Molecular interactions of dental resins with human oral and intestinal cells

有机成分从许多树脂基修复材料中释放出来。与其临床使用的快速增加相反，在其对主要靶组织（如口腔粘膜）的长期临床服务期间，对低浓度释放的物质的生物学效应和细胞机制知之甚少。共聚单体如甲基丙烯酸2-羟乙酯（HEMA）和聚合反应的（光）引发剂如蒽醌（CQ）和过氧化二苯甲酰（DBPO）在各种细胞类型中诱导细胞内氧化还原稳态应变。我们的研究结果表明，CQ诱导凋亡和CQ和DBPO产生氧化损伤的人口腔角质形成细胞的基因组DNA。此外，两种引发剂均影响涉及氧化还原敏感性转录因子Nrf 2的细胞代谢（低水平效应）。我们假设，这样的细胞氧化还原稳态的修改影响的细胞外基质（ECM）的组成，因此，潜在的组织稳态。我们提案的目的是调查，（1）用牙科材料处理的人角质形成细胞是否显示出在单层（2D）中培养的细胞和使用3D细胞成像仪与成纤维细胞共培养的细胞之间在参与氧化应激反应的蛋白质的基因表达和ECM组成方面的差异。（3维）-口腔粘膜的ECM模型和（2）是否和在多大程度上材料-氧化还原敏感性转录因子的诱导激活影响ECM组分的表达。因此，我们将比较CQ、DBPO和HEMA对口腔粘膜的人细胞（角质形成细胞、成纤维细胞）、单层培养和3D-ECM模型中的组织稳态的相关参数的影响。我们将使用完善的方法来研究细胞增殖，分化和凋亡。ECM的分子组分的表达（例如，膜受体、基质金属蛋白酶及其抑制剂、细胞因子）和氧化应激途径（Nrf 2、NF κ B和靶基因）将通过真实的时间PCR和蛋白质分析（蛋白质印迹、ELISA、活性测定）进行分析。我们的研究结果将提供有价值的数据有关的材料调制的氧化细胞代谢及其意义的组织稳态。这项研究将大大扩展我们对牙科树脂毒理学效应的认识，并将为现有材料的风险评估和牙科修复中新生物相容性材料的开发提供重要数据。

Organic components are released from many resin-based restorative materials. Contrary to the rapid increase in their clinical use, very little is known about the biological effects and the cellular mechanisms of substances released at low concentrations during their long-term clinical service on primary target tissues, such as the oral mucosa. Comonomers, like 2-hydroxyethyl methacrylate (HEMA), and (photo)initiators of the polymerization reaction, like camphorquinone (CQ) and dibenzoyl peroxide (DBPO), induce intracellular redox homeostasis strain in various cell types. Our results showed that CQ induces apoptosis and CQ and DBPO generate oxidative damages in genomic DNA of human oral keratinocytes. Moreover, both initiators affect the cellular metabolism involving the redox sensitive transcription factor Nrf2 (low level effect). We postulate, that such a modification of the cellular redox homeostasis influences the composition of the extra cellular matrix (ECM) and, therefore, potentially the tissue homeostasis. Purpose of our proposal is to investigate, (1) whether human keratinocytes treated with dental materials show differences in gene expression of proteins involved in oxidative stress response and in ECM composition between cells cultured in monolayers (2D) and in co-culture with fibroblasts using a 3D (three dimensional)-ECM model of the oral mucosa and (2) whether and to what extent a material-induced activation of redox sensitive transcription factors influences the expression of components of the ECM. Therefore, we will compare the effects of CQ, DBPO, and HEMA on relevant parameters of tissue homeostasis between human cells of the oral mucosa (keratinocytes, fibroblasts), cultivated in monolayers and in a 3D-ECM-model. We will use well-established methods to investigate cell proliferation, differentiation, and apoptosis. The expression of molecular components of the ECM (e.g., membrane receptors, matrix metalloproteinases and their inhibitors, cytokines) and oxidative stress pathways (Nrf2, NFkappaB, and target genes) will be analyzed by real time PCR and protein analysis (western blot, ELISA, activity assays). Our results will provide valuable data concerning the material-modulated oxidative cellular metabolism and its significance for tissue homeostasis. This study will significantly extend our body of knowledge about the toxicological effects of dental resins and will generate important data for the risk assessment of present materials and the development of new biocompatible materials in restorative dentistry.