Retinoic acid (RA) signal is involved in tooth development and osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) are one of the useful MSCs in tissue regeneration. However, the function of RA in osteo/odontogenic differentiation of DPSCs remains unclear. Here, we investigated the expression pattern of RA in miniature pig tooth germ and intervened in the RA signal during osteo/odontogenic differentiation of human DPSCs. Deciduous canine (DC) germs of miniature pigs were observed morphologically, and the expression patterns of RA were studied by in situ hybridization (ISH). Human DPSCs were isolated and cultured in osteogenic induction medium with or without RA or BMS 493, an inverse agonist of the pan-retinoic acid receptors (pan-RARs). Alkaline phosphatase (ALP) activity assays, alizarin red staining, quantitative calcium analysis, CCK8 assay, osteogenesis-related gene expression, and in vivo transplantation were conducted to determine the osteo/odontogenic differentiation potential and proliferation potential of DPSCs. We found that the expression of RARβ and CRABP2 decreased during crown calcification of DCs of miniature pigs. Activation of RA signal in vitro inhibited ALP activities and mineralization of human DPSCs and decreased the mRNA expression of ALP, osteocalcin, osteopontin, and a transcription factor, osterix. With BMS 493 treatment, the results were opposite. Interference in RA signal decreased the proliferation of DPSCs. In vivo transplantation experiments suggested that osteo/odontogenic differentiation potential of DPSCs was enhanced by inversing RA signal. Our results demonstrated that downregulation of RA signal promoted osteo/odontogenic differentiation of DPSCs and indicated a potential target pathway to improve tissue regeneration.

中文翻译：

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维甲酸信号负调节牙髓干细胞的成骨/成牙细胞分化。

维甲酸（RA）信号参与间充质干细胞（MSCs）的牙齿发育和成骨分化。牙髓干细胞（DPSC）是组织再生中有用的MSC之一。然而，RA在DPSC的骨/牙源性分化中的功能仍不清楚。在这里，我们调查了人类DPSCs的骨/牙源性分化过程中RA在微型猪牙胚中的表达模式并干预了RA信号。形态学观察小型猪的落叶犬（DC）细菌，并通过原位研究RA的表达模式杂交（ISH）。分离人DPSC，并在有或没有RA或BMS 493（泛视黄酸受体（pan-RARs）的反向激动剂）的成骨诱导培养基中培养。进行了碱性磷酸酶（ALP）活性测定，茜素红染色，定量钙分析，CCK8测定，成骨相关基因表达以及体内移植，以确定DPSC的成骨/成牙分化潜能和增殖潜能。我们发现在小型猪的DC冠状钙化过程中RARβ和CRABP2的表达降低。体外RA信号的激活抑制人DPSC的ALP活性和矿化并降低ALP的mRNA表达，骨钙素，骨桥蛋白和转录因子osterix。用BMS 493处理，结果相反。RA信号的干扰降低了DPSC的增殖。体内移植实验表明，通过逆转RA信号可以增强DPSC的骨/牙源性分化潜能。我们的结果表明，RA信号的下调促进了DPSC的骨/牙源性分化，并表明了改善组织再生的潜在靶途径。