FGF-2 was obtained from R&D Systems (Minneapolis, MN, USA). Anti-CD31 monoclonal antibody for the Western blot analysis was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The dental pulp (DP) tissues were obtained from the crown and root of healthy human deciduous teeth from three donors, aged 7–8 years. Informed consent was obtained from the donors’ parents prior to tooth extraction, which was carried out in our hospital during the course of orthodontic treatment. The study protocol was approved by the Ethics Committee of Iwate Medical University, School of Dentistry (no. 01101).

DP tissues were cut into pieces using a surgical blade and digested with collagenase (2 mg/ml) at 37°C for 30 min. The tissues were then washed with Dulbecco’s phosphate-buffered saline (PBS), placed on culture dishes and maintained in α-modified minimum essential medium (α-MEM) (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL). Fibroblastic cells that outgrew from the DP tissues were used as DP cells. When the cells reached confluence, they were detached with 0.2% trypsin and 0.02% EDTA (4 Na) in PBS and subcultured at a 1:4 split ratio. Experiments were performed using 4th passage cells cultured in α-MEM supplemented with 10% FBS in the absence or presence of 10 ng/ml FGF-2 for 2 days. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air.

Total RNA was extracted from the cultured DP cells using Isogen (Nippon Gene, Tokyo, Japan) as previously described (11,12). The pellet of total RNA was washed briefly with 75% ethanol, resuspended in 30 μl of diethylpyrocarbonate (DEPC)-treated water, and stored at −80°C. The concentration of total RNA was determined spectrophotometrically by measuring the optical density at 260 nm.

Using a PrimeScript RT reagent kit (Takara Shuzo, Kyoto, Japan), 1 μg of RNA sample was reverse-transcribed to first-strand cDNA according to the manufacturer’s protocol. A Thermal Cycler Dice Real-Time system (Takara Shuzo) was used for the two-step reverse transcription-polymerase chain reaction. cDNA was amplified with SYBR Premix ExTaq and specific oligonucleotide primers for target sequences encoding parts of VE-cadherin, VEGFR2 and CD31. The primers (listed in Table 1) were designed based on the cDNA sequences of human mRNA for VE-cadherin, VEGFR2, CD31 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Amplification conditions consisted of 10 sec at 95°C followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec, with a final 15 sec at 95°C and 30 sec at 60°C in the Thermal Cycler Dice Real-Time system.

After treatment with FGF-2 for 21 days, DP cells were washed twice with PBS and then treated with lysis buffer [10 mM HEPES-KOH (pH 7.5), 100 mM KCL and 0.1% NP-40]. Protein concentration in the cell lysate was measured using a BioRad Protein Assay kit (BioRad, Hercules, CA). Each sample containing equal amounts of protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After being blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), the membrane was incubated with mouse anti-human CD31 antibodies and subsequently with anti-mouse secondary antibodies (Zymed Laboratories Inc., San Francisco, CA). Specific protein bands on the membrane were detected using an enhanced AP conjugate substrate kit (BioRad Laboratories, CA) as previously described (11,12).

Results are expressed as the means ± SEM. Statistical significance was determined by one-way analysis of variance and Bonferroni comparisons between pairs of groups. Data with P-values <0.01 were considered statistically significant.

Using phase-contrast microscopy, DP cells were observed to differentiate into various types of cells 10 days after isolation from DP tissues (Fig. 1). Most of the DP cells derived from deciduous teeth exhibited a fibroblastic cell morphology (Fig. 1A). Some of the cells showed polygonal-like epithelial cell and mature osteoblast morphologies (Fig. 1A, arrow). A few cells showed a senescent fibroblastic cell-like morphology (Fig. 1A, arrowhead). After reaching confluence and undergoing subculture, it was no longer possible to distinguish between these cell morphologies (Fig. 1B).

DP cells were cultured in control media (Fig. 2A) or in the presence of FGF-2 (Fig. 2B), and reached subconfluence after 2 days of culture. FGF-2-treated DP cells reached confluence and exhibited an altered morphology of long and thin spindle-shaped fibroblasts (Fig. 2B).

In DP cells cultured in the presence of FGF-2, VE-cadherin and VEGFR2 mRNA expression was increased, though not significantly (Fig. 3A and B). However, CD31 expression was significantly induced by treatment with FGF-2 (Fig. 3C).

After three weeks of culture with FGF-2, DP cells reached confluence and exhibited a long and thin spindle-shaped fibroblastic morphology (Fig. 4B) compared to the cells cultured in control media (Fig. 4A).

To determine whether CD31 protein was induced in DP cells cultured with FGF-2 (Fig. 5), Western blot analysis was conducted. CD31 expression was not detected after 2 days of treatment with FGF-2 (data not shown), nor after 2 weeks of culture. After treatment with FGF-2 for 3 weeks, CD31 expression was detected by Western blot analysis as compared with the control (Fig. 5A). Therefore, the production of CD31 was induced in DP cells by FGF-2 treatment (Fig. 5B).