Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Prosthodontics, Geriatric Dentistry and Craniomandibular Disorders, Assmanshauser Straße 4-6, 14197 Berlin, Germany.
Department of Oral Biotechnology, Center for Dental Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany.
Dent Mater. 2022 Sep;38(9):1532-1546. doi: 10.1016/j.dental.2022.07.006. Epub 2022 Jul 30.
The aim of this study was to investigate the influence of hybrid CAD/CAM-blocks on immortalized human gingival keratinocytes (HGK).
Samples of two different hybrid CAD/CAM materials [Lava™ Ultimate (3 M); VITA Enamic® (VITA Zahnfabrik)], a composite material [ceram.x® universal (Dentsply Sirona)] and a CAD/CAM ceramic [VITABLOCS® (VITA Zahnfabrik)] were stored in cell culture medium for 72 h to prepare eluates according to ISO-10993-12:2012. HGK were exposed to eluates for 6, 24 and 48 h. Cell monitoring was performed by RTCA iCELLigence™ system. The morphological changes were evaluated using phase contrast imaging. Specific biomarkers of apoptosis and terminal differentiation (Caspase-3, Involucrin) were analyzed semi quantitatively by indirect immunofluorescence (IIF). Protein levels and activation of MAP kinases ERK1/2 (p44/42) were quantified by Western blot. Data were statistically analyzed by unpaired t-test (p < 0.05).
Regarding Vita Enamic® and Lava™ Ultimate, results of RTCA iCELLigence™ and Western blots showed no statistically significant differences (p > 0.05) compared to the negative control (HGK in native keratinocyte growth medium). No aberrant expression of Caspase-3 and Involucrin was detected in cells incubated with Vita® Enamic eluates Cells incubated with Lava™ Ultimate showed a higher expression of Involucrin after 24 h of incubation compared to the negative control. Statistically significant differences (p < 0.01) were found between cells incubated with ceram.x® universal and the negative control in RTCA iCELLigence™ assay and in quantitative measurements of Western blots after 6 h against phospho-p44/42 (p = 0.044). Increased expression of Caspase-3 and Involucrin were detected by IIF in cells after incubation with eluates of ceram.x® universal.
The present data show no significant effect of hybrid materials on analyzed functions of cell behavior. A cytotoxic influence of ceram.x® universal eluates was observed in HGK in terms of a strong modulation of proliferation, morphology and protein expression.
本研究旨在探讨混合 CAD/CAM 块对永生化人牙龈角质细胞（HGK）的影响。
将两种不同的混合 CAD/CAM 材料（Lava™ Ultimate [3M]；VITA Enamic® [VITA Zahnfabrik]）、一种复合材料（ceram.x® universal [Dentsply Sirona]）和一种 CAD/CAM 陶瓷（VITABLOCS® [VITA Zahnfabrik]）的样本储存在细胞培养基中 72 小时，根据 ISO-10993-12:2012 制备浸提液。将 HGK 暴露于浸提液中 6、24 和 48 小时。通过 RTCA iCELLigence™ 系统进行细胞监测。使用相差成像评估形态变化。通过间接免疫荧光（IIF）半定量分析凋亡和终末分化的特异性生物标志物（半胱天冬酶-3，兜甲蛋白）。通过 Western blot 定量测定 MAP 激酶 ERK1/2（p44/42）的蛋白水平和激活。通过未配对 t 检验（p<0.05）对数据进行统计学分析。
与 Vita Enamic®和 Lava™ Ultimate 相比，RTCA iCELLigence™ 和 Western blot 的结果显示与阴性对照（天然角质细胞生长培养基中的 HGK）相比无统计学差异（p>0.05）。在用 Vita®Enamic 浸提液孵育的细胞中未检测到 Caspase-3 和兜甲蛋白的异常表达。在用 Lava™ Ultimate 孵育 24 小时后，与阴性对照相比，细胞中兜甲蛋白的表达更高。在 RTCA iCELLigence™ 测定和 Western blot 定量测量后 6 小时，用 ceram.x® universal 孵育的细胞与阴性对照之间在磷酸化 p44/42 方面存在统计学显著差异（p=0.044）。用 ceram.x® universal 浸提液孵育的细胞中通过 IIF 检测到 Caspase-3 和兜甲蛋白的表达增加。
本研究数据显示混合材料对细胞行为分析功能无显著影响。ceram.x® universal 浸提液对 HGK 具有细胞毒性作用，表现为增殖、形态和蛋白表达的强烈调节。
