OBJECTIVE Two-dimensional (2D) in vitro models have been extensively utilized for cytotoxicity assessment of dental materials, but with certain limitations in terms of direct in vitro-in vivo extrapolation (IVIVE). Three-dimensional (3D) models seem more appropriate, recapitulating the structure of human tissues. This study established a 3D dentin/pulp analogue, as advanced cytotoxicity assessment tool of dental restorative materials (DentCytoTool). METHODS DentCytoTool comprised two compartments: the upper, representing the dentin component, with a layer of odontoblast-like cells expanded on microporous membrane of a cell culture insert and covered by a treated dentin matrix; and the lower, representing a pulp analogue, incorporating HUVEC/SCAP co-cultures into collagen I/fibrin hydrogels. Representative resinous monomers (HEMA: 1-8mM; TEGDMA: 0.5-5mM) and bacterial components (LPS: 1μg/ml) were applied into the construct. Cytotoxicity was assessed by MTT and LDH assays, live/dead staining and real-time PCR for odontogenesis- and angiogenesis-related markers. RESULTS DentCytoTool supported cell viability and promoted capillary-like network formation inside the pulp analogue. LPS induced expression of odontogenesis-related markers (RUNX2, ALP, DSPP) without compromising viability of the odontoblast-like cells, while co-treatment with LPS and resin monomers induced cytotoxic effects (live/dead staining, MTT and LDH assays) in cells of both upper and lower compartments and reduced expression angiogenesis-related markers (VEGF, VEGFR2, ANGPT-1, Tie-2, PECAM-1) in a concentration- and time- dependent manner. LPS treatment aggravated TEGDMA-induced and -in certain concentrations (2-4mM)- HEMA-induced cytotoxicity. SIGNIFICANCE DentCytoTool represents a promising tissue-engineering-based cytotoxicity assessment tool, providing more insight into the mechanistic aspects of interactions of dental materials to the dentin/pulp complex.

中文翻译：

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基于三维组织工程的Dentin / Pulp组织类似物作为牙科修复材料的高级生物相容性评估工具。

目的二维（2D）体外模型已广泛用于牙科材料的细胞毒性评估，但在直接体外-体内外推法（IVIVE）方面有一定的局限性。三维（3D）模型似乎更合适，可以概括人体组织的结构。这项研究建立了3D牙本质/牙髓类似物，作为牙科修复材料的高级细胞毒性评估工具（DentCytoTool）。方法DentCytoTool包括两个部分：上部，代表牙本质成分，在细胞培养插入物的微孔膜上扩展成牙本质细胞样细胞层，并被处理过的牙本质基质覆盖。下半部代表果肉类似物，将HUVEC / SCAP共培养物掺入胶原I /纤维蛋白水凝胶中。代表性的树脂单体（HEMA：1-8mM; TEGDMA：0。5-5mM）和细菌成分（LPS：1μg/ ml）被应用到构建体中。通过MTT和LDH分析，活/死染色和实时PCR评估牙源性和血管生成相关标记的细胞毒性。结果DentCytoTool支持细胞活力并促进果肉类似物内部的毛细血管状网络形成。LPS诱导牙源性生成相关标记（RUNX2，ALP，DSPP）的表达而不会损害成牙本质细胞样细胞的活力，而与LPS和树脂单体共同处理可诱导细胞毒性作用（活/死染色，MTT和LDH分析）上下隔室和表达减少的血管生成相关标记（VEGF，VEGFR2，ANGPT-1，Tie-2，PECAM-1）均呈浓度和时间依赖性。LPS治疗加重了TEGDMA诱导的HEMA诱导的细胞毒性，并在一定浓度（2-4mM）下加剧了HEMA诱导的细胞毒性。重要性DentCytoTool代表了一种有前途的基于组织工程的细胞毒性评估工具，可提供有关牙科材料与牙本质/牙髓复合物相互作用的机械方面的更多见解。