Cell culture medium and reagents were purchased from Gibco-BRL (Grand Island, NY, USA). Other experimental reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA), unless otherwise specified.

RP cells were obtained from the palatal tissues of 5-week-old male Sprague-Dawley (SD) rats. Isolated palatal tissues were washed with phosphate-buffered saline (PBS), minced into sections, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotic solution (100 U/ml of penicillin-G and 100 μg/ml of streptomycin) at 37°C in a 5% CO₂ humidified incubator. After 20 days of culture with medium changes every 3 days, RP cells were collected and subcultured under the same conditions. Passages 5–8 were used for the present study.

Adhesion and proliferation of RP cells on the polystyrene surface of culture plates were observed in the presence of Tβ₄. For the adhesion assay, 1×10⁴ palatal cells were incubated in the wells of a 96-well plate with Tβ₄ at various concentrations of 1–1,000 ng/ml for 4 h. The wells were gently washed three times with PBS, and the number of attached cells was quantified using 2-(2-methoxy-4-nitropenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8; Dojindo Laboratories, Kumamoto, Japan). The cells were incubated in 100 μl of WST-8 solution for 1 h at 37°C in a humidified atmosphere (5% CO₂/95% air). The absorbance was measured at a wavelength of 450 nm using a plate reader (Sunrise; Tecan Austria GmbH, Salzburg, Austria). To observe proliferation, palatal cells were treated with Tβ₄ for 24 h, and the number of cells was measured by using WST-8.

For the in vitro wound closure assay, a culture insert (ibidi GmbH, Martinsried, Germany) was employed to create a wound in cell culture. The culture insert was placed on a culture dish, and 70 μl of RP cell suspension (5×10⁵ cells/ml) was added into the two wells of the insert. The RP cells were incubated at 37°C for 18 h, and were serum-starved for 24 h. Following serum starvation, the culture insert was carefully removed, and the cells were exposed to Tβ₄ at various concentrations of 0–1,000 ng/ml. The wound closure was observed and recorded at intervals under a phase contrast microscope (Olympus, Tokyo, Japan). This experiment was replicated three times.

To investigate the effect of Tβ₄ on the mRNA expression of genes related to cell migration and angiogenesis, mRNA expression of MMP2 and VEGF was analyzed by quantitative polymerase chain reaction (RT-qPCR) assay. Following serum starvation for 24 h, the palatal cells were treated with Tβ₄ for 6 and 24 h and the total RNA was isolated using RNA extraction reagent (WelPrep Total RNA isolation reagent; Welgene Inc., Daegu, Korea). From the total RNA, cDNA was prepared using a cDNA synthesis kit (Power cDNA Synthesis kit; Intron Biotechnology, Seongnam, Korea) and RT-qPCR was performed in an ABI PRISM 7500 Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA) with 20 μl reaction volumes containing 10 μl SYBR Premix Ex Taq (Takara Bio, Otsu, Japan), 0.4 μl ROX reference dye II (Takara Bio), cDNA, and primers. The primers for gene amplification were: VEGF, sense: 5′-GAGTATATCTTCAAGCCGTCCTGT-3′ and antisense: 5′-ATCTGCATAGTGACGTTGCTCTC-3′; MMP2, sense: 5′-CAGGGAATGAGTACTGGGTCTATT-3′ and antisense: 5′-ACTCCAGTTAAAGGCAGCATCTAC-3′; GAPDH, sense: 5′-TGTGTCCGTCGTGGATCTGA-3′ and antisense: 5′-CCTGCTTCACCACCTTCTTGAT-3′. The PCR conditions were 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C (34 sec) for MMP2 and 63°C (34 sec) for VEGF. The reactions were run in triplicate. Gene expression was evaluated based on the threshold cycle (Ct value) and normalized to the amount of GAPDH transcript.

Western blot analysis was performed to examine the protein expression of MMP2 and VEGF in Tβ₄-treated palatal cells. After treatment with Tβ₄ for 6 h, the cells were centrifuged and re-suspended in an extraction buffer containing 50 mM Tris base-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, and 1 tablet of protease inhibitor cocktail (1 tablet/10 ml; Roche Applied Science, Mannheim, Germany) for 45 min on ice. Extracts containing equal amounts of protein were run on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were incubated with rabbit polyclonal antibodies against VEGF, MMP2 or GAPDH in PBST (PBS containing 0.1% Tween-20) for 1.5 h, washed three times with PBST, and then probed with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase. The antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The blots were developed using a chemiluminescence kit (West-Zol plus western blot analysis detection system; Intron Biotechnology). Chemiluminescence was detected using the LAS 1000 Plus Luminescent Image Analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).

The effect of Tβ₄ on the wound healing of palatal tissue was investigated in a RP wound model. Thirteen-week-old SD male rats, weighing 300–350 g, were used in this study. Animal experiments were performed under the control of the animal welfare committee of Seoul National University Institutional Animal Care and Use Committee. Under general anesthesia, punch wounds were made on a central area of hard palate with a disposable 3-mm diameter biopsy punch (Kai Industries Co., Ltd., Gifu, Japan), exposing a circular area of bare bone. The wound area was covered with 30% CMC ointment containing 0 or 1 mg/ml Tβ₄. Six rats were used for each group. After the surgery, the animals were fed a standard diet of pellets and water with enrofloxacin. The agents were re-applied on day 2 and 4 to reduce stress by anesthesia, and the rats were sacrificed on day 7. The maxillae were separated, and wound area was observed by stereoscopic microscope (Nikon, Tokyo, Japan) and by histological analysis. The wound areas in the microscopic images were calculated using CellSense Dimension 1.6 software (Olympus).

The palatal specimens were fixed in 10% formalin for at least 24 h, decalcified in Calci-Clear Rapid solution (National Diagnostics, Atlanta, GA, USA) for 35 h, and processed for histological analysis. Serial sections, 5 μm apart, were cut across the wound, perpendicular to the palatal midline at the widest diameter of the wound, and stained with hematoxylin and eosin (H&E). The sections were examined under a light microscope (Olympus), and the distance of wound margins in each section was measured with a calibrated ocular micrometer (Olympus).

Each experiment was performed in triplicate unless otherwise specified. Data were presented as the mean ± SD. Statistical analyses were performed by the Student’s t-test. P<0.05 was considered statistically significant.

To investigate the effects of Tβ₄ on the adhesion and proliferation of palatal cells, the cells were incubated for 4 and 24 h in the presence of Tβ₄. At concentrations of 1–1,000 ng/ml, Tβ₄ did not exert any significant effects on the adhesion and proliferation of palatal cells (data not shown), whereas cell migration was affected (Fig. 1). Untreated control cells did not exhibit any movement during 24 h, possibly due to the serum-starved test conditions. However, cell movement was observed when Tβ₄ was present at concentrations >100 ng/ml. The migration effect of Tβ₄ was more rapid at the higher concentrations and cell motility was observed at 12 h when incubated with 1,000 ng/ml Tβ₄.

The mRNA expression of MMP2 and VEGF was quantified to investigate the effects of Tβ₄ on cell migration and angiogenesis at molecular levels. As shown in Fig. 2, Tβ₄ enhanced the gene expression of MMP2 and VEGF after 6 h. The expression of MMP2 in the Tβ₄-treated group was significantly higher than that in the untreated cells. Although the expression did not increase in a dose-dependent manner, the highest concentration of Tβ₄ (1,000 ng/ml) led to the strongest induction of MMP2. Tβ₄ also enhanced mRNA expression of the VEGF gene in RP cells, inducing an ~1.4-fold increase at 1,000 ng/ml at 6 h. A further increase of VEGF mRNA was obtained when exposed for 24 h. However, the expression level of the MMP2 gene was not maintained over the 24-h period, with expression levels decreasing below those of the untreated control (Fig. 3).

The protein expression of MMP2 and VEGF was determined by western blot analysis. RP cells were treated with Tβ₄ for 6 h at various concentrations. The results showed that Tβ₄ upregulated the expression of VEGF and MMP2 proteins in a dose-dependent manner (Fig. 4). The quantitative measurement of VEGF and MMP2 protein showed that treatment with Tβ₄ (1,000 ng/ml) significantly enhanced the level of VEGF protein by 2.1-fold, and enhanced the level of pro-MMP2 and active MMP2 by 3.8- and 1.3-fold, respectively, when compared to the control (Fig. 4).

The wound healing effect of Tβ₄ was observed seven days after surgery, whereas the palatal wound gap was completely closed <2 weeks in the untreated rats (Fig. 5A). As shown in Fig. 5B, the wound area was not completely epithelized in either the control or the test groups. However, microscopically smaller wound areas were observed in the Tβ₄-treated test group, indicating that more advanced epithelization at the wound margin had occurred in the Tβ₄-treated rats (Fig. 5B). The mean area of unepithelized surface in the Tβ₄-treated rats was significantly smaller than that in the control group (Fig. 5C). The histological examination also demonstrated an accelerated epithelization by Tβ₄ (Fig. 5D). The mean distance between the epithelized margin at the center of a palatal wound was ~1,280 μm in Tβ₄-treated rats, while 1,830 μm of the wound remained unepithelized, on average, in the control group (Fig. 5E). Therefore, we have clearly demonstrated that Tβ₄ significantly accelerated the epithelization of RP wounds.