Introduction

Tooth is made up with complex tissues - hard and soft tissues - especially hard tissues are composed of enamel, dentin and cementum. Dentin which occupies most of calcified part of tooth plays a pivotal role in the functional aspect of teeth and is designed to mutually resist the masticatory pressure occurring when chewing or biting food. Unlike enamel, dentin responds very sensitively to stimulation and is less hard than enamel but it has flexibility to compensate for the fragile prop- erties of enamel [1]. There are a myriad of microscopic tubes called dentinal tubules in dentin which penetrate the entire dentin from the pulp cavity wall to the surface of the dentin [2]. Odontoblasts are well-arranged and organized cells along the dentin-pulp dimensional gap. The origin of odontoblasts differentiates from undifferentiated mesenchymal cells which originates from neural crest cells [3]. At cap stage - embryonic day 14 (E14) of mouse, the three layers of epithelium are presented including outer enamel epithelium (OEE), inner enamel epithelium (IEE), and the stellate reticulum. Mesenchymal cells showed the condensed structure to be differentiated into odontoblast, which locate intactly with epithelium, and pulp cells [4]. Odontoblasts, which produce dentin matrices, deposit matrix and migrate towards the dental papilla and once the coronal dentin is formed to the cementum-enamel junction, it begins to form a root dentin [5]. Dentin-pulp complex is of great importance for protecting teeth in two ways: immunologically defending against harmful stimuli and responding to trauma or infection through healing and regeneration [6].

The types of dentin are classified into primary, secondary and tertiary dentin depending on the order of occurrence. Primary dentin refers to the dentin formed from odontoblasts at very beginning until root formation is completed. It occupies most of the dentin of normal teeth. Most of the primary dentin is peripulmonary dentin and the outer thin layer is the mantle dentin [7-9]. Secondary dentin refers to dentin formed addition to primary dentin due to physiological conditions. Secondary dentin is gradually formed and added over the entire surface of the pulp gradually while the pulp is viable [9,10]. Tertiary dentin is built beneath the irritated area of pulp, an irritation such as attrition, abrasion or dental restorative treatment applied to the tooth. Tertiary dentin is divided into reactive dentin and reparative dentin. Reactive dentin is dentin formed by existing odontoblasts when applied stimulation is weak [11]. Reparative dentin is formed due to strong stimulation of which existing odontoblasts lose their vitality, instead of odontoblasts, odontoblast-like cells are differentiated and produce reparative dentin [12].

Endoplasmic reticulum (ER) is a designated organelle which regulates proteostasis – protein homeostasis – through processes of protein synthesis, folding, assembly and trafficking [13]. Also ER is important for structural maturation of one third of all proteins which is produced in cell [14]. However, not all the proteins translocated into ER are assembled or folded. The success rate for proper folding is under 20% because incomplete folded forms of proteins are not tolerated by cell and by the process called ER-associated degradation (ERAD), they are removed to cytosol. The protein-folding capacity of ER is in balance with demand through the corrective responses of signal transduction pathway called unfolded protein response (UPR) [15,16]. Despite of corrective responses, when there is an excess of workload imposed on ER protein-folding machinery over its capacity, ER stress occurs [17]. ER stress leads to accumulation of misfolded proteins in ER which includes nutrient deprivation, hypoxia and point mutation [18-20]. In addition, recent studies revealed that modulation of ER stress is important in hard tissue formation, especially dentin formation [21-23].

Small molecules and compounds are studied for their ability to influence various components of UPR recently [24]. Among those molecules, chemical chaperones such as Tauroursodeoxycholic acid (TUDCA) was reported to reduce ER stress [25,26]. Especially TUDCA, the United States Food and Drug Administration approved drug for treating biliary cirrhosis has been reported recently about its antioxidant and antiapoptotic activity [27,28] and it is also known to induce activation of UPR to attenuate ER stress [26]. However, the effect of TUDCA on developing tooth regarding modulation of ER stress is not studied yet. In this study, we examined altered cellular events in secretory stage of developing mice molar after treatment of TUDCA, a chemical chaperone known to alleviate ER stress, to understand the developmental mechanisms of tooth morphogenesis.

Materials and Methods

All animal experiments were ethically approved and conducted in accordance with the guidelines of the Intramural Animal Use and Care Committee of Kyungpook National University School of Dentistry (KNU 2020-0107).

1. Animals

Embryos of mouse were obtained from healthy time-mated pregnant ICR mouse which was maintained in an optimal environment. Embryonic day 0 (E0) was designated as the day of vaginal plug was confirmed. Embryos of stages E14 mice were used in this study. Healthy 7 weeks-old male ICR mice were used for kidney capsule transplantation. Adult ICR mice were housed in a temperature-controlled room (22℃) under artificial illumination (lights on from 05:00 to 17:00), at 55% relative humidity, with access to food and water ad libitum.

2. In vitro organ cultivation, kidney transplantation and drug treatment

The embryonic mice molar tooth buds at E14 were dissected out from the lower jaws in phosphate-buffered saline under a stereo-microscope. The dissected teeth were cultivated in 100 M TUDCA (Sigma-Aldrich, St. Louis, MO, USA) and 0.05% dimethyl sulfoxide with Dulbecco Modified Eagle Medium (Hy- Clone, Logan, UT, USA; cat. no. -SH30243.01) and 10% fetal bovine serum (HyClone) for 48 hours using a modified Trowell’ s culture method as previously described [29]. The cultured tooth germs were transplanted into renal subcapsular layer of the adult male mice as previously described and the host mice were sacrificed after 1 weeks as calcified teeth were obtained [30].

3. Histology and immunohistochemistry

Calcified teeth were sectioned after decalcified using 0.5 M ethylenediaminetetraacetic acid for 3 weeks. Histological analyses were carried out routinely with hematoxylin and eosin (H&E) staining and immunohistochemistry was done as previously described [31]. Sections were rehydrated first and processed with antigen retrieval for immunostaining. 1X western blocking solution (Roche, Mannheim, Germany; Ref. 11921673001) was treated for blocking as sections were incubated for one hour in room temperature. Primary antibodies used in this study are Nestin (Abcam, Cambridge, UK, 1:400; cat. no. ab11306), Amelogenin (AMELX; Abcam, 1:500; cat. no. ab153915), Glucose regulatory protein 78 (GRP78; Abcam, 1:400; cat. no. ab21685), HRD1 (Novus Biologicals, Centennial, CO, USA, 1:200; cat. no. NB100-2526), IRE1 alpha (Novus Biologicals, 1:500; cat. no. NB100-2323) and Ki67 (Thermo Scientific, Waltham, MA, USA, 1:400; cat. no. RM-9106-s). Secondary antibodies were used as biotinylated anti-rabbit or anti-rat immunoglobulin G. Binding of the primary antibody to the sections was visualized using the diaminobenzidine tetrahydrochloride reagent kit (GBI Labs, Bothell, WA, USA, cat no. C09-12).

4. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

TUNEL assay was achieved as previously described [31] using in situ cell apoptosis detection kit (Trevigen, Gaithersburg, MD, USA, cat. no. 4810-30-K) according to the manufacturer’s instructions.

Result

1. ER stress modulated calcified tooth continues to secrete matrices

Histological changes of calcified teeth after treatment of chemical chaperone, TUDCA, were observed using H&E staining routinely. In control group, enamel space between accumulated dentin and a layer of ameloblasts was found (Fig. 1A and A’), however, in TUDCA treated group, enamel space was not found (Fig. 1B and B’). Meanwhile, in tooth root area, it was observed that the double layer of OEE and IEE continued to elongate in TUDCA treated group (Fig. 1B’’) while the root area of control group started to be enclosed by invagination of OEE (Fig. 1A’’). The localization pattern of active odontoblasts in TUDCA treated calcified teeth was more intense than the control group (Fig. 1C–D). Moreover, the cusp and root area showed denser alignment of odontoblasts in experimental group than that of the control (Fig. 1C’–D’’). On the other hand, the localization pattern of Amelogenin in experimental group (Fig. 1F’ and F’’) was comparably stronger than the control group (Fig. 1E’ and E’’).

2. ER stress in developing teeth was alleviated after treatment of chemical chaperon

To evaluate ER stress related signalings, we examined the localization patterns of ER stress related molecules including GRP78, HRD1 and IRE1 alpha. The localization of GRP78, an universal marker for ER stress, in control group showed stronger positive reaction in ameloblasts and odontoblasts than of the experimental group (Fig. 2A and B). Especially localization pattern of GRP78 in odontoblasts at tooth root area showed stronger positive reaction (Fig. 2A’’). HRD1, a marker for ERAD which is one pathway of UPR system, localized only at ameloblasts of cusp area in control group (Fig. 2C), however in TUDCA treated group, most of ameloblasts and some odontoblasts showed the positive reaction against HRD1 (Fig. 2D). The IRE1 alpha localized both at nuclei of odontoblasts and ameloblasts strongly in experimental group when compared with control (Fig. 2E and F).

3. Modulation of ER stress promotes the elongation of tooth root

To evaluate the altered mechanisms in developing calcified teeth induced by modulation of ER stress, we examined cell proliferation and apoptotic events in calcified teeth. First, localization pattern of Ki67, a cell proliferation marker, was observed. Generally in in vivo, proliferating cells in developing calcified tooth would be found at the limited area, such as OEE of root forming regions. Control groups showed proliferating cells only at restricted part of Hertwig’s epithelial root sheath, especially only at a layer of OEE (Fig. 3A) which correlated with in vivo results. However, in TUDCA treated groups showed broader localized region at the area of root forming area (Fig. 3B) compared to the control. Additionally, to elucidate apoptotic events in calcified teeth, TUNEL assay was employed. In control group, apoptotic event occurred at the area of tooth root forming (Fig. 4A), but in TUDCA treated group, there was noticeable decrease of apoptotic cells all throughout the calcified teeth (Fig. 4B).

Discussion

In order to examine the function of TUDCA in secretory stage of tooth development, we designed the experimental plan with 2 days cultivation at E14, with or without drug treatment and kidney transplantation for 1 week. The in vitro cultivated molars which were performed organ cultivation with renal capsule transplantation at E14 would fully mimic in vivo developing PN3 molar (Fig. 1A) [32]. Enamel space is found when ameloblasts complete their secretory and maturating functions [5], however, there was only intact layers of dentin and ameloblasts in TUDCA treated group which would explain that there is a continuation of secretion and maturation of matrices (Fig. 1B and B’). Alteration in active odontoblasts and ameloblasts after treatment of TUDCA were observed after immunohistochemistry of Nestin and Amelogenin (Fig. 1C–F). It was obvious that control group started to form reduced enamel epithelium and enamel space (Fig. 1E) which indicates enamel layers are matured fully [5] while ER stress inhibited teeth showed continuous secretion of enamel. The histological and immunostaining results suggested that TUDCA treated calcified groups continue to secrete enamel when ER stress is attenuated with treatment of TUDCA.

Whereas, the localization pattern of GRP78, the universal marker for ER stress [33] showed that ER stress in root forming area would be unavoidable to complete tooth roots, however, when ER stress is modulated by treatment of chemical chaperone, TUDCA, tooth roots would continue to maintain its thickened epithelial structures (Figs. 1B” and 3B”). As examined with ERAD, one of the ER stress rescue system – UPR [34, 35], would activate all throughout the calcified tooth forming period. ER stress is modulated as shown in GRP78 localization. It can be also observed in localization pattern of IRE1 alpha, the major molecule in UPR system [36].

These localization patterns of ER stress related signaling molecules in calcified teeth after treatment of TUDCA would confirm that ER stress in developing teeth can be modulated by chemical chaperone; TUDCA. In addition modulated ER stress in calcified teeth allows to retain root development. These results confirm that modulation of ER stress inhibits the apoptotic events [17] and promotes cell proliferation in tooth root development. Moreover, the results ascertain that ER stress in crown formation period would involve in and modulate tooth morphogenesis [22,23], including tooth root formation. This experimental model system, designed and performed in this study, would permit us to examine the precise efficacy of various drug or small molecules in dental hard tissue formation and revealed developmental mechanisms would be a plausible answer to regenerate the hard tissues.