## 4 Discussion  
In our recent study, 4 HR was found to accelerate orthodontic tooth movement by increasing both bone formation and resorption in ovariectomized rats [23]. Since the tooth eruption process requires dental hard tissue formation as well as bone turnover, the current study aimed to explore whether 4 HR could increase both dental hard tissue formation and rate of eruption, using in vivo experiments and evaluating the expression of relevant markers. In vitro experiments using primary cultured rat incisor pulp cells demonstrated that 4 HR administration increased the level of protein expression associated with tooth formation (Figure 1). Accordingly, the total amount of incisor eruption was significantly higher in Group B (12.8 mg/kg of 4 HR administered) than that in the untreated control  
Figure 4: Results of immunohistochemistry. (**a**) Schematic drawings of the cut plane. The specimen was cut behind the third molar. The cross-section had ameloblasts and odontoblasts with pulp tissue. The boxed area in the right figure is shown in (**b**) for immunohistochemical staining. (**b**) Immunohistochemistryichemical staining for DSPP, OSX, Runx2, TGF-\(\beta\)1, BMP-2/4, and PTHrP-R. TGF-\(\beta\)1 was mainly expressed in the ameloblasts and OSX in the odontoblasts. Runx2 was expressed in both ameloblasts and odontoblasts, though more intensely in the ameloblasts. However, PTHrP-R was not highly expressed in this region. (\(p<0.05\), Figure 2). In the mandibular tissue samples, expression of proteins associated with tooth formation was higher, and the results were consistent with those of cellular experiments (Figure 3). Interestingly, the localization of their expression was specific in accordance with their role in tooth formation (Figure 4). Bone is a representative calcified tissue in the body, and 4 HR may influence the remodeling of bone via immunomodulation [21]; 4 HR is an M2 type macrophage-polarizing agent [21; 26]. Tooth is also a kind of calcified tissue, whose eruption rate may be influenced by 4 HR administration. Rat incisors erupt continuously throughout the animal's lifetime [18; 20]. Indeed, 4 HR administration accelerated mandibular incisor eruption in the 4 HR-administered groups (Figure 2a). Administration of 4 HR increased the rate of incisor eruption in both sexes of rats (Figure 2b). The rate of incisor eruption increased with an increase in the dose of 4 HR; however, no statistically significant difference was observed between Groups A and B (\(p>0.05\)). When the rate of incisor eruption was compared between the sexes within the same group, the total amount of incisor eruption was found to be greater in the females of Group B (Figure 2b), although the difference was statistically insignificant (\(p>0.05\)). According to a previous report, a cut at the mandibular incisor produces hypofunctional eruption conditions, under which the mandibular incisor shows increased eruption rates [3]. The eruption rate of the hypofunctional incisor has been reported to be approximately 1 mm/day [3] or 1.5 mm/day [20]. In this study, eruption rates of 1.45 mm/day for males and 1.46 mm/day for females were recorded in the untreated control (Figure 2b). Accelerated mandibular incisor eruption after 4 HR administration may be due to the increased level of expression of proteins associated with tooth formation. 4 HR administration increased TGF-\(\beta\)1, BMP-2/4, Runx2, OSX, DSPP, and PTHrP-R expression in both cell culture and tissue samples (Figures 1 and 3). It increased the expression of TGF-\(\beta\)1 in both RAW264.7 cells [27] and human umbilical vein endothelial cells (HUVECs) [28]. Since TGF-\(\beta\)1 plays an important role in cell proliferation and differentiation, as well as in bone and tooth metabolism [5], an increased expression of this cytokine may contribute to the activation of various cells related to tooth eruption.