ABSTRACT
Objective Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells. Material and Methods Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein. Conclusion The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues. .
Subject(s)
Humans , Animals , Mice , Dental Enamel Proteins/pharmacology , Epithelial Cells/drug effects , Periodontium/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Chromatography, High Pressure Liquid , Electrophoresis, Gel, Two-Dimensional , Epithelial Cells/cytology , Gingiva/cytology , Gingiva/drug effects , Guided Tissue Regeneration, Periodontal/methods , Periodontitis/drug therapy , Reference Values , Reproducibility of Results , Silver Staining , Time FactorsABSTRACT
OBJECTIVE: In the present study, we examined whether 2, 2-bis [4-(2-hydroxy-3-methacryloxypropoxy) phenyl] propane (Bis-GMA) has effects on LSC2 cells, human dental pulp cell line. MATERIAL AND METHODS: The viability, cell cycle, and morphology of LSC2 cells were analyzed after exposure to several different concentrations of Bis-GMA. The recovery of viability of Bis-GMA exposed cells was also analyzed in the condition without Bis-GMA. Further, penetration of Bis-GMA to dentin disc was examined using isocratic high-performance liquid chromatography. RESULTS: There was a concentration-dependent decrease in cell proliferation and an increase in cell number in the sub-G1 population after exposure to Bis-GMA. Furthermore, the cells showed typical characteristics of apoptotic cells after the exposure to high concentration of Bis-GMA. In contrast, cells exposed to lower concentrations of Bis-GMA recovered their viability after being cultured without Bis-GMA. We also found that Bis-GMA is capable of penetrating 1-mm-thick dentin discs, though the penetrated concentration was lower than that showing cytotoxicity. CONCLUSION: These results suggest that Bis-GMA has cytotoxic effects, though dental pulp exposed to lower concentrations is able to recover their viability when Bis-GMA is removed.