Dynamic alternations of RANKL/OPG ratio expressed by cementocytes in response to orthodontic‑induced external apical root resorption in a rat model

Abstract

The aim of the present study was to investigate the alterations in the formation of cementocytes in response to orthodontic forces and to evaluate the contribution of these cells in the biological changes of tooth movement and associated root resorption. A total of 90 Sprague Dawley rats were randomly assigned to the control, high force, and low force groups. Intrusion forces of 10 and 50 g were applied on the rat molar to induce tooth intrusion. The tooth movement was observed from 0 to 14 days by micro-computed tomography, bone histometric analysis, tartrate-resistant acid phosphatase staining, as well as reverse transcription-quantitative PCR and immunofluorescence staining assays. The results suggested that under low force conditions, osteoclasts were distributed at a higher frequency on the bone side than on the root side. Under high force conditions, both sides suffered osteoclast infiltration. In the low force group, the cementocytes exhibited downregulated sclerostin ( SOST ) and osteoprotegerin ( OPG ) mRNA levels and a lower receptor activator of nuclear factor-κB ligand ( RANKL )/ OPG ratio over a certain period of time. The expression levels of these genes were lower compared with those of the osteocytes at each time-point. In the high force group, both cementocytes and osteocytes upregulated the SOST and RANKL/OPG ratio on days 7 and 14, while the cementocytes expressed higher levels of SOST mRNA than those noted in the osteocytes. These data suggested that cementocytes responded to the orthodontic force via modulation of the RANKL/OPG ratio and SOST expression. The biological response of cementocytes contributed to the mechanotransduction and homoeostasis of the roots under compression. Excessive forces may act as a negative factor of this regulatory role. These results expand our knowledge on the function of cementocytes.

Biological alterations associated with the orthodontic treatment with conventional appliances and aligners: A systematic review of clinical and preclinical evidence

“…Regarding bone formation mediators, the most studied one (investigated by 87 % of the animal studies) is osteoprotegerin (OPG). Several studies reported a decrease in OPG expression and/or levels at distinct time points in animal models after both 10- and 50-g intrusion [ 40 ] and 80- to 100-g to produce inclination [ 27 , 34 ].…”

“…Besides the abovementioned alterations, the main alternative biological response occurring along the mechanical loading was an increase of coarse and irregular fibers and blood capillary compression in rodents [ 16 , 25 , 28 , 29 ]. Also, it should be noted that three studies observed that heavier forces were not associated with a greater amount of tooth movement when two or more levels of forces (3–100 g) were assessed [ 29 , 33 , 37 , 40 ], when producing different types of movements. On the other hand, one study detected a significantly higher OTM under 100-g force compared to 30 g [ 23 ].…”

“…The most common problem verified when conducting the quality assessment of the animal articles was associated with performance and detection bias, mainly due to the lack of blinding of the investigators regarding the experimental groups [ 12 , 16 , 23 , [24] , [25] , [26] , [27] , 29 , 31 , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] ]. Similarly, randomization issues were detected, which compromises the quality and verisimilitude of the data collected from such studies.…”

“… Noguchi et al (2022) [ 39 ] - C57BL6/J (WT) mice - TNFRs deficient (TNFRsKO) mice Intervention: Ni–Ti closed coil spring applying 10-g force to the maxillary left 1st molar (inclination movement) Duration: 12 days Inflammation: -↑ VEGF-positive cells and mRNA levels in the alveolar bone surface of the orthodontically-treated WT mice compared to WT mice control (real-time PCR); -↑ VEGF-positive cells and mRNA levels in the alveolar bone surface of the orthodontically-treated TNFRsKO mice compared to TNFRsKO mice control (real-time PCR); -↑ VEGF-positive cells and mRNA levels in the alveolar bone surface of the orthodontically-treated WT mice compared to the orthodontically-treated TNFRsKO mice (real-time PCR). Wei et al (2022) [ 40 ] Sprague-Dawley rats Intervention: stainless-steel wire applying 10-g and 50-g force to right maxillary 1st molar (intrusion movement) Duration: 0, 2, 6, 24 h, 4, 7 and 14 days Bone formation & resorption: -↓ BV/TV ratio, Tb.N and bone mineral density after 10- and 50-g interventions compared to control, and after 50-g intervention compared to the 10-g group (bone histometric analysis); -↑ Tb.Sp levels after 10- and 50-g interventions compared to control (bone histometric analysis); -no significant changes in Tb.Th between groups for all treatment durations (bone histometric analysis); -↑ TRAP + osteoclasts in the AB side after the 50-g intervention delivered for 24 h, 4, 7 and 14 days compared to all groups (TRAP staining); -no significant differences in TRAP + osteoclasts number in the AB side between 10- and 50-g interventions for all treatment durations (TRAP staining); -↑ SOST mRNA expression in the AB side compared to the CC side after 10-g intervention delivered for 24 h, 4, 7 and 14 days (RT-qPCR); -↑ SOST mRNA expression in the AB side compared to the baseline in the same side after 10-g intervention delivered for 24 h and 4 days, and after 50-g intervention delivered for 4, 7 and 14 days (RT-qPCR); -↑ co-localization of SOST protein with osteocytes after both 10- and 50-g interventions delivered for 4, 7 and 14 days compared to the baseline (IHC); -↑ SOST expression in the AB side compared to the CC side the 50-g intervention delivered for 7 and 14 days compared to the baseline (IHC); -↑ RANKL mRNA expression in the AB side compared to the CC side after 10-g intervention for all treatment durations (RT-qPCR); -↑ RANKL mRNA expression in the AB side compared to the CC side after 50-g intervention delivered for 0, 6, 24 h and 4 days (RT-qPCR); -↑ RANKL mRNA expression in the AB side compared to the baseline in the same side after 10-g intervention delivered for 4 days, and after 50-g intervention de...…”

Osteocyte Mechanotransduction in Orthodontic Tooth Movement

“…RANKL is produced by osteoblasts and osteocytes, and binds to its receptor RANK on the surface of osteoclasts [ 107 ]. This interaction activates a signaling cascade that leads to the differentiation, activation, and survival of osteoclasts, resulting in bone resorption [ 108 ]. During orthodontic tooth movement, RANKL expression increases in gingival crevicular fluid in juvenile patients [ 109 ].…”