Biological Effects Of Phenytoin On Cultured Human Periodontal Ligament Cells In Vitro

Objective:Phenytoin (diphenylhydantoin), well known for its clinical use as anticonvulsant, anti-arrhythmic and antineuralgic drug, was first reported to be useful for the promotion of wound healing by Shapiro in 1958. Since then, other investigators have extended the use of topical phenytoin in wound healing to skin ulcers of various etiology, such as decubitus ulcers, venous stasis ulcers, diabetic ulcers, and leprosy trophic ulcers. A number of studies also indicate that phenytoin promotes the proliferation and differentiation of macrophages, osteoblasts in animals, and human fibroblasts. These results may suggest the possibility of its accelerating periodontal regeneration in some degree.Human periodontal ligament cells (hPDLCs) as the main functional cells in periodontium, have multipotential for differentiation and play a key role in wound healing and periodontal tissue regeneration. The purpose of this present study was to evaluate the effects of different concentrations of phenytoin on biological characteristics of hPDLCs in vitro, and explore the possibility of its accelerating periodontal regeneration. Methods:hPDLCs were obtained from healthy premolars extracted for orthodontic reasons. All samples were placed into Dulbecco's Modified Eagle's Medium(DMEM), cultured and subcultured at confluence. Experiments were carried out with cells from the third to fifth passages.1. Cells were plated into 96-well plate at an initial concentration of 2 × 10~4cells/ml. After incubated for 24 hours, multiple concentrations of phenytoin (0, 1, 5, 20, 100, 500, 2500μg/ml) were added to the wells. The cells were then incubated with drugs for 3 days to measure proliferation by MTT assay, the total amount of protein was detected with Coumassie Bright Blue method and alkaline phosphatase (ALP) activity was measured by PNPP.2. The fourth passage hPDLCs were plated into 24-well plate at an initial concentrationof 5×10～4 cells/ml. After incubated for 24 hours, mineralized medium (15%FBS, 10mmol/L β-glycerophosphate, 10～(-4)mmol/L dexamethasone, 50mg/L vitamin C and DMEM) and phenytoin (20, 100, 500μg/ml) were added into the test group, and mineralized solution without phenytoin was added into the medium of the control. After co-incubated for 4 weeks, the mineralized nodules formation was detected with Von Kossa'silver nitrate staining for calcium.3. The third passage hPDLCs were plated into 24-well plate at an initial concentration of1 × 10~6 cells/ml. After incubated for 24 hours, phenytoin (20 and 100μg/ml) was added into the medium. The cells were then incubated with drugs for 24h, and cell solution were collected, prepared, and analyzed for BMP-2 concentration by enzyme linked immunosorbent sandwich assay (ELISA).Results:Phenytoin could enhance hPDLCs proliferation, protein synthesis and the activity of ALPat a range of concentrations. It could also increase the capability of mineration and BMP-2expression of hPDLCs at a certain range of concentrations.1. Over a concentration range of 1 to 100μg/ml, cells demonstrated a normal appearance, spindle or fusiform shaped. Within a certain range of concentrations(20～100μg/ml), phenytoin significantly enhanced hPDLCs' proliferation activity, and the maximal effect could be observed at the concentration of 100μg/ml (P<0.01). When the concentration reached 500μg/ml, proliferation effect began to decrease significantly in contrast with the concentration of 100μg/ml and 20μg/ml (P<0.01). And at the concentrations of 2500μg/ml, cell proliferation effect decreased dramatically, lower than any other test group(1, 5, 20, 100, 500μg/ml) and the controlled group, which indicated that high dosage of phenytoin had cytocidal effect on hPDLCs, thus inhibited cell proliferation (P<0.01). Phenytoin began to promote ALP activity of hPDLCs at the concentration of 20μg/ml (P<0.01), and the effect improved with the increase of concentrations, reaching its highest effect at 100μg/ml. The inhibition became highly significant at concentrations of 2500μg/ml when compared with the other test groups (P<0.01). At doses not exceeding 100μg/ml, phenytoin enhanced protein synthesis of hPDLCs in a dose-dependent manner and the concentrations of 100μg/ml exhibited the maximal effect (P<0.05). When it reached to 500μg/ml, protein synthesis began to decrease, but the difference between 500μg/ml and 100μg/ml did not reach the level of statistically significance. Meanwhile, because of its toxic effect on cells, 2500μg/ml of phenytoin decreased protein synthesis accordingly.2. After co-incubated for 4 weeks, there was statistical significance among the four groups in the rate of mineralization. The capability of mineralization of hPDLCs increased significantly (P<0.01) in 100μg/ml group when compared with that in the control group. When it reached to 500μg/ml, the capability of mineralization began to decrease significantly in contrast with the concentration of 100μg/ml. But the difference between 20μg/ml and 100μg/ml did not reach the level of statistical significance.3. When treated with 20 and 100μg/ml phenytoin sodium for 24 h, the capability of BMP-2 expression of hPDLCs increased significantly, compared to controls (P<0.05 and P<0.01, respectively).Conclusion:The results suggested that proper doses of phenytoin could promote proliferation and biosynthesis and also enhance osteogenesis by increasing the differentiation, mineralization and BMP-2 expression of hPDLCs while higher concentrations of phenytoin had cytotoxic effect.