Department of Endodontics, Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China; National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Endodontics, Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Department of oral surgery, Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
J Endod. 2020 Jul;46(7):964-972. doi: 10.1016/j.joen.2020.03.013. Epub 2020 May 7.
The antimicrobial peptide LL-37, in addition to its broad spectrum of antibacterial function, can promote odontogenesis and osteogenesis. Stem cells from the apical papilla (SCAPs) are essential for the formation of dentin/bonelike tissues. However, little information on these cells is available in regenerative endodontics. This study aimed to evaluate the effects of LL-37 on the proliferation, migration, and differentiation of SCAPs.
SCAPs were isolated, cultured, and characterized. Cell viability was analyzed by Cell Counting Kit-8 assays (Dojindo, Kumamoto, Japan). Cell migration was investigated by transwell assays. Dentin sialophosphoprotein, dentin matrix protein 1, runt-related transcription factor 2, and osterix were assessed by quantitative polymerase chain reaction and Western blots. Alkaline phosphatase (ALP) activity and ALP staining were assessed to determine the in vitro potential for osteogenic differentiation. The involvement of the Akt/Wnt/β-catenin signaling pathway was also studied.
In the 2.5-μg/mL LL-37 -treated group, cell proliferation and migration were up-regulated. Quantitative polymerase chain reaction and Western blot assays both revealed that LL-37 at 2.5 μg/mL up-regulated odonto/osteogenic markers (dentin sialophosphoprotein, dentin matrix protein 1, runt-related transcription factor 2, and osterix). LL-37 at 2.5 μg/mL significantly promoted ALP activity and increased the staining in SCAPs. In addition, the p-Akt and p-glycogen synthase kinase-3β levels were increased in LL-37-treated SCAPs. The migratory and odonto/osteogenic differentiation capacities of SCAPs were inhibited after treatment with inhibitors LY294002 and XAV-939.
Our study showed that LL-37 at 2.5 μg/mL promoted the migration and odonto/osteogenic differentiation of SCAPs by activating the Akt/Wnt/β-catenin signaling pathway.
抗菌肽 LL-37 除了具有广谱的抗菌功能外，还能促进牙发生和骨生成。根尖乳头干细胞（SCAPs）是牙本质/骨样组织形成所必需的。然而，在再生牙髓学中，关于这些细胞的信息很少。本研究旨在评估 LL-37 对 SCAPs 增殖、迁移和分化的影响。
分离、培养和鉴定 SCAPs。通过 Cell Counting Kit-8 检测试剂盒（Dojindo，熊本，日本）分析细胞活力。通过 Transwell 检测细胞迁移。通过定量聚合酶链反应和 Western blot 检测牙本质涎磷蛋白、牙本质基质蛋白 1、Runt 相关转录因子 2 和骨桥蛋白的表达。通过碱性磷酸酶（ALP）活性和 ALP 染色评估体外成骨分化潜能。还研究了 Akt/Wnt/β-catenin 信号通路的参与。
在 2.5μg/mL LL-37 处理组中，细胞增殖和迁移得到上调。定量聚合酶链反应和 Western blot 分析均表明，2.5μg/mL 的 LL-37 上调牙/骨形成标志物（牙本质涎磷蛋白、牙本质基质蛋白 1、Runt 相关转录因子 2 和骨桥蛋白）。2.5μg/mL 的 LL-37 显著促进 ALP 活性并增加 SCAPs 的染色。此外，LL-37 处理的 SCAPs 中 p-Akt 和糖原合酶激酶-3β 水平升高。用抑制剂 LY294002 和 XAV-939 处理后，SCAPs 的迁移和牙/骨向分化能力受到抑制。
本研究表明，2.5μg/mL 的 LL-37 通过激活 Akt/Wnt/β-catenin 信号通路促进 SCAPs 的迁移和牙/骨向分化。
