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.

Keywords: 12/15-lipoxygenase; Hyaline degeneration; Odontoclasts; Orthodontic tooth movement; Osteoclasts; Root resorption.

Figure 1

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.

Figure 2

Measurements of tooth movement.

Figure 3

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.

Figure 4

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.

Figure 5

(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.

Figure 6

(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.