofunctional teeth than in control teeth under normal occlusal conditions during orthodontic tooth movement in rats [3]. Moreover, root size and the structure of the PDL may be reduced because of disuse atrophy resulting from defects in occlusal function [4]. The effects of the loss of occlusal stimuli loss on alveolar bone formation have also been reported [5].
Low Level Laser (LLL) has recently been shown to have a positive influence on diseases of the joints, connective tissue, neuronal tissue, bone formation, and vasculature [6,7]. LLL was previously used to enhance bone healing after fractures [8,9], and to stimulate condylar growth [10]. Moreover, LLL may accelerate the process of fracture repair or increase bone mineral density (BMD) [11]. These findings suggest that low level laser therapy (LLLT) may have a positive effect on newly formed bone. The use of low-level diode lasers in periodontal therapy has recently been considered to improve wound healing in gingival tissue and accelerates gingival healing after LLLT in sites undergoing gingivectomy [12,13]. Due to their wavelength characteristics, low-level diode lasers are able to reach not only epithelial tissues, but also subepithelial tissues, and laser irradiation is also expected to induce the proliferation of osteoblasts [14] and periodontal ligament fibroblasts [15].
Numerous studies have reported structural changes in the PDL under hypofunctional conditions, and an association with cytokine growth factors that may affect the biological properties of hypofunctional teeth. Occlusal stimuli have been shown to regulate interleukin-1 beta and basic fibroblast growth factor (bFGF) [16]. On the other hand, recent studies have indicated that LLL irradiation is able to stimulate the release of growth factors, such as vascular endothelial growth factor (VEGF) [17], bFGF, and insulin-like growth factor-1 [18]. bFGF is known to promote the proliferation of various cells associated with wound healing, and plays important roles in the differentiation of mesenchymal cells into fibroblasts and osteoblasts, angiogenesis, and formation of the extracellular matrix [19,20]. VEGF is the primary mediator of angiogenesis [21] and has various biological functions, such as increasing vascular permeability [22]. It has also been shown to be involved in bone remodeling [23,24]. Therefore, bFGF and VEGF may play important roles in maintaining homeostasis in periodontal tissue. Thus, we focused on bFGF and VEGF when we evaluated the condition of the PDL in hypofunctional teeth.
The aim of the present study was to elucidate the relationship between LLL and periodontal tissue in hypofunctional teeth, and clarify the participation of bFGF and VEGF.

Ethics Statement
All experiments were approved by the Animal Experimentation Committee at Hiroshima University, and conformed to the ARRIVE guidelines for animal research [25] and Rules for Animal Experiments of Hiroshima University. Animals Twenty-four 5-week-old male Wistar rats (Charles River Labs, Yokohama, Japan) were used. Rats were randomly divided into hypofunctional and control groups. In the hypofunctional group, an appliance consisting of a metal cap made of band material (3M Unitek Co., Tokyo, Japan) and an anterior bite plate made of a new ST lock base (Dentsply-Sankin, Tokyo, Japan) were bonded with composite resin (Clearfil Majesty LV; Kuraray Co., Ltd., Kurashiki, Japan) onto the maxillary and mandibular incisors, respectively [4] (Fig. 1A). The appliance was used for 2 weeks in the hypofunctional group. LLL irradiation was applied to the maxillary first molar through the gingival sulcus in half of the rats (Lumix2 TM HEPL, Fisioline s.r.l.; Verduno, Cuneo, Italy) (48.6 J; frequency: 30 kHz). Rats were then divided into four groups; control, control+LLL, hypofunctional, and hypofunctional+LLL. Exposure was performed for 3 minutes every other day for 2 weeks. Animals were subjected to Micro-CT (SkyScan1176; SkyScan, Kontich, Belgium) at 5 and 7 weeks, and were then scarified. Rats in the hypofunctional group were subjected to soft X-ray radiography to confirm occlusal conditions at age 5 and 7 weeks (Fig. 1B). Next the heads were then resected and examined histologically and immunohistologically. The experimental itinerary is summarized in Figure 1C.
All animals were fed a powder diet (Rodent Diet CE-2; Japan CLEA Inc., Tokyo, Japan), and water ad libitum under a 12-hour light/dark environment at a constant temperature of 23uC. Rats were weighed once a week during the experimental period.

Laser Device and Irradiation
We used a low-level diode laser (Lumix2 TM HEPL, Fisioline s.r.l.) that emits pulse waves at a wavelength of 904-910 nm with a peak power of 45 W, maximum pulse repetition rate of 30 kHz, and pulse duration of 200 ns.
LLL was applied with a probe (diameter, 8.0 mm) possessing a free program mode around the maxillary first molar through the gingival sulcus in the control+LLL and hypofunctional+LLL groups (48.6 J; frequency: 30 kHz). Exposure was performed for 3 minutes every other day for 2 weeks.

Morphological Analysis of PDL Thickness and BMD of the Maxillary Alveolar Bone by Micro-CT
The thickness of the PDL at the distal palatal root of the maxillary first molars was measured using of Micro-CT at 5 and 7 weeks of age. Image reconstruction on an appropriate crosssection was performed using software (Nrecon; SkyScan), and the thickness of the PDL on the buccal side of the distal palatal root of the maxillary first molars was measured (Data Viewer; SkyScan) ( Fig. 2A). The BMD of the maxillary alveolar bone on the palatal side was analyzed (500 mm 3 ) (CT-An; SkyScan), and measurement items are summarized in Figure 2B. The same researcher performed all measurements. Measurements were repeated 3 times, and mean values were used.

Tissue Preparation
Animals were deeply anesthetized in diethyl ether, followed by an intraperitoneal injection of chloral hydrate (400 mg/kg), and were then were subjected to soft X-ray radiography to confirm the occlusal condition prior to being sacrificed by means of transcardiac perfusion with 4% paraformaldehyde. Maxillae were immediately immersed in the same fixative solution overnight at 4uC. Tissue blocks were subsequently decalcified in 14% ethlylene diamine tetraacetic acid (EDTA) at 4uC for 4-6 weeks and prepared for the paraffin-embedded method. Serial sections of 5.0 mm in thickness were prepared along the frontal fault, perpendicular to the long axis of the distal root of the maxillary first molar. Sections were prepared for hematoxylin-eosin (H-E) and immunohistochemical staining.

Immunohistochemical Staining
After deparaffinization, sections from 7-week-old rats that included the root canal were treated with 3% hydrogen peroxide in absolute methanol to block endogenous peroxidase. Sections were immunostained with a 1:50 dilution of primary anti-rat bFGF rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc., CA), followed by the anti-rabbit secondary IgG antibody (Hystofine simple stain rat MAX-PO(R); Nichirei, Tokyo, Japan), and were immunostained with a 1:150 dilution of primary anti-rat VEGF chicken polyclonal antibodies (Abcam, Inc., CA), followed by an anti-chicken secondary IgG antibody (Abcam). Immunoreactive sites were finally visualized with 3, 39-diaminobenzidine (DAB). Counterstaining was performed using hematoxylin. Sections incubated without the primary antibody were used as a negative control.

Number of bFGF-and VEGF-immunopositive PDL Cells
The middle one-third buccal aspect of the PDL on the distal palatal root was photographed using an optical microscope (Biozero; Keyence). Quantitative images were measured using image analysis software (BZ analyzer; Keyence). The number of bFGF-and VEGF-positive PDL cells was counted in a rectangular area (3006400 mm) (Fig. 2B). Three representative sections from each of the five samples of all groups were measured in a blinded manner.

Statistical Analysis
PDL thickness, BMD of the maxillary alveolar bone, and number of bFGF-and VEGF-immunopositive PDL cells were measured in each group, and characteristic values in the experimental groups were compared with those in the control groups. To determine the significance of differences among groups of rats, we performed a repeated one-way analysis of variance (ANOVA) and the Tukey-Kramer test using a Statview Confidence level of p,0.05.

Body Weight
All animals exhibited normal growth, and no significant difference was observed among the experimental groups (data not shown).

Occlusal Condition
In order to confirm occlusal contact in the molar region, soft Xray images were taken at 5 and 7 weeks of age before sacrifice in each group (Fig. 1B). Occlusal hypofunction was confirmed in the molars of all rats in the hypofunctional and hypofunctional+LLL groups.

Morphometric Findings from Micro-CT Analyses
No significant difference was observed in PDL thickness between the four groups at 5 weeks of age (data are not shown). However, the PDL in the experimental groups exhibited morphological changes at 7 weeks of age (Fig. 3A). No significant difference was observed in PDL thickness between the control, hypofunctional+LLL, and control+LLL groups at 7 weeks of age. In contrast, PDL thickness was significantly smaller in the hypofunctional group than in the other groups (Fig. 3B).

BMD in the Maxillary Alveolar Bone
No significant differences were observed in BMD in the maxillary alveolar bone between the four groups at 5 weeks of age (data not shown). BMD in the maxillary alveolar bone for the four groups is shown in Figure 3C. BMD was significantly lower in the hypofunctional group than in the controls. On the other hand, BMD was higher in the hypofunctional+LLL group than in the hypofunctional group, and No significant difference was observed between the control and hypofunctional+LLL groups. Figure 4A shows H-E-stained distal palatal root sections from the four groups. In the hypofunctional group, changes in stricture on the PDL were observed, and PDL thickness was thinner than in the other groups. In addition, PDL thickness in the hypofunc-tional+LLL group was similar to that in the control and control+ LLL groups.

Immunohistochemical Findings of bFGF and VEGF Expression
The number of bFGF and VEGF immunopositive cells, particularly fibroblastic cells, cementum, and alveolar bone lining Effects of a Low Level Laser on Periodontal Tissue in Hypofunction PLOS ONE | www.plosone.org cells was significantly lower in the hypofunctional group than in the other groups (P,0.01; Fig. 4B, 4C).
However, the number of bFGF and VEGF immunopositive cells was significantly higher in the hypofunctional+LLL group than in the hypofunctional group, and was similar to that in the control group (P,0.01; Fig. 4B, 4C).

Discussion
The present study was designed to investigate the influence of LLL on hypofunctional teeth. We developed an experimental hypofunctional model in the molar region using a bite-raising appliance [4]. This method made it possible to simulate hypofunctional conditions in the molar region.
In the present study, occlusal hypofunction using a bite-raising appliance resulted in changes in the periodontal tissue; PDL thickness and BMD in the maxillary alveolar bone were thinner and lower, respectively, than these in the controls. Numerous studies have reported structural changes in the PDL [1,2] and loss of BMD in alveolar bone [5] with occlusal hypofunction. However, PDL thickness and BMD in the maxillary alveolar bone were thicker and higher, respectively, in the hypofunctional+ LLL group than in the hypofunctional group, and no significant difference was observed from the control group. Occlusal stimuli have been shown to affect periodontal tissue; root size and the structure of the PDL may be reduced because of disuse atrophy resulting from defects in occlusal function, and atrophy may recover after occlusal stimuli are regained [4]. The effects of occlusal stimuli on alveolar bone formation have also been reported previously [5]. On the other hand, LLL may increase BMD [11]. Due to its wavelength characteristics, low-level diode lasers are able to reach periodontal tissues, and laser irradiation is expected to induce the proliferation of osteoblasts [14] and periodontal ligament fibroblasts [15]. Therefore, LLL affects periodontal tissue, leading to extensive changes in the PDL and increases in BMD in alveolar bone with occlusal hypofunction.
The number of bFGF-and VEGF-immunopositive cells was significantly lower in the hypofunctional group, particularly for fibroblastic cells, cementum, and alveolar bone lining cells, than in the other groups. However, when LLL was applied, the number of bFGF-and VEGF-immunopositive cells was significantly higher than that in the hypofunctional group, and levels were similar to those in the control group. Recent in vitro studies indicated that LLL irradiation was able to stimulate the release of growth factors, such as VEGF [17] and bFGF [18]. LLL has also been shown to have a positive effect on bone formation, and the vasculature [6,7]. On the other hand, occlusal stimuli regulate bFGF in the rat PDL [16]. PDL homeostasis is a complex mechanism involving inflammation, neovascularization, neurogenesis, bone formation and matrix remodeling. bFGF is a potent angiogenic factor, and angiogenesis may be involved in the periodontal regeneration promoted by bFGF. VEGF also increases vascular permeability [22] and is involved in bone remodeling [23,24]. Constitutive VEGF expression may contribute to periodontal tissue homeostasis by regulating local blood circulation and bone metabolism.
Thus, bFGF and VEGF are essential for periodontal remodeling and vascular permeability in PDL. Therefore, the expression of bFGF and VEGF after LLL stimulation may in increase vascular permeability in the PDL of hypofunctional teeth with a reduction in blood vessels.
Malocclusions, such as an open bite, are often encountered in orthodontic practice and that include hypofunctional teeth in orthodontic practice. Root resorption was shown to be more prominent in hypofunctional teeth than in normal teeth during orthodontic tooth movement [3,26]. Therefore, it is important for hypofunctional teeth with atrophied periodontal tissues to recover their physiological structure and function. The results of the present study suggests that the recovery of periodontal tissue in hypofunctional teeth is possible with LLL prior to orthodontic tooth movement, and this may reduce or prevent root resorption.
In conclusion, occlusal hypofunction during the growth period may weaken periodontal tissue, leading to PDL stricture and decreased BMD in the alveolar bone crest; however, LLL irradiation to hypofunctional teeth led to a periodontal condition similar to that in normal teeth. bFGF-and VEGF-positive fibroblasts and odontoclasts were also observed in the PDL in the hypofunctional+LLL group. Thus, periodontal tissues, in terms of bFGF and VEGF, were enhanced by LLL, which stimulated the structure and function of periodontal tissues.