用于牙髓细胞和组织的牙材料毒性筛选的人类三维牙髓类器官模型。

Human three-dimensional dental pulp organoid model for toxicity screening of dental materials on dental pulp cells and tissue.

机构信息

Stomatological Hospital of Chongqing Medical University, Chongqing, China.

Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, China.

出版信息

Int Endod J. 2022 Jan;55(1):79-88. doi: 10.1111/iej.13641. Epub 2021 Oct 16.

DOI:10.1111/iej.13641

PMID:34587308

Abstract

AIM

To establish a 3D model for screening the biocompatibility of dental materials/drugs on dental pulp cells and tissue.

METHODOLOGY

Human dental pulp cells (hDPC) and endothelial cells (EC) were mixed with or without human dental pulp derived extracellular matrix (hDP-ECM) according to several protocols and cultured in 3D plates to fabricate 3D organoids. Cell viability and proliferation in organoids were evaluated using Live/Dead cell viability assay and ATPase assay. Organoids were fixed, cut and stained with a H&E staining kit. The expressions of DSPP, DMP-1, CD31, vWF and COL1A in 3D organoids were evaluated using immunofluorescence. To assess the feasibility of 3D organoids on drug/material toxicity screening, the organoids were treated with lipopolysaccharides (LPS) or iRoot BP. Then, cell viability and apoptosis were assessed. The expressions of IL-6, TNF-α, and IL-1β were compared in LPS-treated and non-treated organoids. Alizarin Red S staining was used to evaluate calcium deposit formation in organoids. Data were analysed using one-way anova followed by Tukey's post hoc comparison.

RESULTS

The 3D spheres/organoids were formed at day 1 or day 2. Cells in 3D organoids maintained a high viability rate and low proliferation activity. The level of CD31 increased significantly (p < .05) when EC were added to coculture with hDPC. The expressions of odontogenesis-associated proteins (DSPP, COL1A) upregulated (p < .05) with the addition of hDP-ECM. Level of IL-6 expression and rates of dead and apoptotic cells in 3D organoids were increased significantly (p < .05) in response to LPS. Calcium deposit formation was observed in iRoot BP-treated organoids.

CONCLUSIONS

Coculture of hDPC and EC in the presence of hDP-ECM formed functional dental pulp organoids. The experimental model provides an alternative tool for toxicity screening of dental pulp capping agents and dental pulp regeneration research.

摘要

目的

建立一种筛选牙体牙髓细胞和组织中牙科材料/药物生物相容性的 3D 模型。

方法

根据几种方案将人牙髓细胞（hDPC）和内皮细胞（EC）与或不与牙髓来源细胞外基质（hDP-ECM）混合，并在 3D 板中培养以构建 3D 类器官。通过活/死细胞活力检测和 ATP 酶检测评估类器官中的细胞活力和增殖。用 H&E 染色试剂盒固定、切割和染色类器官。用免疫荧光法评估 3D 类器官中 DSPP、DMP-1、CD31、vWF 和 COL1A 的表达。为了评估 3D 类器官在药物/材料毒性筛选中的可行性，用脂多糖（LPS）或 iRoot BP 处理类器官。然后，评估细胞活力和细胞凋亡。比较 LPS 处理和未处理类器官中 IL-6、TNF-α 和 IL-1β 的表达。用茜素红 S 染色评估类器官中钙沉积的形成。使用单因素方差分析（ANOVA），然后用 Tukey 事后比较进行数据分析。

结果

第 1 天或第 2 天形成 3D 球体/类器官。3D 类器官中的细胞保持高活力和低增殖活性。当 EC 加入与 hDPC 的共培养中时，CD31 的水平显著增加（p<0.05）。加入 hDP-ECM 后，牙发生相关蛋白（DSPP、COL1A）的表达上调（p<0.05）。3D 类器官中 IL-6 表达水平和死亡及凋亡细胞比例在 LPS 刺激下显著增加（p<0.05）。在 iRoot BP 处理的类器官中观察到钙沉积的形成。