Recombinant human insulin-like growth factor-1 promotes osteoclast formation and accelerates orthodontic tooth movement in rats

Abstract Background: IGF-1 may be an important factor in bone remodeling, but its mechanism of action on osteoclasts during orthodontic tooth movement is complex and unclear. Methodology: The closed-coil spring was placed between the left maxillary first molar and upper incisors with a force of 50 g to establish an orthodontic movement model. Eighty SD rats were randomized to receive phosphate buffer saline or 400 ng rhIGF-1 in the lateral buccal mucosa of the left maxillary first molar every two days. Tissue sections were stained for tartrate-resistant acidic phosphatase (TRAP), the number of TRAP-positive cells was estimated and tooth movement measured. Results: The rhIGF-1 group exhibited evidential bone resorption and lacuna appeared on the alveolar bone compared to the control group. Moreover, the number of osteoclasts in compression side of the periodontal ligament in the rhIGF-1 group peaked at day 4 (11.37±0.95 compared to 5.28±0.47 in the control group) after the orthodontic force was applied and was significantly higher than that of the control group (p<0.01). Furthermore, the distance of tooth movement in the rhIGF-1 group was significantly larger than that of the control group from day 4 to day 14 (p<0.01), suggesting that rhIGF-1 accelerated orthodontic tooth movement. Conclusion: Our study has showed that rhIGF-1 could stimulate the formation of osteoclasts in the periodontal ligament, and accelerate bone remodeling and orthodontic tooth movement.


Introduction
Currently, the research on immune biomarkers of tooth root surrounding tissues is a hot spot. [1][2][3] Orthodontic tooth movement depends on the remodeling of tissues surrounding the roots. The periodontium is composed of three closely related structures: alveolar bone, periodontal ligament (PDL) and cementum, which plays an important role in orthodontic tooth movement 4 .
The periodontal ligament connects the cementum to the alveolar bone by Sharpey's fibers and undergoes remodeling in homeostasis and orthodontic tooth movement. Osteoclasts derive from the hematopoietic/ monocyte lineage and are replaced every few months in the periodontal ligament and alveolar bone. 5 Accelerating orthodontic tooth movement could shorten treatment duration and in combination with corticectomy-assisted tooth movement could promote post-treatment stability and has been intensively investigated. 6 The remodeling of the periodontal tissue relies on the physiological process of balancing osteoblasts and osteoclasts under the regulation of various biological factors and complicated molecular mechanisms to lead to tooth movement due to mechanical force. Hu, et al. 7 (2016) investigated the effects of recombinant growth hormone on orthodontic tooth movement in rats and found that recombinant growth hormone treatment increased the number of insulin-like growth factor-1 (IGF-1) positive osteoclasts in the periodontal ligament and accelerated tooth movement. 7 Xu, et al. 8 (2014) showed that local injection in rats of recombinant human TGF-α1 noticeably increased the number of osteoclasts and stimulated tooth movement. IGF-1 is crucial for bone cell function and skeletal development and maintenance and is the major mediator of growth hormone-induced bone growth.
Osteocytes express high quantities of IGF-1, 9 which is one of the earliest bone responses to mechanical loading. 10 IGF-1 may be an important factor in bone remodeling, and changes in IGF-1 content during tooth movement in orthodontic patients may be involved in alveolar bone remodeling. 11 The mechanical load of PDL is closely related to the autocrine/paracrine expression of IGF components, which leads to a longterm organized remodeling of alveolar bone. 12 IGF-1 may also modulate the bone remodeling process by its actions on osteoblasts and osteoclasts. Osteoclasts are the major cell for bone resorption, which plays a pivotal role in remodeling the alveolar bone during the movement of orthodontic tooth. 13,14 The study showed that recombinant human growth hormone could stimulate IGF-1 expression in PDL, and accelerate bone remodeling and tooth movement. 7 Recombinant human IGF-1 (rhIGF-1) is now in the research spotlight because of the role of IGF-1 in tooth bone remodeling and movement. Our study aimed at investigating the effect of rhIGF-1 on the number of osteoclast formation in periodontal tissue and changes of orthodontic tooth movement in SD rats.

Methodology Animals
The study protocol was approved by the local ethics committee at the authors' affiliated hospital.
Animal study was conducted in strict accordance with the established institutional guidelines on the use of experimental animals. The two maxillary incisors acted as the anchorage teeth, with a designed force of 50 g with the use of GAC Ni-Ti spiral spring (Shanghai, China) between the upper incisor and the first molar to move the left maxillary first molar mesially. 7,15 Moreover, the lower anterior teeth were ground to prevent the breakage of appliance ( Figure 1). The appliances were monitored regularly in case there was any breakdown. All rats were fed with softer foods on the first two days after the appliances were used.
The rats were then randomized to receive phosphate buffer saline (PBS) (the control group) or 400 ng rhIGF-1 (Pepro-Tech, USA) (the rhIGF-1 group) in the lateral buccal mucosa of the left maxillary first J Appl Oral Sci. 2021;29:e20200791 3/7 molar every two days. 16

TRAP staining
The rats were sacrificed by transcranial perfusion at days 1, 4, 7, 10, and 14 after the orthodontic force was applied, and subjected to fixation with 4% paraformaldehyde. The left ventricle was thoroughly rinsed with 250 mL normal saline at 37°C. The left maxillary was preserved in 4% paraformaldehyde for 18 to 24 hours at 4°C and then decalcified at 4°C for up to a month and a half in 15% ethylenediaminetetraacetic acid (EDTA, pH 7.4), which was replenished every week until the preserved sample was penetrated easily with a pin. The palatal side of the sample was

Measurement of tooth movement
A single tray of potassium alginate impression material was used to take the maxillary impression of rats before and after the pressure side dental operation, and the impression was perfused to form a plaster model. 17 The models were placed horizontally on the observation platform of the XTL-3400C stereomicroscope (Shanghai, China). The conjunctive plane was parallel to the ground, and was of the same height as the observation platform.
Images were captured using a JVC digital camera with 40× magnification to set the length scale. The model image was acquired using a computer, and the proximal middle lingual groove of the first molar and the distal middle surface of the second molar were marked. The distance between the two points was determined at least three times independently using YR-MV1.0 microscopic image measurement software 18 and the mean value was used. The distance difference between two points before and after the operation is the distance of the first molar on the afterburner side.

Statistical analysis
Data were presented as mean ± standard deviation  Moreover, the number of osteoclasts in compression side of the periodontal ligament in the rhIGF-1 group peaked at day 4 (11.37±0.95, compared to 5.28±0.47 in the control group) after the orthodontic force was applied, and was significantly higher than that of the control group at day 1, 4, 7 and 10 (p<0.01) ( Figure   3). Afterwards, the number of TRAP-positive cells decreased in the rhIGF-1 group and remained stable.
However, the activation and accumulation of TRAPpositive cells were observed at day 7 in the control group.

rhIGF-1 accelerates orthodontic tooth movement
We determined orthodontic tooth movement at different time points. Both groups showed timedependent increase in the distance of orthodontic tooth movement (Table 1). Furthermore, the distance of tooth movement in the rhIGF-1 group was significantly larger than that of the control group from Day 4 to   However, the potential mechanism of IGF-1 that promotes the proliferation and differentiation of osteoclasts is complex and unknown. Some studies found that osteoprotegerin (OPG) was associated with tooth movement. 27,28 Hill, Reinolds and Meikle 29 (1995) found that IGF modulated bone absorption by regulating OPG. Kobayashi, et al. 30 (2000) showed that the expression of RANKL mRNA increased evidently in the maxillary first molar by applying the force on orthodontic tooth. RANKL is an essential factor for osteoclast differentiation, which is expressed on the surface of osteoblasts by binding to osteoclast or the RANK receptor on the surface of its precursor cells to stimulate the formation and differentiation of osteoclasts. 31,32 In the process of tooth movement, the OPG/RANK/RANKL system 33,34 is the key in [2017]5).

Author disclosure statement
The authors declare no conflicts of interest.