Effects of HEMA on Nrf2-related gene expression using a newly developed 3D co-culture model of the oral mucosa.
Effects of HEMA on Nrf2-related gene expression using a newly developed 3D co-culture model of the oral mucosa.
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DOI：
10.1016/j.dental.2019.05.006
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发表时间：
2019-05
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影响因子：
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通讯作者：
Renke Perduns;J. Volk;M. Plum;F. Gutzki;V. Kaever;W. Geurtsen
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文献类型：
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作者：
Renke Perduns;J. Volk;M. Plum;F. Gutzki;V. Kaever;W. Geurtsen
甲基丙烯酸羟乙酯（HEMA）是许多树脂改性材料的一种成分，从牙科冲洗液中洗脱到口腔中。我们的调查的目的是确定口腔角质形成细胞（OKF 6/TERT 2）和牙龈成纤维细胞（HGFs）在一个新建立的3D共培养模型（3D-CCM）的影响，并分析的渗透性OKF 6/TERT 2细胞的HEMA. MethodsWell特征的3D-CCM，包括融合的OKF 6/TERT 2细胞的细胞培养插入上述含HGF的胶原蛋白凝胶，处理超上皮与HEMA。24小时后，使用质谱法测量HEMA在上皮上和上皮下的分布。通过qRT-PCR和western blot分析检测HEMA对核因子红细胞2相关因子2（Nrf 2）靶基因的影响。分析的Nrf 2靶基因在mRNA水平上在两种细胞类型中均被诱导，但在HGF中不太明显。在蛋白质水平上，两种细胞类型显示出相似的作用：在5 mM HEMA时，血红素加氧酶-1在OKF 6/TERT 2细胞中诱导5.1倍，在HGF中诱导4.1倍。NAD（P）H醌脱氢酶-1在两种细胞类型中诱导约1.85倍。显著性我们的3D-CCM适合于分析牙科材料的生物相容性，因为与单层培养物相比，它改善了对口腔粘膜的模拟。我们的研究结果表明，HEMA能够穿透角质形成细胞的致密层，并激活细胞的氧化防御反应。这可能是由于两种细胞类型中Nrf 2-途径的激活。
Objective2-Hydroxyethyl methacrylate (HEMA) is a component of many resin-modified materials and elutes from dental restorations into the oral cavity. Objective of our investigation was to determine the impact of HEMA on oral keratinocytes (OKF6/TERT2) and gingival fibroblasts (HGFs) in a newly established 3D co-culture model (3D-CCM) and to analyze the permeability of OKF6/TERT2 cells for HEMA.MethodsWell-characterized 3D-CCMs, consisting of confluent OKF6/TERT2 cells on cell culture inserts above HGF-containing collagen gels, were treated supra-epithelial with HEMA. Mass spectrometry was used to measure the supra- and sub-epithelial distribution of HEMA after 24 h. The impact of HEMA on nuclear factor erythroid 2-related factor 2 (Nrf2) target genes was measured by qRT-PCR and western blot analysis.ResultsMass spectrometry showed that HEMA was evenly distributed above and below the keratinocyte layer after 24 h. Analyzed target genes of Nrf2 were induced in both cell types on the mRNA-level but less pronounced in HGFs. On the protein-level, both cell types showed similar effects: At 5 mM HEMA, heme oxygenase-1 was induced 5.1-fold in OKF6/TERT2 cells and 4.1-fold in HGFs. NAD(P)H quinone dehydrogenase-1 was approximately induced 1.85-fold in both cell types.SignificanceOur 3D-CCM is suitable to analyze the biocompatibility of dental materials due to an improved simulation of the oral mucosa compared to monolayer cultures. Our results indicate that HEMA is able to penetrate a dense layer of keratinocytes and to activate the cellular oxidative defense response. This may be due to the activation of the Nrf2-pathway in both cell types.