• Introduction
• Materials and Methods
• Culture of human dental pulp cells
• RAW264.7 Cell culture
• FACS analysis
• Alkaline phosphatase activity assay
• Alizarin red S staining
• RT-PCR
• DD-PCR
• Results
• Stem cell population in culture of human dental stem cells
• Differentiation potential of human dental pulp stem cells with additives
• Morphological phenotypes in human dental pulp stem cells with additives
• mRNA expression patterns of osteoblastic/odontobla-stic markers in human dental pulp stem cells
• Comparison of hDPSC and odontoblast-like cells samp-les with DD-PCR
• Discussion
• Acknowledgements

Introduction

 During mammalian tooth development, a complex series of interactions occur between the oral epithelium and neural crest-derived ectomesenchyme [1-3]. In the initial stage of tooth development, the process begins with odontogenic diffe-rentiation of ectomesenchyme, which is induced by signals from the oral epithelium. Thereafter, tooth morphogenesis is regulated by sequential and reciprocal interactions between the epithelial and mesenchymal tissues. Such interactive pro-cesses between oral epithelium and ectomesenchyme are mediated by a variety of macromolecules of the extracel-lular matrix together with diffusible signaling molecules, and continue until a tooth is formed [2-4]. Accumulating evi-dence from in vitro and  in vivo experiments has demons-trated that paracrine signaling molecules, including bone mor-phogenetic proteins (BMPs), fibroblast growth factors (FGFs), sonic hedgehog (Shh), Wnts, tumor necrosis factors (TNFs) and activin, play important roles in the sophisticated epit-helial–-mesenchymal interaction of the tooth development [2-7].

 After injury to the tooth, the dentin-pulp complex may need to undergo complete regeneration, including the dif-ferentiation of various cell types and the induction of new proteins. For the regenerative processes, odontoblast differen-tiation in the pulp tissue may be induced followed reparative dentinogenesis [8,9]. Dentin regeneration is mediated by the differentiation of a new generation of odontoblasts from a precursor population of dental pulp stem cells(DPSCs) [10]. DPSCs are a mesenchymal stem cell (MSC) population that is present in the cell-rich zone and core of the pulp. These cells have the ability to differentiate into odontoblast-like cells, pulpal fibroblasts, adipocytes, and neural-like cells [11]. With conventional molecular biological approaches, some signa-ling pathways, gene expression, and protein activities have been identified in the process of dentinogenesis [12-17].

 Primary hDPSCs maintain their stem cell properties and continue to express several CD markers [18-21]. In the pre-sence of dexamethasone, β-glycerophosphate (β-GP), and inor-ganic phosphate, hDPSCs are capable of differentiating into an odontoblast-like lineage and expressing alkaline phosp-hatase. This transition is accompanied by deposition and mine-ralization of collagenous matrix [22-23].

 While hDPSCs have great potential for dentin regenera-tion and tooth repair, the molecular mechanisms controlling odontoblast differentiation are yet to be fully understood. The identification of the regulatory factors governing these pro-cesses will enhance our understanding and open up potential treatment strategies for dental pulp regeneration.

 In this study, the final goal was to identify differentially ex-pressed genes at the early differentiation stage of odontoge-nesis to find candidate genes involved in odontoblast differen-tiation. As an initial step, the application of the technique of DD-PCR allowed the comparison of gene expression patterns between multiple mRNA populations.

Materials and Methods

Culture of human dental pulp cells

 Healthy human third molars were collected from patients aged 20-25 years at the Department of Oral and Maxillofa-cial Surgery, under approved guidelines in IRB of the Wonk-wang Dental Hospital. Soon after the extraction, tooth sur-faces were cleaned and cut around the cementoenamel junc-tion by using sterilized dental fissure burs to reveal the pulp chamber. The pulp tissue was gently separated from the crown and root and then digested in a solution of 3 mg/ml collagenase type I (Wako Pure Chemicals, Osaka, Japan) and 4 mg/ml dispase I (Sigma, St. Louis, MO, USA) for 1 h at 37℃. The dental pulp sample from each individual was poo-led, and single-cell suspensions were obtained by passing the cells through a 70-µm strainer (Falcon, Franklin Lakes, NJ, USA). Single-cell suspensions (2.5×10⁵ cells/well) of dental pulp were seeded into 10-cm culture dishes (Coster, Cam-bridge, MA, USA) with α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 20% FBS (Invitrogen, Carlsbad, CA, USA) and 100 units/ml penicillin/ 100 µg/ml strepto-mycin (Invitrogen, Carlsbad, CA, USA) and then incubated at 37℃ in 5% CO₂. The Aggregates of clonogenic cell popu-lations were isolated and seeded into 6-well plates (Coster, Cambridge, MA, USA). The adherent cells were grown to 70% confluency and were defined as passage zero. When cells were grown 70% confluency, they were sub-cultured at 1/5 dilution for later passaging. Media was replaced every 3 days till growing to proper confluency. For differentiation, 5mM β-glycerophosphate (Sigma, St. Louis, MO, USA) and 100 µm ascorbic acid (Sigma, St. Louis, MO, USA) were added into culture media as additives, and cells were incubated for 14-42 days.

RAW264.7 Cell culture

 RAW264.7, a murine macrophage/monocyte lineage cell line, was obtained from ATCC (ATCC no.: TIB-71), and cultu-red with α-MEM (Invitrogen, Carlsbad, CA, USA) supple-mented with 10% heat-inactivated FCS (HyClone, Logan, UT, USA) and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).

FACS analysis

 Approximately 1 × 10⁶ hDPSCs were collected, washed with 1x PBS. The hDPSCs and antibodies against FITC-conjugated CD24 and FITC-conjugated CD44 incubated at room temperature for 30 min. The antibody-cell mixture was washed with 1X PBS and centrifuged at 400 x  g for 5 min. The hDPSCs was then resuspended in 1x PBS. FACS analysis was carried out using BECKMAN COULTER XL system II according to the manufacturer's instructions (Beck-man Coulter, Fullerton, CA, USA).

Alkaline phosphatase activity assay

 hDPSCs were plated at 1×10⁴ cells per cm² in 24-well plates (Coster, Cambridge, MA, USA) in  α-MEM (Invit-rogen, Carlsbad, CA, USA)  containing 10% FBS (Invit-rogen, Carlsbad, CA, USA) and 100 units/ml penicillin/ 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) until 60% confluence and serum-starved for 24 hours. The ad-ditives was added in the experimental groups, while the controls were not treated with the additives. Cells were cul-tured for 7, 14, 21 and 42 days. Alkaline phosphatase (AL-Pase) activity was determined using cell lysates. Aliquots of each sample were incubated with ALPase substrate (Sigma, St. Louis, MO, USA) for 30 minutes at room temperature. The absorption at 405 nm was measured with a spectrop-hotometer (Bio-Rad Laboratories, Hercules, CA, USA). Total cellular protein was determined by BCA protein assay kit (Pierce Chemical Co., Rockford, IL, USA). ALPase activity (IU/L) was defined as the release of 1 nmol p-nitrophenol per minute per g of total cellular protein.

Alizarin red S staining

 Cells were cultured and stimulated for differentiation as des-cribed. Cells were fixed with 70% ethanol for 5 min at room temperature, and then allowed to dry completely. For stai-ning, cells were treated with 2% alizarin red S (pH 4.5) for 1 min, then washed with water.

RT-PCR

 The total RNA was extracted from hDPSCs using Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. Aliquots of the same RNA used for the DD-PCR were used for reverse transcription-polyme-rase chain reaction (RT-PCR). For the RT and the PCR reac-tion, AccPower RT PreMix (Bioneer, Daejeon, Korea) and AccPower PCR PreMix (Bioneer, Daejeon, Korea) were used. The RT and PCR reaction was carried out on a Takara PCR thermal cycler (Takara, Shiga, Japan). Table 1 lists the pri-mer sequences and expected product sizes. The PCR pro-ducts were visualized with ethidium bromide and sequenced for confirmation. The product size was checked using a 100 bp marker (Takara, Shiga, Japan).

Table 1. Primer sequences for PCR

DD-PCR

A DD-PCR was performed using a GeneFishing DEG kit 101 (Seegene, Rockville, MD, USA). Briefly, combinations of deoxythymidine-Annealing Control primer 2 (dT-ACP2) with 120 ACPs were made arbitrarily. For the reverse transcription of the first-strand cDNA, the reaction was started by incubating the reagents composed of the total RNA and dT-ACP1 at 80℃  for 3 min. The reactant was added to the mixture of dNTP, RNase inhibitor and Mo-loney Murine Lekemia Virus (M-MLV) reverse transcrip-tase, followed by incubation at 42℃ for 90 min and subse-quent heating at 92℃  for 2 min. The DD-PCR was per-formed on a Takara PCR thermal cycler (Takara, Shiga, Japan). Mixtures of first-strand cDNA, 1x buffer without MgCl₂, 2.5 mM MgCl₂, 0.2 M arbitrary ACP, 0.2 M dT-ACP2, and 0.2 mM dNTP, in a final concentration, were made up to a 50 ㎕ volume. Hot-start PCR was performed by preheating the mix-tures at 94℃ with the subsequent addition of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The PCR reac-tion was started by incubation for 3 min at 94℃, followed in succession by 3 min at 50℃, and 1 min at 72℃. Subse-quently, 40 cycles of 40 s at 94℃, 40 s at 65℃, and 40 s at 72℃ were then performed. The products were resolved on a 1.2% agarose gel and stained with ethidium bromide.

Results

Stem cell population in culture of human dental stem cells

 Fluorescence-activated cell sorting (FACS) analysis was performed to determine the pattern of expression of the stem cell surface marker proteins such as CD24 and CD 44 in sub-cultured dental pulp cells (5th passage). CD24 is known as one of the representative  stem cell surface marker pro-teins in stem cells from dental apical papilla and CD44 is a general cell surface marker protein to define mesenchymal stem cells. As shown Fig. 1B, a phenotypical analysis of hDPSCs cultivated in 20% FBS medium showed high positivity for CD24 (99.6 %) and CD44 (99.6 %) (Fig. 1B). Raw 264.7 cells were used as negative controls (Fig. 1A).

Fig. 1. Stem cell surface marker protein expression of Raw 264.7 cells and hDPSCs. In FACS analysis of (A) Raw 264.7 cells and (B) hDPSCs for the expression of stem cell surface marker protein, CD24 and CD44, hDPSCs were positive for the stem cell markers CD24 and CD44, however Raw 264.7 cells were negative for CD24 and CD44 (FI ; Fluorescence Intensity)

Differentiation potential of human dental pulp stem cells with additives

 To investigate the potential of hDPSCs undergoing odon-toblast-like differentiation under treatment of differentiation additives, the ALP staining and ALP activity were assessed. ALP staining and ALP activity were markedly stimulated by treatment of differentiation additives; a four-fold increase was observed after 14 days. This stimulation reached a maxi-mum value after 21 days with a significant increase of about 6 fold (Fig. 2).

Fig. 2. ALP assay in hDPSC after odontogenic differentiation. The ALP activity and ALP staining induced by the differentiation media at 14 days of culture. The cells were grown in the presence of ascorbic acid and β-glycerophosphate for inducing the mineralization.

Morphological phenotypes in human dental pulp stem cells with additives

 To examine whether the nodule-like structure and depo-sits materials produced in long-term cultures in the presence of additives were consisted with high amount of calcium or not, hDPSCs were stained with alizarin red solution. As ex-pected, calcified nodules were scattered throughout the culture as several mineralized zone at 42 days (Fig. 3).

Fig. 3. Mineralized nodule formation in hDPSC after odon-togenic differentiation. The mineralized nodule formation in-duced by the differentiation media at 42 days of culture. The cells were grown in the presence of ascorbic acid and β-gly-cerophosphate for inducing the mineralization. The nodules were detected by Alizaline red-S staining.

mRNA expression patterns of osteoblastic/odontobla-stic markers in human dental pulp stem cells

 The changes of transcriptional expression of the cellular markers (dentin sialophosphoprotein and osteocalcin; DSPP and OC) representing osteogenic/odontoblastic differentia-tion were analyzed by RT-PCR. After the odontogenic media treatment in hDPSCs, the expression of the OC known as the early expression gene among osteogenic mar-kers was highly up-regulated at 14 days after differentiation. The transcriptional expresson of dentinogenic marker, DSPP was slightly increased at 21 days when hDPSCs were stimu-lated with additives (Fig. 4).

Fig. 4. DSPP and OC mRNA expression in hDPSCs after odon-togenic differentiation. Thranscripts for DSPP, OC and gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) were detec-ted by RT-PCR by using total RNA isolated from in hDPSCs at each day. the level of DSPP mRNA expression was slightly increased at 21 days after odontogenic induction. OC was also up-regulated at 14 days after odontogenic induction (0; 0 day after treatment with differentiation media, 7; 7 days after treat-ment with differentiation media, 14; 14 days after treatment with differentiation media, 21; 21 days after treatment with dif-ferentiation media)

Comparison of hDPSC and odontoblast-like cells samp-les with DD-PCR

 DD-PCR was performed to search for genes expressed differentially between hDPSCs and odontoblast-like cells. Several differentially expressed genes were detected bet-ween hDPSCs and odontoblast-like cells using combina-tions of ACPs. These genes were divided in three groups; Group 1 genes were up-regulated during odontogenic diffe-rentiation, wherease group 2 genes were down-regulated in hDPSCs (Fig. 5). Group 3 genes were unchanged during odontogenic differentiation. In group 1, One of them, which was detected using the specific primer ACP 92, was stron-gly expressed in odontoblast-like cells and was approxi-mately 400bp in size (Fig. 5B). On the contrary, when the specific primer ACP 81 was used, a band of approximately 350bp was detected in both hDPSCs and odontoblast-like cells. This band was exhibited in the decreasing mRNA expres-sion patterns during odontogenic differentiation (Group 2, Fig. 5A).

Fig. 5.The Gel image Acquired from DD-PCR using the specfic primer ACP 81 and ACP92. (A) A detected band using the specific primer ACP 81 was down-regulating tendency between hDPSCs and odontoblast-like cells. A expressed band was approximately 350bp in size (arrow). (B) A detected band using the specific primer ACP 92 was strongly found in odontoblast-like cells and was approximately 400bp in size(arrow). The molecular marker is a 100bp ladder. (0; 0 day after treatment with differentiation media, 14; 14 days after treatment with differentiation media, 21; 21 days after treatment with differentiation media)

Discussion

 The aim of this study is to identify expressed genes at the early differentiation stage of odontogenesis. The present study exhibit, for the first time, the differential mRNA expression patterns of hDPSC using the technique of DD-PCR on 14 and 21 days of odontogenic induction compared with non- induced control. It is possible that expression changes of mRNA may be the involvement of various regulation mecha-nism in odontoblast-like differentiation including cell cycle, protein synthesis, signal transduction and so on.

 At the 5th passage in hDPSC culture, FACS analysis was used to determine the expression pattern of CD24 and CD44. The data was shown that hDPSCs cultivated in  α-MEM supplemented with 20% FBS showed high positivity for stem cell markers (CD 24 and CD 44) (Fig. 1). Through FACS analysis, hDPSCs was available for odontoblast-like differentiation as stem cells.

 To determine whether hDPSCs following treatment with the additives such as β-GP and AA could be differentiated into odontoblast-like cells, next experiment explored the ALP activity assay and AR-S staining. As expected, the ALP acti-vity in hDPSCs after odontogenic differentiation was mar-kedly increased (Fig. 2) and calcified nodules were scattered throughout the culture as several mineralized zones (Fig. 3). Therefore, this result was consistent with the concept that treatment of the additives in hDPSC promotes the odonto-genic differentiation in cell culture.

 In order to characterize odontoblast-like cells further, osteo-blastic and/orodontoblastic differentiation markers in hDPSC after odontogenic differentiation was investigated. In RT- PCR analysis, hDPSC after odontogenic differentiation expres-sed transcripts for the DSPP and OC (Fig. 4). DSPP in pulp is dentin-specfic and expressed mostly by odontoblast cells. Fur-thermore, DSPP is considered as a terminal phenotypic hal-lmark of mature odontoblasts. Therefore, this data suggest the possibility that hDPSC at 14 and 21 days after odon-togenic differentiation might be odontoblast-like cells.

 Several methods for comparison of two samples are used for finding potential biomarkers and studying the mechanis-ms involved in cell differentiation. In this study, DD-PCR approach was used to examine the differential mRNA expres-sion in the early process of odontoblast-like differentiation of hDPSCs. The changes of mRNA expression were displayed by DD-PCR, which were either upregulated or downregulated during odontogenic differentiation (Fig. 5). These results show that DD-PCR tool is capable of finding the meaningful gene in the process of odontoblast-like differentiation.

Therefore, through the identification and characterization of these genes, further studies will lead to a more in-depth understanding of the biological processes related to odonto-blast-like differentiation and reparative dentinogenesis. 

Acknowledgements

 This paper was supported by Wonkwang university in 2012.