Osteoprotegerin-deficient mice develop severe high-turnover osteoporosis with porous low-density trabecular bone from an age-related increase in osteoclast activity and are useful alveolar bone models of osteoporosis or frail periodontal tissue. Bisphosphonate (BP), a first-line drug for osteoporosis, is bone-avid, causing side effects such as brittle and fragile bones and jaw osteonecrosis after tooth extraction. In orthodontics, active movement is precisely controlled by temporarily suppressing and resuming movement. BP impedes such control because of its long half-life of several years in bone. Therefore, we investigated the novel osteoclast-specific inhibitor reveromycin A (RMA), which has a short half-life in bone. We hypothesized that tooth movement could be precisely controlled through temporary discontinuation and re-administration of RMA.
Osteoprotegerin-deficient mice and wild-type mice were developed as tooth movement models under constant orthodontic force. A constant orthodontic force of 10 g was induced using a nickel-titanium closed coil spring to move the maxillary first molar for 14 days. We administered BP (1.25 mg/kg) or RMA (1.0 mg/kg) continuously and then discontinued it to reveal how the subsequent movement of teeth and surrounding alveolar bone was affected.
Continuous BP or RMA administration suppressed osteoclast activity and preserved alveolar bone around the roots, apparently normalizing bone metabolism. Tooth movement remained suppressed after BP discontinuation but resumed at a higher rate after discontinuation of RMA.
RMA appears useful for controlling orthodontic tooth movement because it can be suppressed and resumed through administration and discontinuation, respectively.
Osteoprotegerin-deficient mice are alveolar bone models of osteoporosis or frail periodontal tissue.
Mice were developed as tooth movement models under constant orthodontic force.
Continuous reveromycin A (RMA) administration suppressed osteoclast activity.
RMA has a short half-life in bone.
RMA appears useful for controlling orthodontic tooth movement.
In bone tissue, bone resorption by osteoclasts and bone formation by osteoblasts occurs continually without interruption. Similarly, in orthodontic tooth movement, bone is resorbed by osteoclasts at the site where compression is applied while bone is formed by osteoblasts at the tension site, so the alveolar bone is continuously remodeled. Osteoclasts are the only cells capable of destroying and resorbing calcified bone tissue, and their differentiation, maturation, and function are strictly regulated by the receptor activator of NF-κB ligand (RANKL) present on the membrane of osteoblasts or bone-marrow stromal cells. Upon the recognition of RANKL, osteoclasts and osteoclast progenitors differentiate into mature osteoclasts. Osteoprotegerin (OPG) synthesized by osteoblasts is a member of the tumor necrosis factor receptor superfamily and functions as a decoy receptor for RANKL. OPG inhibits the differentiation and function of osteoclasts by strongly suppressing the interaction between RANKL and RANK. Overexpression of OPG in mice causes severe osteopetrosis through the suppression of bone resorption. In contrast, bone resorption in osteoprotegerin-deficient (OPG KO) mice is increased because of the upregulated production of osteoclasts. In OPG KO mice, despite having normal bone tissue at birth, bone loss becomes apparent in trabecular bone at 1 week of age, and in cortical bone, mainly consisting of cancellous bone, at 4 weeks of age. Because of enhanced osteoclastic activity during development, mature OPG KO mice have severe high-turnover osteoporosis with porous and low-density trabecular bone compared with wild-type (WT) mice. , Therefore, OPG KO mice are an instrumental animal model to simulate the condition of alveolar bone in patients with osteoporosis or fragile periodontal tissue.
Increasingly often in recent years, young individuals who are still developing, as well as older adults and elderly individuals, now undergo orthodontic procedures for prosthetic pretreatment or prevention of periodontal disease. However, many adult patients who request orthodontic treatment have fragile periodontal tissue or show signs of systemic osteoporosis.
Osteoporosis is classified into low-turnover osteoporosis, caused by aging-related downregulation of bone formation, and high-turnover osteoporosis, caused by upregulation of bone resorption due, for example, to menopause. , Because bisphosphonate (BP) is the drug of choice for osteoporosis, the number of orthodontic patients on BP is expected to increase. However, BP remains in bone for many years after administration and is associated with side effects such as dense and fragile bones and jaw osteonecrosis after tooth extraction.
In orthodontic treatment, during active movement, precise control is also necessary by temporarily suppressing and then resuming the movement. BP is a hydrolysis-resistant PP1 derivative that has a high affinity for bone and inhibits osteoclastic bone resorption. BP is used clinically for the treatment of osteoporosis. However, because of the long half-life of BP, it remains in bone for several years after administration, which would likely interrupt the precision of orthodontic tooth movement. We, therefore, focused on the osteoclast-specific inhibitor reveromycin A (RMA), an acid polyketide recently isolated from actinomycetes. RMA is not readily taken up by most cells, but active osteoclasts, which dissolve bone by secreting acid, can take up RMA in acidic environments. RMA has a high potential to inhibit bone resorption by inducing osteoclast apoptosis via the suppression of isoleucyl-tRNA synthetase in osteoclasts. In addition, RMA has an extremely short half-life and is taken up readily by osteoclasts that are actively resorbing bone but not by osteoclast progenitors or osteoclasts not actively resorbing it (inactive osteoclasts). After uptake, RMA induces apoptosis in the osteoclasts, thereby inhibiting bone resorption. By taking advantage of these properties, we hypothesize that orthodontic tooth movement can be precisely controlled through repeated cycles of administration, discontinuation, and resumption of RMA.
In this study, using OPG KO mice with high-turnover osteoporosis, we developed an animal model of experimental tooth movement under constant orthodontic force to investigate the movement of teeth and the surrounding alveolar bone. To verify our hypothesis that tooth movement is suppressed by BP even after discontinuation because of its long half-life and bone avidity but is resumed after discontinuation of RMA because of its short half-life, we administered BP or RMA continuously and then discontinued their administration to reveal their effects on tooth movement and the surrounding alveolar bone.
Material and methods
Eight-week-old male OPG KO mice (n = 24, experimental group) and WT C57BL/6J mice (n = 24, control group) were used in this study. All mice were purchased from CLEA Japan (Tokyo, Japan) and were reared in the Animal Facility of Aichi Gakuin University School of Dentistry under the same environmental conditions (room temperature, 22 ± 2°C; humidity, 50 ± 10%; and 12-h light/dark cycle). Animals were fed CE-2 solid diet (CE-2; CLEA Japan, Tokyo, Japan), and tap water was provided ad libitum. This study was approved by the Institutional Animal Care and Use Committee of our institution and was conducted following the Institutional Animal Care and Use Committee policies and procedures. This report complies with the Animals in Research: Reporting In Vivo Experiments guidelines.
To move teeth experimentally, general anesthesia was induced using inhalational diethyl ether in 8-week-old OPG KO or WT mice. A nickel-titanium closed coil spring with a force of 10 g was placed on the maxillary incisors and left first molar (M1) to move the molar mesially for 14 days in experimental animals (loaded side). The right M1 was used as control (unloaded side; Figs 1 , A and B ). Experimental reagents were RMA 3Na salt and alendronate sodium hydrate (Teiroc; Teijin Pharma, Tokyo, Japan).
Mice were divided into 3 groups (n = 4 each) for the continuous administration of physiological saline (saline) for 14 days (SA group), RMA (1.0 mg/kg body weight) for the first 7 days followed by saline for the remaining 7 days (RMA+/− group), or RMA for the entire 14 days (RMA+ group). Saline or RMA was administered intraperitoneally twice daily starting 3 days before the placement of the 10 g of force with nickel-titanium closed coil spring. BP (1.25 mg/kg body weight) was administered intraperitoneally to 3 groups of animals (SA, BP+/−, and BP+), but the administration was once daily starting 5 days before placement of the 10 g of force with nickel-titanium closed coil spring ( Fig 1 , C ). Regarding the dose of RMA and BP, this experiment was conducted with reference to studies by Tanaka et al and Shoji et al, respectively. These studies demonstrated that RMA and BP are both effective in reducing tooth movement.
Microcomputed tomography (CT) was performed using a 3-dimensional micro x-ray CT device developed for use in small laboratory animals (R_mCT; Rigaku, Tokyo, Japan), with tube voltage and current set at 90 kV and 88 μA, respectively, run time at 2 min, and pixel size at 20 × 20 × 20 μm. Under inhalational anesthesia, animals underwent imaging 7 days after initiation of experimental tooth movement, and the maxillary bone was excised and scanned after 14 days of experimental tooth movement ( Figs 1 , C and 2 , A ). Movement distance ( Figs 2 , B and C ) was measured using TRI/3D-BON software (Ratoc System Engineering Co, Ltd, Tokyo, Japan) to determine how far the teeth had moved. The images were rotated and adjusted to ensure that the occlusal view with the narrowest gap between M1 and M2 was observed. To observe the state of the alveolar crest of the left M1 interradicular septum, we observed the roots of M1 and the surrounding alveolar bone in a sagittal view at 14 days after the appliance was inserted ( Fig 3 , A ).