Background
Oral soft tissues are inevitably damaged during autogenous gingival grafting, periodontal surgery, or dental implant surgery. Recovery of soft tissue from injuries depends on wound size, location, condition of oral hygiene, and the level of immunity. Oral administration or topical application of antibiotics is recommended as a choice of oral wound care to prevent bacterial infection. In the case of large-sized defects that can be caused by autogenous gingival grafts, dressing materials are used to protect damaged donor sites and to help the wound healing process. Intended rapid wound healing can also reduce the opportunity of infection and discomfort of patients. In a previous study, acceleration of palatal mucosa repair was achieved with a collagen-gelatin scaffold retaining basic fibroblast growth factor (bFGF), a potent angiogenesis inducer [
1]. Thymosin β
4 (Tβ
4), an oligopeptide that can sequester G-actin monomers, also promotes wound healing of rat mucosa [
2]. Enhancement of cell migration and angiogenesis is likely to be involved in wound healing by Tβ
4. Umeki et al. have shown that wound healing of oral mucosa is accelerated by leptin, a circulating anti-obesity hormone, via enhancement of angiogenesis [
3]. These studies imply that enhancement of angiogenesis is an effective way to accelerate oral wound repair.
Angiogenesis can be stimulated by various growth factors such as fibroblast growth factor (FGF) [
4], vascular endothelial growth factor (VEFG) [
5], platelet-derived growth factor (PDGF) [
6,
7], and transforming growth factor β (TGF-β) [
8,
9]. Several peptides have also been reported to promote angiogenesis [
10‐
12]. Considering the importance of angiogenesis in wound healing, the application of angiogenic proteins and peptides to wounds is expected to expedite the repair of oral tissue damage if the molecules are properly delivered to the wound sites. However, the stability and activity of proteins and peptides cannot be assured in the oral environment where temperature and pH are changeable due to eating and drinking. Instead, angiogenic small molecules which are chemically stable can be more appropriate for the harsh conditions of the oral cavity.
Dimethyloxalylglycine (DMOG), with small molecular weight, is a cell-permeable unspecific inhibitor of prolyl hydroxylases (PHDs) [
13]. Under normoxic conditions, PHD2 is known to hydroxylate specific proline residues in HIF-1α, which then leads to degradation of HIF-1α by the ubiquitin-proteasome pathway [
14‐
16]. Therefore, inhibition of PHD2 by DMOG can prevent HIF-1α degradation, creating an environment similar to that found in hypoxic cells. Furthermore, various genes related to tissue repair such as angiogenesis are upregulated. A previous study showed that DMOG enhanced the production of VEGF in periodontal fibroblast cells [
17]. Furthermore, Botusan et al. demonstrated that DMOG stabilizes HIF-1α and enhances dermal wound healing in diabetic mice [
18]. In this study, the effect of DMOG on oral wound healing was investigated in a rat palatal wound model. We also evaluated the effects of DMOG on cell migration and HIF-1α expression of rat palatal (RP) cells. Furthermore, mRNA and protein expression of vascular endothelial growth factor (VEGF) were analyzed in DMOG-treated RP cells.
Methods
Chemical reagents and cell culture
Cell culture medium and antibiotics were purchased from WELGENE Inc. (Daegu, Korea). Fetal bovine serum (FBS) was purchased from Lonza (Basel, Switzerland), and other experimental reagents were obtained from Sigma-Aldrich Co. (Saint Louis, MO, USA), unless otherwise specified.
RP cells were isolated from the palatal tissues of 5-week old male Sprague–Dawley (SD) rats. Isolated palatal tissues were washed with phosphate buffered saline (PBS) (PH 7.4), aseptically minced into pieces, and then immersed in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and antibiotic solution (100 U/ml of penicillin-G and 100 μg/ml of streptomycin) at 37 °C in a humidified incubator (5% CO2/95% air). After 20 days of culture with medium changes at 3-day intervals, RP cells were collected and sub-cultured under the same conditions. Passages three through five were used for this study. Although we did not identify the type of palatal cells at the molecular level, morphological features of the isolated cells under the microscope indicated distinct dominance of fibroblast-like cells.
Cytotoxicity
RP cells incubated until 80% confluent were removed and sub-cultured at 0.8 × 105 cells per ml in 96-well plates and incubated at 37 °C with 5% CO2 in air for 24 h, and then the cells were treated with DMOG at various concentrations ranging from 0.1 to 10 mM. After treatment for 24 h, the cell viabilities were quantified using the Cell Counting Kit-8 (WST-8) (Dojindo Laboratories, Kumamoto, Japan). 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, Salzburg, Austria). The optical density of untreated cells was designated as 100%, and cell viability of treated cells was expressed as the percentage of untreated negative control.
Cell migration assay
For the in vitro cell migration assay, a culture insert (ibidi GmbH, Martinsried, Germany) was used 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 both wells of the insert. The RP cells were incubated at 37 °C for 48 h and then exposed to DMOG (0, 0.1, 0.5, 1, 2 mM) in culture media containing 2% FBS for the cell migration analysis. Wound closure was observed and recorded at intervals under a phase contrast microscope (Olympus, Tokyo, Japan). To quantify cell migration, the uncovered area where no cells were present was measured by using ImageJ program, and the ratio of uncovered area between untreated control and treated groups was obtained.
mRNA expression analysis by real-time PCR
The effect of DMOG on the expression of VEGF mRNA was investigated by real-time polymerase chain reaction (RT-PCR) assay. After treatment with DMOG at 0, 0.1, 0.5, 1, and 2 mM for 24 h, total RNA was isolated using RNA extraction reagent (WelPrep Total RNA Isolation Reagent, WELGENE Inc.). From the total RNA, cDNA was prepared using a cDNA synthesis kit (Power cDNA Synthesis Kit, iNtRON Biotechnology, Sungnam, Korea), and RT-PCR 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 as follows: VEGF sense, 5’-GAGTATATCTTCAAGCCGTCCTGT-3’; VEGF antisense, 5’-ATCTGCATAGTGACGTTGCTCTC-3’; GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) sense, 5’-TGTGTCCGTCGTGGATCTGA-3’; GAPDH antisense, 5’-CCTGCTTCACCACCTTCTTGAT-3’. The PCR conditions were 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 63 °C (34 s) for VEGF. All reactions were run in triplicate. Gene expression was evaluated on the basis of the threshold cycle (CT value) and normalized to the expression of the GAPDH gene.
Western blot analysis
Western blot analysis was performed to examine the protein expression of HIF-1α and VEGF in DMOG-treated palatal cells. After treatment with DMOG at various concentrations for 24 h, cells were lysed in extraction buffer containing 50 mM Tris base-HCl (PH 7.5), 150 mM NaCl, 0.5% Triton-X 100, and one 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, HIF-1α, 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 protein bands were visualized using a chemiluminescence kit (WEST-ZOL plus Western Blot Detection System, iNtRON Biotechnology). Chemiluminescence was detected using the LAS 1000 Plus Luminescent Image Analyzer (Fuji Photo Film, Tokyo, Japan). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Rat palatal wound healing assay
After confirming the angiogenesis effect of DMOG on RP cells, the effect of DMOG on wound healing of palatal tissue was performed in a rat palatal wound model. All rats were housed under specific pathogen-free (SPF) conditions at the animal experimental center of Seoul National University Dental School. Eighteen 13-week-old male SD rats (six rats for each group), weighing 300–350 g, were used in the present study. All animal experiments were approved by the Institutional Animal Care and Use Committee (SNU-130123-8-1) of Seoul National University (Seoul, Korea). Under general anesthesia, punch wounds were made on a central area of hard palate with a disposable 3-mm diameter biopsy punch (Kai Industries Ltd., Gifu, Japan), exposing a circular area of bare bone. Hyaluronic acid (HA) ointment (20 mg/ml) containing 0, 0.5, or 1 mg/ml (5.7 mM) DMOG was applied to the wound area. After the surgery, animals were fed a standard diet of pellets and water with enrofloxacin. The agents were re-applied on days 2 and 4 under anesthesia to reduce stress, and the rats were sacrificed on day 7. The hard palate was separated, and the wound area was observed with a stereoscopic microscope (Nikon, Tokyo, Japan) and by histological analysis. The wound area on the microscopic images was calculated using CellSense Dimension 1.6 software (Olympus, Tokyo, Japan). Palatal specimens were taken for histomorphometric evaluation and samples were stained with haematoxylin and eosin (H&E) staining. More than ten slides for each sample were prepared, and we selected a section that exhibited the widest diameter of wound for histological analysis. 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.
Statistical analysis
For statistical analysis, each experiment was performed in triplicate unless otherwise specified. The data are expressed as the mean ± Standard Deviation. Statistical analyses were determined by one-way ANOVA for in vivo study and cell migration assay. The unpaired Student’s t-test was used for the other in vitro results. A P-value < 0.05 was considered statistically significant.
Discussion
HIF prolyl hydroxylase inhibitors have been investigated to find a new drug for treatment of anemia, kidney disease, and heart failure [
19,
20]. Promotion of angiogenesis is a targeted therapeutic effect of the newly developing drugs, because angiogenesis is an inevitably necessary step in tissue regeneration or wound healing.
We observed the effect of DMOG, a HIF-1α prolyl hydroxylase inhibitor, on wound healing of rat palatal mucosa. For
in vitro studies, cytotoxicity and the ability to regulate VEGF and HIF-1α in cells originating from rat palatal tissues were investigated. The cytotoxicity of DMOG against rat palatal cells appeared at relatively higher concentrations (Fig.
1). Accumulation of HIF-1α protein is not likely a direct cause of cell death because the intracellular level of HIF-1α was already saturated at 0.5 mM and was not altered until 2.0 mM. A previous study reported cell cycle arrest by PHD inhibitors [
21], which provides a possible explanation for the toxic effects of DMOG. Since cytotoxicity undoubtedly can disturb tissue repair, DMOG concentrations for in vivo studies should be carefully chosen to elicit a wound healing effect. Marchbank et al. demonstrated that DMOG stimulates migration of human stomach and colonic carcinoma cells [
22]. However, a decrease in single cell migration has been observed in the hypoxia state of certain fibroblast cells [
23]. Therefore, the effect of hypoxia or HIF-1α accumulation on cell migration is cell-type specific. The motility of rat palatal cells in this study was not promoted by DMOG (Fig.
2), which suggests that migration of fibroblast-like palatal cells was not a determining factor in wound healing of DMOG-treated palatal cells.
Previously, it has been demonstrated that PHD inhibitors up-regulate the expression of VEGF in various kinds of cells, including endothelial cells [
24], epithelial cells [
25], gingival fibroblasts, and periodontal ligaments [
17]. In agreement with the results of these studies, the present study also confirmed the ability of DMOG to up-regulate the expression of VEGF (Fig.
3) in rat palatal fibroblast-like cells. DMOG also induced stabilization of HIF-1α protein, and the range of effective concentrations was overlapped except for 0.1 mM at which only HIF-1α protein but not VEGF was up-regulated. This indicates that VEGF cannot be induced in the absence of obvious increases in the amount of HIF-1α. Additionally, the stabilization of HIF-1α is also known to induce glucose transporter-1 and phosphoglycerate kinase-1, which can improve the wound healing by enhancing the re-epithelialization [
26].
Because of the moist condition of oral cavity in the animal experiment, it was required to employ a vehicle to extend the residual period of a drug at the wound area. As a vehicle for delivery of DMOG to oral mucosa in this study, was used a hyaluronic acid hydrogel due to its high viscosity and excellent biocompatibility; less cytotoxic and no inflammatory or allergic effects. However, the oral environment is extremely dynamic. Food, saliva, and drinking water can quickly remove topically applied hydrogels. In the present study, the porous structure of the hyaluronic acid hydrogel was not observed on the H-E stained sections, indicating that hyaluronic acid and incorporated DMOG had been washed away from the wound area before sacrifice on day 7. Therefore, it was not possible to estimate a valid amount of DMOG for wound healing. In this study, DMOG was applied at 0.5 and 1 mg/ml. A modest but statistically significant reduction in the wound area and distance occurred only at 1 mg/ml (Figs.
4 and
5). A more durable vehicle and increased concentration of DMOG could further accelerate healing of the palatal wound. However, the results of this study did demonstrate that HIF-1α is a potent target of wound healing in oral tissues.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YHC contributed to planning and designing the study. ZT performed most of the laboratory work and data analysis. PHC and SKM participated in the animal study. All authors read and approved the final manuscript.