## 4 Discussion
In immunohistochemical study, tissue expression of TGF-\(\beta\)1 was mainly localized in the ameloblasts, whereas OSX expression was mainly localized in odontoblasts (Figure 4). Runx2 expression was observed in both odontoblasts and ameloblasts (Figure 4). TGF-\(\beta\)1 is important in the maturation stage of ameloblasts [29], and loss of TGF-\(\beta\)1 in epithelial cells impairs enamel formation in mice [30]. TGF-\(\beta\)1 could also affect enamel mineralization by modulating Runx2 [8]; thus, enamel mineralization in ameloblasts is regulated by TGF-\(\beta\)1 and Runx2 [31]. Runx2 and OSX are independently involved in osteoblast differentiation and odontoblast/ameloblast differentiation, to regulate expression of many bone- and tooth-related genes [6; 7; 8]. Cross-talk between ameloblasts and odontoblasts is important in tooth development. Accordingly, differential expression patterns of TGF-\(\beta\)1, OSX, and Runx2 in tooth development may be associated with programmed differentiation of odontoblasts and ameloblasts. The expression of BMP-2/BMP-4 was observed not only in odontoblasts, but also in ameloblasts and associated cells (Figure 4). According to previous studies, BMP-2 is required for odontoblast differentiation [32] and is expressed more in the basal part of the dental follicle to promote eruption [33]. BMP-2 also induces DSPP [34], which is important for odontoblast differentiation and dentin formation, forming dental roots [8]. The expression of DSPP was mainly observed in the pulp tissue (Figure 4). The expression level of PTHrP was increased in both cell culture and tissue samples (Figures 1 and 3); however, PTHrP expression was poorly localized in the tissue sections (Figure 4). PTHrP in the dental follicle is known to recruit mononuclear cells by enhancing colony stimulating factor-1 (CSF-1) [35] and vascular endothelial growth factor (VEGF) [36]; thus, the expression of PTHrP may be localized in other areas related to osteoclastogenesis. 4 HR-induced TGF-\(\beta\)1 and BMP-2 expression is also observed in osteoblast-like cells [23]. It activates the TGF-\(\beta\)/SMAD/VEGF pathway in HUVECs [37]. Therefore, increased expression of TGF-\(\beta\)1, BMP-2/4, Runx2, OSX, and DSPP in Groups A and B, in this study, may indicate the overall increase in differentiation of dental follicle cells and acceleration of dental hard tissue formation required for tooth eruption by 4 HR. There are some limitations in this study. First, since the incisors of rats, in this study, had already erupted, the eruption rate was mainly affected by the formation of dental hard tissue, and the effect on intraosseous eruption was not evident. In our previous study [23], 4 HR increased both bone resorption and bone formation in orthodontic tooth movement; however, further studies may be required to identify the effects of 4 HR on intraosseous eruption. Second, this study was limited to a selected age group only. There may be some differences in the eruption rate depending on age; in fact, the eruption rate in the same group may also be slightly different, such as 1 mm/day [3] or 1.5 mm/day [20]. Third, the effect of 4 HR administration on dental hard tissue formation was mainly shown indirectly by increased incisor eruption rate and expression of related markers in cell culture and tissue samples; it mainly focused on the amount of tooth formation, rather than the degree of calcification. Fourth, humans do not have continuously erupting teeth. Though humans may have unerupted teeth [38], their etiology may not be due to calcification disorder. Accordingly, translated clinical applications based on current results will be limited. In the future, 4 HR may be administered to specific gene knock-out models and its effect on tooth development and eruption may be examined.