title: Cytotoxicity and anti-inflammatory effects of zinc ions and eugenol during setting of ZOE in immortalized human oral keratinocytes grown as three-dimensional spheroids.
abstract: OBJECTIVE
  The objective of this study is to assess the cytotoxic and anti-inflammatory effects of ZOE cement during setting in two-dimensional (2D) or three-dimensional (3D) cultures of immortalized human oral keratinocytes (IHOKs) with determining the extract components responsible for these effects.
  
  
  METHODS
  Extracts of mixed ZOE at different stages of setting were analyzed by a digital pH meter, ICP-MS, and GC-MS. Serial concentrations of extract and their mixture of ZnCl2, ZnSO4·H2O, and eugenol liquid were added to the 2D and 3D IHOK cultures to determine the half maximal effective concentration in investigating the cause of cytotoxicity by means of WST assay and to investigate mRNA expression levels of inflammatory cytokines by RT-PCR.
  
  
  RESULTS
  Zn(2+) and eugenol (4-19 ppm) were detected in the extracts. In the early setting stage, significant cytotoxicity was observed in the 2D and 3D IHOK cultures (P<0.05). The EC50 of Zn(2+) from ZnCl2 was 5-44 ppm in both cultures, whereas the EC50 of eugenol was not detectable under 100 ppm. Along with the lower levels of inflammatory cytokine gene expressions in the extract, treatment of the 2D IHOKs with Zn(2+) alone and treatment of the 3D IHOKs with Zn(2+) plus eugenol resulted in significantly lower expression levels of IL-1β, IL-6, and IL-8 (P<0.05).
  
  
  CONCLUSIONS
  The cytotoxic effect of ZOE on IHOKs was greater during the setting stage owing to the presence of Zn(2+). The anti-inflammatory response to ZOE was induced by a combination of Zn(2+) and eugenol. Cytotoxic and anti-inflammatory effects differed between the 2D and 3D IHOK cultures.
authors.affiliation: Department and Research Institute of Dental Biomaterials and Bioengineering, BK21 PLUS Project, College of Dentistry, Yonsei University, Seoul, Republic of Korea; Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan, Republic of Korea; Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, Republic of Korea.
authors.forename: Jung-Hwan
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan, Republic of Korea; Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, Republic of Korea.
authors.forename: Hae-Hyoung
authors.initials: HH
authors.lastname: Lee
authors.affiliation: Department and Research Institute of Dental Biomaterials and Bioengineering, BK21 PLUS Project, College of Dentistry, Yonsei University, Seoul, Republic of Korea.
authors.forename: Kyoung-Nam
authors.initials: KN
authors.lastname: Kim
authors.affiliation: Department and Research Institute of Dental Biomaterials and Bioengineering, BK21 PLUS Project, College of Dentistry, Yonsei University, Seoul, Republic of Korea. Electronic address: kmkim@yuhs.ac.
authors.forename: Kwang-Mahn
authors.initials: KM
authors.lastname: Kim
mesh_terms: D005054:Eugenol / Q000494:pharmacology*; D006801:Humans; D007477:Ions; D015603:Keratinocytes; D009055:Mouth; D015032:Zinc; D015035:Zinc Oxide-Eugenol Cement / Q000494:pharmacology*
chemical_list: D007477:Ions; D015035:Zinc Oxide-Eugenol Cement; D005054:Eugenol; D015032:Zinc
keywords: 3D spheroid culture system; Cytotoxicity; Eugenol; Immortalized human oral keratinocytes; Zinc ion; Zinc oxide–eugenol