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Introduction

Tooth development is a complicated process involving cell differentiation, proliferation, movement and death that is regulated by strict genetic control [1,2]. The tooth is formed by the ectoderm-derived oral epithelium and neural crest­ derived ectomesenchymal cells. The first sign of tooth devel­opment is a thickening of the oral epithelium, which invagi­nates into the underlying mesenchyme and forms the bud, cap and bell of the enamel organ [3]. At the late bell stage of tooth development, ectomesenchymal cells of the dental papilla differentiate into polarized odontoblasts and secrete the dentin matrix, whereas ameloblasts derived from the inner enamel epithelium synthesize the enamel matrix [4,5].

A large number of studies have examined the genes involved in tooth development and elucidated their functions in the biological research field. The major focus of these studies was on determining the mechanism of the interac­tions involved in tooth development [4]. However, the genes investigated thus far are not enough to explain all physio­logical phenomena in tooth development [6,7]. In addition, many studies have used in vitro system to identify genes involved in the development and therefore, they cannot represent exactly in vivo state of tooth development.

The aim of this study was to detect the differentially expressed genes involved in dental hard tissue formation. One of the genes differentially expressed was a part of osteonectin [8]. Localization of osteonectin and changes in expression during tooth development was further studied.

Materials and methods

Animals and tooth germs

Newborn rat pups (Sprague-Dawley) were housed in laboratory animal care-approved facilities. All procedures were performed in accordance with the ethical standards for­mulated by the animal care and use committee in Chonnam National University. Histological observation revealed that the 2nd molar germ at postnatal days 3, 6, and 9 was at the bell stage, crown stage and root formation stage, respectively and the 3rd molar germ was at the cap or early bell stage at postnatal day 9. 

Histological preparation

Rats at postnatal days 3, 6 and 9 were sacrificed and tooth germ-containing portions of the maxilla were isolated surgi­cally. They were immersion-fixed in 4% paraformaldehyde solution overnight and decalcified in ethylene diamine tetra-acetic acid (pH 7.4) over several weeks. They were then routinely processed for embedding in paraffin. Sagittal sections were cut 5 µm-thick for hematoxylin and eosin stain and immunofluorescency. 

RNA preparation and Differential display polymerase chain reaction (DD-PCR)

After rats at postnatal days 3, 6 and 9 were sacrificed, the overlaying gingivae and alveolar bone were removed care­fully to expose the maxillary 2nd and 3rd molar germs. Molar germs were extracted with surrounding follicular tissues from their crypts. The total RNA was extracted from the prepared molar germs using Trizol^® Reagent (Gibco BRL, MD, USA). Quantitative analysis of the RNA samples was performed using a UV spectrophotometer (Amersham-Pharmacia Biotech, Arlington Heights, IL, USA). DD-PCR was performed according to the manual provided using a GeneFishing^TM DEG kit 101 (Seegene, Del Mar, CA, USA). The positive control was performed according to the manual provided and identified from the agarose gel pattern. For a reverse transcription of the first-strand cDNA, the reaction was initialized by incubating the total RNA and dT-ACP1 at 80^oC for 3 min. After chilling the reagents on ice for 2 min, dNTP, RNase inhibitor and M-MLV reverse transcriptase were added and incubated at 42^oC for 90 min. The reagents were then heated to 94^oC and chilled on ice for 2 min. The first-strand cDNA was amplified using a Palm-Cycler thermocycler (Corbett Life Science, Sydney, Australia). The reagents, mixtures to perform PCR, were composed of first-strand cDNA, MgCl₂, arbitrary ACP, dT-ACP46 and dNTP. "Hot-started" PCR was carried out by preheating the reagents to 94^oC for 2 min. After preheating, Taq DNA polymerase was added to the reagents. The starting conditions for PCR were 94^oC for 3 min, 50^oC for 3 min and 72^oC for 1 min. This was followed by 40 cycles consisting of 94^oC for 40 sec, 65^oC for 40 sec, and 72^oC for 40 sec. The reagents were finally reacted at 72^oC for 5 min. The PCR products were separated by gel electrophoresis on 2% agarose gel and identified using a UV spectrophotometer (Amersham-Pharmacia Biotech, Arlington Heights, IL, USA).  

DNA Elution, Subcloning and Sequencing

The differentially expressed DNA bands at each develop­mental stage were eluted from agarose gel using a QIAquick gel extraction kit (Qiagen Inc., CA, USA). The DNAs were ligated with the T-vector using T4 DNA ligase. The ligated products were transformed with DH5á and then smeared on a MacConkey plate containing ampicillin. The plate was incubated overnight at 37^oC. The correct transformation was confirmed by isolating the plasmid DNAs using SolGent Plasmid Mini-Prep kit (SolGent Co., Ltd. Daejeon Korea). The isolated plasmid DNAs were cut by EcoRI (Takara, Otsu, Shiga, Japan), separated by gel electro­phoresis on 1.5% agarose gel for 30 min, and then identified using a UV spectrophotometer (Amersham-Pharmacia Biotech, Arlington Heights, IL, USA). The plasmid DNAs were sequenced. 

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Primers and their products size were summarized in Table 1. GAPDH gene was used for the reference gene. RT-PCR was performed using AccPower^® RT PreMix (Bioneer, Daejoen, Korea). The reagents composed of a RT buffer, dNTP, RNase inhibitor and M-MLV reverse transcriptase were added to the mixtures of the total RNA and Oligo-dT. The tube was incubated at 42^oC for 90 min to make the first-strand cDNA. The PCR reaction was performed using Palm-Cycler thermocycler (Corbett Life Science, Sydney, Australia) using the following procedures: 30 cycles con­sisting of denaturation at 94^oC for 30 sec, annealing at 60^oC for 30 sec, and extension at 72^oC for 30 sec with a final extension at 72^oC for 5 min. The products were separated by gel electrophoresis on 1.5% agarose gel and then visualized using a UV spectrophotometer (Amersham-Pharmacia Bio­tech, Arlington Heights, IL, USA).

Real time RT-PCR

The level of osteonectin expression was also determined by real-time RT-PCR. The cDNA was synthesized from the total RNA extracted from the 2^nd molar germs at postnatal days 3, 6 and 9, and the 3^rd molar germs at postnatal day 9 using a SuperScript^TM First-strand Synthesis System (Invitrogen^TM Life Technologies, Carlsbad, CA, USA). The target genes were amplified from equal amounts of cDNA using a Rotor-Gene RG-3000 (Corbett Research, Morklake, Australia). The amplified cDNA was detected using a SYBR Green PCR Master Mix Reagent kit (Qiagen, Maryland, CA, USA). The data was analyzed using the Corbett Robotics Rotorgene software (Rotorgene 6 version 6.1, Build 90 software). The ratios of the intensities of the target genes and β-actin signals were used as a relative measure of the expression level of target genes. A reaction mixture lacking cDNA was used as the negative control in each run.

Table 1. Sequences of the primers for RT-PCR

Western blotting analysis

The proteins were extracted from the 2nd and 3rd molars using a Ready prep protein extraction kit (Bio-RAD, Hercules, CA, USA). The extracted proteins were separated by gel electrophoresis on a 12% resolving gel and a stacking gel for 2 hr and 30 min. After gel electrophoresis, the stacking gel was removed. The proteins of the remaining resolving gel were transferred to a Protran nitrocellulose membrane (Whatman GmbH, Dassel, Germany) at 100V for 2 hrs. The transferred Protran nitrocellulose membrane con­taining the proteins was identified using a Ponceau solution. The membrane was blocked with mixtures containing 5% skim milk and a TBS-T buffer composed of 10 mM Tris­buffered isotonic saline (pH 7.0), 0.1% merthiolate, and 0.1% Tween-20 at room temperature for 1 hr. The membrane was incubated with the 1:500 purified mouse polyclonal primary antibody at 4^oC overnight with constant shaking. The membrane was washed 10 times with TBS-T for 6 min, and then incubated with 1:3000 anti-mouse osteonectin antibody and GAPDH for 2 hrs. The bound antibody was reacted with the Lumiglo reagent (Millipore, Billerica, MA, USA). The results were identified using LAS 4000 mini loaded with ImageReader LAS-4000 software (Fuji fillm, Minatoku, Tokyo, Japan). 

Immunofluorescent stain

Immunofluorescence staining was performed using a TSATM Kit (Invitrogen, Carlsbad, CA, USA). The same purified osteonectin antibody, which was used for the immunoblot, was used as the primary antibody. Briefly, after blocking the endogenous peroxidase with 1% H2O2, routinely processed sections were reacted with the primary antibody for 2 days and then with the HRP-conjugated secondary antibody. They were then incubated in an Alexa Fluor 488® tyramide working solution and counterstained with propidium iodide to examine the nuclear morphology. The reactants were visualized and photographed using a LSM confocal microscope (Carl Zeiss, Gottingen, Germany). For the immunological specificity test, the primary antibody was substituted with normal serum.  

Results

Osteonectin is identified in developing molars

 DD-PCR was used to search the differentially expressed genes in the 2nd and 3rd molar germs in Sprague-Dawley rats at postnatal day 9. Many DNA bands were detected by the combinations of ACPs. One of the bands detected by dT­ACP46 was expressed only in the 2nd molar germs (Fig. 1). The size of the DNA band was approximately 350 bp. The detected band in Fig. 1 was selected from agarose gel, eluted, ligated and transformed for identification. The plasmid DNA obtained from subcloning showed homology to osteonectin (GenBank accession no. NM_012656.1) and 344 bp in size.

Fig. 1. An agarose gel image from DD-PCR using dT-ACP46. The differentially expressed gene (arrow) was detected between the 2nd and 3rd molar germs at postnatal day 9. The band was a little big-ger than 300 bp in size. The molecular marker (M) was a 100 bp ladder.

mRNA level is changed during development

 RT-PCR was performed using specific primers to deter­mine osteonectin mRNA level in each developmental stage. The PCR products were 220 bp in size as expected. The level of osteonectin mRNA was higher in the 2nd molar germs (root formation stage) than in the 3rd molar germs (cap stage) (Fig. 2a). This result was also confirmed by real-time PCR, which demonstrated that the level was 30 times higher in the 2nd molar germs. RT-PCR was also performed using the mRNA in the 2nd molar germs at postnatal days 3 (early bell stage), 6 (crown stage), and 9 (root formation stage). The level of mRNA expression was also increased according to the tooth development stages. The level of mRNA expression was highest in the root formation stage at postnatal day 9 and lowest in the bell stage at postnatal day 3. This result was also confirmed by real-time PCR (Fig. 2b).

Protein level is changed during development

 Western blotting was performed to examine the osteonectin protein expression in each developmental stage. The protein was extracted from the 2nd molar germs at postnatal days 3 (early bell stage), 6 (crown stage), 9 (root formation stage). The expression of the osteonectin protein increased ac­ cording to the developmental stages (Fig. 3). These patterns were coincident with the RT-PCR results.

Fig. 2. (a) An agarose gel image from RT-PCR using the mRNAs in the 2nd and 3rd at postnatal day 9. The level of mRNA expres¬sion was higher in the 2nd molar germs (root formation stage) than in the 3rd molar germs (cap stage). GAPDH was used as a refer¬ence. Real-time RT-PCR was performed in triplicate, showing approximately 30 times higher level of the expression in the 2nd molar germs. (b) An agarose gel image resulting from RT-PCR using the mRNAs in the 2nd molar germs at postnatal days 3 (early bell stage), 6 (crown stage) and 9 (root formation stage) shows an increase in a time-dependent manner. This result was also con-firmed by real-time RT-PCR, which was performed in triplicate. The molecular marker (M) was a 100 bp ladder.

Osteonectin is localized in odontoblasts

 To localize osteonectin in the developing molar germs, immunofluorescent staining (green color) was performed and observed under a confocal microscope. Red colored propidium iodide was counterstained for nuclear morph­ology. Strong immunoreactivities against osteonectin were observed in most odontoblasts and preodontoblasts at the root stage molar germs. Weak reactivity was also observed in perifollicular cells (Fig. 4a). Little reactivity was noted in the cap stage 3rd molar germs at postnatal day 9. Any reactivity was not found in the negative (Fig. 4b).

Fig. 3. Western blotting results using the proteins in the 2nd molar germs at postnatal days 3 (bell stage), 6 (crown stage), and 9 (root formation stage). The expression of the osteonectin protein increased according to the developmental stages.

Fig. 4. Localization of osteonectin in developing molars. (a) Immunoreactivity (green color) against osteonectin is seen in odontoblasts. The reactivity is seen in odontoblasts and preodonto¬blasts in the 2nd molar germ. In the 3rd molar germs, the reactivity is hardly found. (b) Any reactivity is not found in the negative con¬trol which substituted the primary antibody with serum. 2nd: 2nd molar, 3rd: 3rd molar, Am: ameloblasts, Od: odontoblasts, D: den¬tin, E: enamel. Propidium iodide (red color) was counterstained for nuclear morphology.

Discussion

The tooth is formed by ectoderm-derived oral epithelium and neural crest-derived ectomesenchymal cells. Its develop­ment undergoes morphological changes from bud, cap, and bell stages to the root formation that are controlled by various regulatory factors [3]. Osteonectin detected in this study can be one of the factors regulating tooth development.  

 Osteonectin, also known as SPARC (secreted protein acidic and rich in cysteine), was first identified by Mason et al. [9] (1986) and is an extracellular matrix-associated glycoprotein. Osteonectin is a "culture shock" glycoprotein separated from proliferative bovine aortic endothelial cells, and is 43 kDa in size [10]. Osteonectin was established to be BM-40, which is produced from the basement membrane and induces EHS tumors [9,11]. The C-terminal (COOH) extracellular Ca2+ ­binding domain of osteonectin binds to the cells and matrix [12,13].

 The established classical function of osteonectin is a Ca­binding protein in the development of ossified and miner­alized tissues [14]. In addition to this function, studies in cultured cells have suggested a potential role in tissue morphogenesis and cellular differentiation [15,16]. Although a cell-surface receptor for osteonectin has not been identified, it can block cell-cell and cell-matrix interactions and inhibit cell migration and chemotaxis. Osteonectin also appears to function inside cells. Intracellular osteonectin is associated with axonemal tubulin in ciliated epithelial cells [17]. In this study, osteonectin expression was detected in the 3rd molar germs at the cap stage (cap stage, before mineralization and differentiation). The level of osteonectin expression was much higher in the 2nd molar germs than in the 3rd molar germs at postnatal day 9. Moreover, the level increased remarkably at postnatal days 9 (root formation stage, during mineralization and differentiation of odontogenic cells) and ­6 (crown stage, during mineralization and differentiation of odontogenic cells), compared to that at postnatal day 3 (early bell stage, before mineralization and differentiation) in the 2nd molar germs. These findings cannot exclude a possibility that osteonectin may be involved in the differentiation of odontogenic cells.

 By immunofluorescency, osteonectin was localized in odontoblasts, suggesting that osteonectin is derived from them. The secreted form of osteonectin can bind to ex­tracellular matrix of dentin via the property of C-terminal extracellular Ca2+-binding domain. Even though the re­activity was not strong, osteonectin was also localized in perifollicular cells in the molar germs at postnatal day 9. This finding is consistent with a suggestion that osteonectin may play a role in maintaining homeostasis of the collagenous extracellular matrix in the periodontal ligament [18].

 This study suggests that osteonectin may regulate tooth development. However, in addition to the involvement of dentin matrix formation, further investigations are needed for the involvement of odontoblasts differentiation.

Acknowledgements

This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0009949) and a grant (CUHRICM­dentistry-2010) from Chonnam National University hospital research institute of clinical medicine.