Sclerostin Promotes Bone Remodeling in the Process of Tooth Movement

<div><p>Tooth movement is a biological process of bone remodeling induced by mechanical force. Sclerostin secreted by osteocytes is mechanosensory and important in bone remodeling. However, little is known regarding the role of sclerostin in tooth movement. In this study, models of experimental tooth movement were established in rats and mice. Sclerostin expression was investigated with immunohistochemistry staining, and osteoclastic activity was analyzed with tartrate-resistant acid phosphatase (TRAP) staining. MLO-Y4 osteocyte-like cells underwent uniaxial compression and tension stress or were cultured in hypoxia conditions. Expression of sclerostin was assessed by RT-qPCR and ELISA. MLO-Y4 cells were cultured with recombinant human sclerostin (rhSCL) interference and then co-cultured with RAW264.7 osteoclast precursor cells. Expressions of RANKL and OPG were analyzed by RT-qPCR, and osteoclastic activity was assessed by TRAP staining. During tooth movement, sclerostin was expressed differently in compression and tension sites. In SOST knock-out mice, there were significantly fewer TRAP-positive cells than in WT mice during tooth movement in compression sites. In-vitro studies showed that the expression of sclerostin in MLO-Y4 osteocyte-like cells was not different under a uniaxial compression and tension force, whereas hypoxia conditions significantly increased sclerostin expression in MLO-Y4 cells. rhSCL interference increased the expression of RANKL and the RANKL/OPG ratio in MLO-Y4 cells and the osteoclastic induction ability of MLO-Y4 cells in experimental osteocyte-osteoclast co-culture. These data suggest that sclerostin plays an important role in the bone remodeling of tooth movement.</p></div

The TRAP staining results showed that the osteoclastic activity was consistent with the sclerostin level at the compression sites during tooth movement.

<p>(A) The TRAP-staining results in the compression sites at 0 d, 1 d, 3 d, 5 d, 7 d, 14 d, 21 d and 28 d during tooth movement. The TRAP-positive cells were identified as red multinuclear cells near the alveolar bone. Alveolar bone (AB), PDL (periodontal ligament), R (root). (B) The TRAP-staining results in the tension sites at 0 d, 1 d, 3 d, 5 d, 7 d, 14 d, 21 d and 28 d during tooth movement. The TRAP-positive cells were identified as red multinuclear cells near the alveolar bone. Alveolar bone (AB), PDL (periodontal ligament), R (root). (C) The quantification of TRAP-positive cells per bone surface (mm^-1) at the compression sites. The number of active osteoclasts increased to the apex in the first three days and gradually decreased with time. *p<0.05 vs 0 d, ***p<0.001 vs 0 d. (D) The quantification of TRAP-positive cells per bone surface (mm^-1) at the tension sites. The number of active osteoclasts increased to the apex in the first three days and gradually decreased with time.</p

Primer sequences of Real-time RT-qPCR.

<p>Primer sequences of Real-time RT-qPCR.</p

The sclerostin expression of the MLO-Y4 cells was increased under hypoxic conditions, whereas it was not different under uniaxial compression and tension stress.

<p>(A) The relative mRNA expression of SOST of the MLO-Y4 cells was decreased under compression stress. ***p<0.001 vs control. (B) The relative mRNA expression of SOST of the MLO-Y4 cells was decreased under compression stress. ***p<0.001 vs control. (C) The relative mRNA expression of the SOST mRNA of the MLO-Y4 cells was increased 48 h after being cultured under hypoxic conditions. ***p<0.001 vs control. (D) The release of sclerostin by the MLO-Y4 cells that was determined using ELISA increased 48 h after being cultured under hypoxic conditions. **p<0.01 vs control.</p

Expression of sclerostin was different between the compression and tension sites during tooth movement.

<p>(A) X-ray images showed the mesial movement of the first molars at 0 d, 7 d and 28 d. (C) IHC of sclerostin at the compression sites. Sclerostin maintained high expression at 1d, 3d, 5d and 7d and gradually diminished until 28 d. Alveolar bone (AB), PDL (periodontal ligament), and R (root). The blue arrow indicated the mesial side in control group and the direction of tooth movement in experiment groups. (D) IHC of sclerostin at the tension site. Sclerostin immediately decreased and was continually maintained at the low level during 28 days of tooth movement. AB (alveolar bone), PDL (periodontal ligament), R (root). (E) Semi-quantification of IHC of sclerostin at the compression sites. n = 3,**p<0.01 vs control, *** p<0.001 vs control. (F) Semi-quantification of IHC of sclerostin at the tension sites. n = 3, ***p<0.001 vs control, ^#p<0.05 vs compression site, ^##p<0.01 vs compression site.</p

Osteoclastic activity was decreased in the SOST deficient mice compared to the WT mice during tooth movement.

<p>(A) TRAP staining at the compression sites in the WT and SOST KO mice at 0 d, 2 d and 4 d. (B) TRAP staining at the compression sites in the WT and SOST KO mice at 6 d, 8 d and 10 d. (C) Quantification of the osteoclasts number per bone surface (mm^-1) at the compression site of mice tooth movement. The osteoclast number/bone perimeter increased to the apex at day 4 and gradually decreased through day 10 in the SOST KO mice and WT mice. The number of osteoclasts was significantly lower in the SOST KO mice than in the WT mice at 4d, 6d, and 8d. *p<0.05 vs WT. ***p<0.001 vs WT.</p