N-Acetylcysteine modulates the effects of composites on human gingival keratinocytes.

OBJECTIVE

The aim of this study was to evaluate, if antioxidants, like N-Acetylcysteine, can modulate effects of composite eluates on human gingival keratinocytes.

METHODS

Composite samples of ceram.x® universal, Filtek™ Supreme XTE, and Admira® Fusion were stored 72h in cell culture medium to prepare eluates, according to ISO 10993-12:2012. Human gingival keratinocytes were exposed to these eluates with or without 3mM N-Acetylcysteine. Following cell observation by iCELLigence®, exposure periods were determined at 1d and 4d. Cell morphological analysis combined with live/dead staining was performed. Tissue-specific biomarkers of terminal differentiation, Involucrin and Filaggrin, were analyzed by indirect immunofluorescence (IIF) and Western blot (WB). qPCR profiling was performed on genes encoding for: inflammation, apoptosis, turn-over of extracellular matrix, adhesion, proliferation and differentiation. For statistical analysis one-way Anova was used (p<0.05).

RESULTS

Cells exposed to N-Acetylcysteine exhibited morphological changes but no cell death. After adding 3mM N-Acetylcysteine to HGK cultures, increased fluorescence intensity and protein amounts of Involucrin and Filaggrin indicated enhanced differentiation (p<0.05). Gene expression was modulated by: (i) composition of the composite eluates, (ii) NAC and (iii) exposure time. Filtek™ Supreme XTE showed a significant increased gene expression in inflammatory genes (p<0.05), which was amplified by the addition of NAC at 1d. Concerning exposure time, modulated gene expression showed eluate dependency, substantiated by Filtek™ Supreme XTE modulation at day 1 and Admira® Fusion at day 4.

SIGNIFICANCE

N-Acetylcysteine-emerging effects on gingival keratinocytes were threefold: (i) increase of differentiation, (ii) modulation of composite-related effects and (iii) in parts counteraction of eluate-induced effects.