Cytotoxicity testing of dental materials: Establishment of a 3D-printed dentin barrier culture system

IF 6.3 1区医学 Q1 DENTISTRY, ORAL SURGERY & MEDICINE

Dental Materials Pub Date : 2026-04-01 Epub Date: 2025-12-02 DOI:10.1016/j.dental.2025.11.016

Ella Ohlsson , Sandra Pohl , Iryna Prots , Carola Bolay , Kerstin M. Galler , Wolfgang Buchalla , Gottfried Schmalz , Matthias Widbiller

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引用次数: 0

Abstract

Introduction

Cytotoxicity testing is critical in assessing the biocompatibility of dental materials with regard to the pulp, however, conventional methods such as the extract test (ISO 10993–5) do not reflect the protective effects of dentin in the dentin-pulp complex. Traditional dentin barrier testing techniques (ISO 7405) address this problem, but face challenges due to their complex and expensive production and the difficult and error-prone handling. Therefore, the aim of this study was to develop a novel, user-friendly and easily accessible dentin barrier culture system (DBCS) to improve cytotoxicity testing of dental materials.

Method

The DBCS was designed and manufactured using stereolithography 3D printing. The components were assembled with dentin discs to create a barrier between an upper and lower compartment (pulp side/restoration side). Dentin discs were pretreated (EDTA or citric acid) and primary human pulp cells (HPC) or mouse fibroblasts (L-929) were cultured in the upper chamber, while a self-adhesive composite (Vertise Flow, Kerr; Scafati, Italy) was applied to the opposite side of the disc after 48 h. Scanning electron microscopy (SEM) was used to visualize dentin structure and cell attachment. Analyses included assessment of cell metabolism (MTT test), membrane integrity (LDH assay), and performing live/dead staining. To validate the model, various dentin disc parameters were evaluated, including species origin (human or bovine), location relative to the pulp (proximal or distal), thickness, and the effects of autoclaving. Statistical evaluation was performed using the Kruskal-Wallis or the Mann-Whitney test (P ≤ 0.05).

Results

Both HPC and L-929 formed confluent cell layers in the DBCS setup. Pretreatment of dentin discs with EDTA or citric acid reliably removed the smear layer, with the latter having a positive effect on cell viability (P ≤ 0.03). The self-adhesive composite had a similar effect on the viability of both cell types, and SEM revealed apoptotic cell morphology. Dentin provided a protection only at a thickness of greater than 400 µm. There were no significant differences in cellular responses to the material depending on dentin origin or autoclaving (P > 0.9999).

Conclusion

This 3D-printable culture system offers a readily available, user-friendly dentin barrier model for evaluating the cytotoxicity of dental materials and can be easily reproduced by anyone with access to a standard 3D printer.

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牙科材料的细胞毒性测试：3d打印牙本质屏障培养系统的建立。

导言：细胞毒性测试是评估牙髓生物相容性的关键，然而，传统的方法，如提取物测试（ISO 10993-5）不能反映牙本质在牙本质-牙髓复合体中的保护作用。传统的牙本质屏障测试技术（ISO 7405）解决了这个问题，但由于其复杂和昂贵的生产以及困难和容易出错的处理，面临着挑战。因此，本研究的目的是开发一种新型的、用户友好的、易于使用的牙本质屏障培养系统（DBCS），以改善牙科材料的细胞毒性检测。方法：采用立体光刻3D打印技术设计制作DBCS。组件与牙本质盘组装，在上下隔室（牙髓侧/修复侧）之间形成屏障。对牙本质盘进行预处理（EDTA或柠檬酸），上腔培养原代人牙髓细胞（HPC）或小鼠成纤维细胞（L-929）， 48 h后将自粘复合材料（Vertise Flow, Kerr; Scafati, Italy）涂于牙本质盘的另侧。扫描电镜观察牙本质结构和细胞附着情况。分析包括评估细胞代谢（MTT试验），膜完整性（LDH测定），并进行活/死染色。为了验证该模型，评估了各种牙本质椎间盘参数，包括物种来源（人类或牛），相对于牙髓的位置（近端或远端），厚度以及高压灭菌的效果。采用Kruskal-Wallis或Mann-Whitney检验进行统计学评价（P ≤ 0.05）。结果：HPC和L-929在DBCS中均形成融合细胞层。EDTA或柠檬酸预处理牙本质盘可靠地去除涂片层，后者对细胞活力有积极影响（P ≤ 0.03）。自粘复合材料对两种细胞的活力有相似的影响，扫描电镜显示凋亡的细胞形态。牙本质仅在厚度大于400 µm时提供保护。根据牙本质来源或高压灭菌，细胞对材料的反应没有显著差异（P > 0.9999）。结论：这种3D打印培养系统提供了一种易于获得的、用户友好的牙本质屏障模型，用于评估牙科材料的细胞毒性，任何人都可以使用标准3D打印机轻松复制。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Dental Materials 工程技术-材料科学：生物材料

CiteScore

9.80

自引率

10.00%

发文量

290

审稿时长

67 days

期刊介绍： Dental Materials publishes original research, review articles, and short communications. Academy of Dental Materials members click here to register for free access to Dental Materials online. The principal aim of Dental Materials is to promote rapid communication of scientific information between academia, industry, and the dental practitioner. Original Manuscripts on clinical and laboratory research of basic and applied character which focus on the properties or performance of dental materials or the reaction of host tissues to materials are given priority publication. Other acceptable topics include application technology in clinical dentistry and dental laboratory technology. Comprehensive reviews and editorial commentaries on pertinent subjects will be considered.