Inhibition of 12/15-lipoxygenase reduces orthodontically induced root resorption in rats
ABSTRACT
Objectives
To investigate whether the inhibition of 12/15-lipoxygenase (12/15-LOX), one of the core enzymes of the arachidonic acid cascade, suppresses orthodontically induced root resorption (OIRR), and examine the involvement of the hyaline degeneration of periodontal ligament cells and odontoclast differentiation.
Materials and Methods
The left maxillary first molars of 10-week-old male Wistar rats were moved mesially for 14 days using a closed-coil spring (25 cN) inserted between the first molar and incisor. The rats were intraperitoneally administered with a 12/15-LOX specific inhibitor (ML-351; 0.05 mmol/kg) daily in the experimental group or vehicle (dimethyl sulfoxide) in the control group. Tooth movement was measured using microcomputed tomography on day 14. The appearance of OIRR, hyaline degeneration, osteoclasts, and odontoclasts was evaluated via histological analysis. Immunohistochemical staining for receptor-activated NF-kB ligand (RANKL) and osteoprotegerin was performed.
Results
OIRR observed on day 14 in the control group was strongly suppressed by ML-351 treatment. Hyaline degeneration observed on the compression side on day 3 and the appearance of osteoclasts and odontoclasts on days 3 and 14 were significantly suppressed by ML-351. RANKL expression on day 3 was significantly suppressed by ML-351. These key processes in OIRR were substantially suppressed by ML-351 treatment.
Conclusions
Inhibition of 12/15-LOX reduced OIRR by suppressing hyaline degeneration and subsequent odontoclast differentiation.
INTRODUCTION
Orthodontic tooth movement (OTM) is caused by bone remodeling when orthodontic forces are applied. It is often associated with orthodontically induced root resorption (OIRR). Histological studies have shown that OIRR, including mild OIRR, occurs in more than 90% of orthodontic patients.1 It is one of the most troublesome problems in orthodontic treatment because it is difficult to predict and there is no preventive method.2
Although various clinical and experimental studies have investigated the processes and mechanisms of OIRR,1–3 its biological mechanisms remain unclear. Macrophages, osteoclasts, and odontoclasts have been widely reported to be involved in OIRR. The site of OIRR has also been reported to correspond to the area of hyaline degeneration of periodontal ligament (PDL) cells compressed by excessive orthodontic force.4,5 OIRR is considered to be initiated by the hyaline degeneration of PDL cells owing to the compressive force of orthodontic treatment. This is followed by osteoclast and odontoclast differentiation and alterations in alveolar bone metabolism, ultimately leading to OIRR.6
12/15-lipoxygenase (12/15-LOX) is one of the core enzymes of the arachidonic acid (AA) cascade, which produces many bioactive lipid metabolites.7 The metabolites of 12/15-LOX are pro-inflammatory and contribute to various human diseases, including severe asthma, aspirin-induced asthma attacks, and other allergic reactions.8 Previous studies have shown that patients with allergic diseases have increased levels of 12/15-LOX9 and a higher rate of OIRR.10
12/15-LOX has been implicated in cell death in models of oxidative stress, and its inhibition has been shown to suppress cell death.11,12 When cells are exposed to oxidative stress, 12/15-LOX binds to and activates the membrane, disrupting the mitochondrial membrane potential.13 Cell death occurs via the translocation of the apoptosis-inducing factor from the mitochondria to the nucleus. In a previous report, the 12/15-LOX specific inhibitor, ML-351, suppressed cerebral infarction in a mouse model of ischemic stroke.14 In addition, 12/15-LOX is involved in the mechanism of natural degradation through organelle degradation, that is, 12/15-LOX can directly bind to and modify organelle membranes, creating pores that release soluble proteins from organelles.15 This was specifically observed in eye lenses. Based on these findings, it has been suggested that 12/15-LOX may be involved in hyaline degeneration.
Furthermore, 12/15-LOX is identified as a susceptibility gene of bone mineral density (BMD) in mice through combined genetic and genomic analyses.16 In addition, the 12/15-LOX gene has been suggested to be one of the genetic determinants of BMD in postmenopausal women.17 Gerhard et al.18 reported that osteoclastogenesis was significantly impaired in 12/15-LOX knockout mice. Conversely, the addition of 12/15-LOX-derived eicosanoids, 12- and 15-hydroxyeicosatetraenoic acid, enhanced the differentiation of precursors into fully mature osteoclasts.
These findings indicate that 12/15-LOX is a key regulator of hyaline degeneration and odontoclast differentiation. Therefore, it was hypothesized that 12/15-LOX induces root resorption and promotes OIRR. This study aimed to investigate whether 12/15-LOX is involved in OIRR by examining the effects of ML-351, a specific inhibitor of 12/15-LOX, on the development of OIRR from the following two aspects: (1) suppression of hyaline degeneration and (2) suppression of odontoclast differentiation in rats.
MATERIALS AND METHODS
This research was approved by the Animal Care and Use Committee of Nagasaki University Graduate School of Biomedical Sciences (No. 2010011668-4).
OIRR Rat Model
A total of 28 male Wistar rats aged 10 weeks (SLC, Shizuoka, Japan, weight, 204.7-231.8 g) were used in the study. The rats were housed in plastic cages in a colony room, fed a standard pellet diet and water ad libitum, and acclimatized for at least 72 hours before the experiments. A 25 cN nickel-titanium closed-coil spring (Sentalloy, Tomy Inc., Fukushima, Japan) was placed between the maxillary first molars and incisors to induce mesial movement of the maxillary first molars (Figure 1A). All surgeries were performed under general anesthesia with an intraperitoneal injection of 0.375 mg/kg medetomidine (Zenoaq, Fukushima, Japan), 2 mg/kg midazolam (Sandoz, Tokyo, Japan), and 2.5 mg/kg butorphanol tartrate (Meiji Seika Pharma Co. Ltd., Tokyo, Japan). After setting up the appliance, the rats were randomly divided into two groups: a 12/15-LOX inhibitor group (LOX) (n = 14) and a control group (CNT) (n = 14). ML-351 (Selleck Biotech Co. Ltd., Tokyo, Japan) dissolved in dimethyl sulfoxide (DMSO) (0.05 mmol/kg/d) was administered intraperitoneally in the LOX group daily for 14 days. An equal volume of DMSO was intraperitoneally administered to the CNT group (Figure 1B).
Citation: The Angle Orthodontist 94, 5; 10.2319/103123-730.1
Microcomputed Tomography Images and Measurements of OTM
Microcomputed tomography (micro-CT; R_mCT, Rigaku, Tokyo, Japan) images were obtained under general anesthesia on days 0 (before the orthodontic appliance was applied) and 14 (Figure 1B) (n = 8 in each group). Image acquisition conditions were as follows: voltage, 90 kV; current, 100 µA; exposure time, 2 minutes; and resolution, 20 µm/pixel. Three parameters were defined to measure OTM: (1) shortest distance (ShD), the shortest distance between the left maxillary first molar and second molar (Figure 1C); (2) distance between contact points (CPD), the distance between the contact points of the left maxillary first and the second molar (Figure 1D); and (3) angle of tooth inclination (TIA), the change in the mesial root inclination of the first molar before and after tooth movement in the sagittal images (Figure 1E).
Preparation for Histological Analysis
Rats in the LOX and CNT groups were euthanized on days 3 (n = 6) and 14 (n = 8), respectively. After euthanasia under deep anesthesia, the maxilla was dissected and immersed in a fixative solution of 4% paraformaldehyde in 50 mM sodium cacodylate buffer (pH: 7.4) for 48 hours. Maxillary bone tissue was decalcified with 17% ethylenediaminetetraacetic acid (pH: 7.4; Osteosoft, Merck Millipore, Darmstadt, Germany) for 4 weeks at room temperature, dehydrated, and embedded in paraffin.
Hematoxylin-Eosin Staining and Tartrate-Resistant Acid Phosphatase Staining
Continuous sections (thickness: 6 µm) were prepared and stained with hematoxylin and eosin to observe the cross-sectional structure (Figure1F–H). The length of hyaline degeneration and OIRR were measured using ImageJ (National Institutes of Health, Bethesda, MD, USA). Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify and count osteoclasts and odontoclasts. Naphthol AS-MX phosphate (Sigma, St. Louis, MO), N, N-dimethylformamide (Fujifilm Wako assay, TOKYO, Japan), 0.2 M acetate buffer, and distilled water with sodium tartrate dihydrate were stirred and filtered and used for staining for 1 hour. Sections were counterstained with hematoxylin for 1 minute. The number of TRAP-positive cells was counted in the mesial periodontium at one-third of the cervical region of the distobuccal root of the first molar of each specimen.
Immunohistochemical Staining
Tissue sections were deparaffinized in xylene, dehydrated in alcohol, and incubated in 10% citric acid buffer for 30 minutes at 90°C to reconstitute protein antigenicity. After rinsing with phosphate buffered saline, endogenous peroxidase activity was inactivated via treatment with 0.3% H2O2/methanol for 30 minutes at room temperature. The tissue sections were washed and blocked with 10% goat serum albumin. They were incubated overnight at 4°C with primary antibodies: rabbit polyclonal anti-receptor-activated NF-kB ligand (RANKL) antibody (1:100; ab4539 Abcam, Cambridge, England) or rabbit polyclonal anti-osteoprotegerin (OPG) antibody (1:100; ab73400 Abcam). Afterward, they were stained using Histofine SAB-PO(R) kit (Nichirei, Tokyo, Japan) according to the manufacturer’s protocol. Visualization of peroxidase activity was performed with 3, 3’-diaminobenzidine (Nichirei), followed by rinsing and counterstaining with hematoxylin for 1 minute.
Statistical Analysis
EZR (Easy R; Jichi Medical University, Saitama Medical Center, Saitama, Japan) was used for statistical analysis. A t-test was used to test for differences between the two groups. All data are expressed as mean ± standard error. Significance was set at P < .05. All measurements were performed in triplicate by the same investigator.
RESULTS
Tooth Movement
Micro-CT scans were used to evaluate tooth movement 14 days after the application of orthodontic force (Figure 2). ShD, CPD, and TIA showed no significant difference between the LOX and the CNT groups (ShD: 239 ± 80 µm vs 170 ± 87 µm, CPD: 298 ± 73 µm vs 215 ± 106 µm, TIA; 5.36 ± 4.22° vs 2.28 ± 1.89°). The results indicated that tooth movement was observed in both groups and that ML-351 administration had no effect on OTM.
Citation: The Angle Orthodontist 94, 5; 10.2319/103123-730.1
Histological Evaluation
OIRR was observed on the compression side on day 14 (Figure 3A,B). The area of OIRR was significantly smaller in the LOX group than in the CNT group (7.97 ± 3.81 × 103 µm2 vs 3.71 ± 1.43 × 103 µm2, P < .01; Figure 3C). Hyaline degeneration was observed on the compressed side on day 3 (Figure 4A,B). The length of hyaline degeneration was significantly shorter in the LOX group than in the CNT group (438 ± 40 µm vs 340 ± 90 µm, P < .05; Figure 4C). These results demonstrated that treatment with ML-351 reduced hyaline degeneration and OIRR.
Citation: The Angle Orthodontist 94, 5; 10.2319/103123-730.1
Citation: The Angle Orthodontist 94, 5; 10.2319/103123-730.1
TPAP-positive cells with three or more nuclei located on the compressive surface of the alveolar bone were defined as osteoclasts, and those located on the compressive side of the root surface were defined as odontoclasts. Representative photographs on days 3 and 14 are shown in Figure 5A. Osteoclasts were observed on days 3 and 14 in the CNT group, whereas ML-351 administration significantly reduced the appearance of osteoclasts throughout the experimental period (P < .01, on days 3 and 14) (Figure 5B). The odontoclasts appeared to be small in number on day 3 in the CNT group and increased substantially on day 14. The appearance of odontoclasts was also significantly suppressed in the LOX group (P < .01, day 3; P < .05, day 14) (Figure 5C). These results indicated that the appearance of osteoclasts precedes the appearance of odontoclasts and that ML-351 suppresses the appearance of osteoclasts and odontoclasts.
Citation: The Angle Orthodontist 94, 5; 10.2319/103123-730.1
Immunohistochemical Staining
Immunohistochemical staining was performed to observe the expression of RANKL (Figure 6A) and OPG (Figure 6C) on the compressed side on days 3 and 14. Both RANKL and OPG were observed in the PDL tissues of the CNT and LOX groups. The kinetics of RANKL expression throughout the experimental period were similar to those observed in osteoclasts. OPG expression was barely observed on day 3 but increased on day 14. The mean optical density (MOD) of RANKL in the LOX group on day 3 was significantly reduced via ML-351 treatment (day 3, P < .01) (Figure 6B). However, no significant differences were observed in the MOD of OPG between the CNT and LOX groups (Figure 6D).
Citation: The Angle Orthodontist 94, 5; 10.2319/103123-730.1
DISCUSSION
OIRR is assumed to occur because of hyaline degeneration and the subsequent appearance of odontoclasts. Since 12/15-LOX has been reported to be involved in natural degeneration and osteoclast differentiation, it was hypothesized that the inhibition of 12/15-LOX might suppress OIRR. The administration of the 12/15-LOX-specific inhibitor, ML-351, suppressed hyaline degeneration and odontoclast differentiation, and subsequently suppressed OIRR. These results suggest that the inhibition of 12/15-LOX suppresses two aspects of the OIRR process: orthodontic force-induced hyaline degeneration of PDL cells and subsequent odontoclast differentiation. Subsequently, alterations in the alveolar bone metabolism are restrained, ultimately leading to OIRR inhibition.
Previous studies on cyclooxygenase (COX), another important enzyme in the AA cascade, in OIRR in rats have shown conflicting results. One COX inhibitor, celecoxib, suppressed OIRR, whereas the other COX inhibitors, aspirin and meloxicam, had no effect on OIRR.19 By contrast, the effect of 12/15-LOX on OIRR has not been conducted. This is the first report that 12/15-LOX inhibition suppresses OIRR and that hyaline degeneration and odontoclast differentiation are involved in the mechanism.
Because RANKL is essential for osteoclast differentiation and function, it may also play an important role in osteoclast and odontoclast activation during OTM. RANKL expression was significantly lower in the LOX group than in the CNT group on day 3. Therefore, it is suggested that reduced RANKL expression in the early phase of orthodontic force application plays an important role in the reduced OIRR in the LOX group by decreasing odontoclast differentiation. OPG also plays a role in osteoclast differentiation by binding to RANKL.20 The transfer of the OPG gene into periodontal tissue inhibits RANKL-mediated osteoclastogenesis and experimental tooth movement.21 In the present study, reduced RANKL expression was observed during histological analysis, while no significant difference was noted in OPG expression. This suggests that the suppression of odontoclasts and osteoclasts via 12/15-LOX inhibition is linked to the altered expression of RANKL and not OPG. However, there have been no previous studies on the relationship between 12/15-LOX, RANKL, and OPG expression. Further in vivo and in vitro studies are required to explore this relationship.
In the present study, OTM was not significantly different between the CNT and LOX groups; however, OIRR was significantly suppressed in the LOX groups. These results suggest that ML-351, a specific 12/15-LOX inhibitor, can suppress OIRR without affecting tooth movement in rats. In clinical practice, orthodontic treatment does not always result in noticeable root resorption, but histological studies have shown that OIRR, including mild cases, is a common occurrence in over 90% of orthodontic patients.1 Several studies have reported that the extent of root resorption depends on the applied force system.1 In this study, an orthodontic force was set at a higher level to reliably and reproducibly induce root resorption. However, even under conditions of excessive orthodontic force application, inhibition of 12/15-LOX reduced OIRR without evident adverse effects. Thus, ML-351 could be used as a prophylactic agent for safe orthodontic treatment of patients especially at risk of OIRR. However, these findings are not directly applicable to practicing orthodontists at this stage as the administration of 12/15-LOX inhibitors is still in the experimental stage. Further studies are needed to determine the biological safety, optimal dosage, and timing of ML-351 administration.
CONCLUSIONS
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Inhibition of 12/15-LOX by ML-351 reduced OIRR by suppressing hyaline degeneration of PDL cells, subsequent odontoclast differentiation, and RANKL expression in rats.
Experimental protocol. (A) Sagittal and axial views of the appliance. The arrows indicate the direction of the orthodontic force. The incisors were fixed with composite resin to prevent detachment from the tooth. (B) Experimental time schedule. (C–E) Method to measure tooth movement: (C) shortest distance, (D) distance between contact points, and (E) angle of tooth inclination. (F) Sagittal microcomputed tomography (CT) image of the left maxillary molars. The dotted line indicates the position of the sliced tissue section, which is located at the cervical third of the distobuccal root of the maxillary first molar (M1). (G) An axial micro-CT image of the left maxillary molars at the level of the dotted line in Figure 1F. (H) Hematoxylin and eosin staining corresponding to the image shown in Figure 1G. The white dotted box indicates the measurement area of the distobuccal root of M1. Appl indicates orthodontic appliance.
Measurements of tooth movement.
Hematoxylin and eosin staining in the control (A) and 12/15-LOX inhibitor (B) groups on day 14 (magnification: 20 × 20). The area marked with a white line indicates root resorption (R). (C) The measured area of root resorption. AB indicates alveolar bone; D, dentin; P, pulp; PDL, periodontal ligament; arrows, direction of orthodontic force; scale bars = 100 µm. **P < .01.
Hematoxylin and eosin staining in the control (A) and 12/15-LOX inhibitor (B) groups on day 3 (magnification: 20 × 20). The white dotted line with arrows at both ends indicates the length of hyalinization degeneration (H). (C) The measured length of hyalinization degeneration in the PDL. AB indicates alveolar bone; D, dentin; P, pulp; PDL, periodontal ligament; arrows, direction of orthodontic force; scale bars = 100 µm. *P < .05.
(A) Tartrate-resistant acid phosphatase (TRAP) staining in the control (CNT) and 12/15-LOX inhibitor (LOX) groups on days 3 and 14 (magnification: 20 × 20). Osteoclasts and odontoclasts are stained in red. Black arrowheads indicate osteoclasts and white arrowheads indicate odontoclasts. (B) The number of TRAP-positive osteoclasts in the CNT and LOX groups on days 3 and 14. (C) The number of TRAP-positive odontoclasts in the CNT and LOX groups on days 3 and 14. AB indicates alveolar bone; D, dentin; P, pulp; PDL, periodontal ligament; arrows, direction of orthodontic force; scale bars = 100 µm. *P < .05, **P < .01.
(A) Immunostaining of receptor-activated NF-kB ligand (RANKL) in the control (CNT) and 12/15-LOX inhibitor (LOX) groups on days 3 and 14 (magnification 20 × 20). (B) The mean optical density (MOD) values of RANKL in the CNT and LOX groups on days 3 and 14. (C) Immunostaining of osteoprotegerin (OPG) in the CNT and LOX groups on days 3 and 14 (magnification 20 × 20). (D) The MOD values of OPG in the CNT and LOX groups on days 3 and 14. AB indicates alveolar bone; D, dentin; P, pulp; PDL, periodontal ligament; arrows, direction of orthodontic force; scale bars = 100 µm. **P < .01.
Contributor Notes