State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases, Shaanxi Key Laboratory of Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Chang-le Road, Xi'an, 710032, People's Republic of China.
Hospital of Stomatology, Zunyi Medical University, 89 Wu-jiang Dong Road, Zunyi, 563003, People's Republic of China.
Stem Cell Res Ther. 2022 Jul 15;13(1):304. doi: 10.1186/s13287-022-02971-4.
Human dental pulp stem cells (hDPSCs) have received widespread attention in the fields of tissue engineering and regenerative medicine. Although amphiregulin (AREG) has been shown to play a vital function in the biological processes of various cell types, its effects on DPSCs remain largely unknown. The aim of this study was to explore the specific role of AREG as a biologically active factor in the regeneration of dental pulp tissue.
The growth of hDPSCs, together with their proliferation and apoptosis, in response to AREG was examined by CCK-8 assay and flow cytometry. We explored the effects of AREG on osteo/odontogenic differentiation in vitro and investigated the regeneration and mineralization of hDPSCs in response to AREG in vivo. The effects of AREG gain- and loss-of-function on DPSC differentiation were investigated following transfection using overexpression plasmids and shRNA, respectively. The involvement of the mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)/Akt pathways in the mineralization process and the expression of odontoblastic marker proteins after AREG induction were investigated by using Alizarin Red S staining and Western blotting, respectively.
AREG (0.01-0.1 µg/mL) treatment of hDPSCs from 1 to 7 days increased hDPSCs growth and affected apoptosis minimally compared with negative controls. AREG exposure significantly promoted hDPSC differentiation, shown by increased mineralized nodule formation and the expression of odontoblastic marker protein expression. In vivo micro-CT imaging and quantitative analysis showed significantly greater formation of highly mineralized tissue in the 0.1 μg/mL AREG exposure group in DPSC/NF-gelatin-scaffold composites. AREG also promoted extracellular matrix production, with collagen fiber, mineralized matrix, and calcium salt deposition on the composites, as shown by H&E, Masson, and Von Kossa staining. Furthermore, AREG overexpression boosted hDPSC differentiation while AREG silencing inhibited it. During the differentiation of hDPSCs, AREG treatment led to phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and PI3K/Akt. Notably, a specific inhibitor of ERK, JNK, and PI3K/Akt signaling markedly reduced AREG-induced differentiation, as well as levels of phosphorylated ERK and JNK in hDPSCs.
The data indicated that AREG promoted odontoblastic differentiation and facilitated regeneration and mineralization processes in hDPSCs.
人牙髓干细胞（hDPSCs）在组织工程和再生医学领域受到广泛关注。尽管表皮生长因子样因子 17（AREG）已被证明在各种细胞类型的生物学过程中发挥着重要作用，但它对 DPSCs 的影响在很大程度上仍是未知的。本研究旨在探讨 AREG 作为一种生物活性因子在牙髓组织再生中的具体作用。
通过 CCK-8 检测和流式细胞术检测 AREG 对 hDPSCs 生长以及增殖和凋亡的影响。我们在体外研究了 AREG 对成骨/成牙本质分化的影响，并研究了 AREG 在体内对 hDPSCs 再生和矿化的影响。通过转染过表达质粒和 shRNA，分别研究了 AREG 对 DPSC 分化的增益和缺失功能的影响。通过茜素红 S 染色和 Western blot 分别研究了 MAPK 或 PI3K/Akt 通路在矿化过程中的参与情况以及 AREG 诱导后牙本质标记蛋白的表达情况。
AREG（0.01-0.1μg/mL）处理 hDPSCs 1-7 天，与阴性对照相比，可增加 hDPSCs 的生长并最小限度地影响细胞凋亡。AREG 暴露可显著促进 hDPSC 分化，表现为矿化结节形成增加和牙本质标记蛋白表达增加。体内 micro-CT 成像和定量分析显示，在 DPSC/NF-明胶支架复合材料中，0.1μg/mL AREG 暴露组形成高度矿化组织的数量明显增加。AREG 还促进细胞外基质的产生，在复合材料上形成胶原纤维、矿化基质和钙盐沉积，H&E、Masson 和 Von Kossa 染色结果表明。此外，AREG 过表达促进 hDPSC 分化，而 AREG 沉默抑制其分化。在 hDPSCs 分化过程中，AREG 处理导致细胞外信号调节激酶（ERK）、c-Jun N-末端激酶（JNK）和 PI3K/Akt 的磷酸化。值得注意的是，ERK、JNK 和 PI3K/Akt 信号通路的特异性抑制剂显著降低了 AREG 诱导的分化以及 hDPSCs 中磷酸化 ERK 和 JNK 的水平。
数据表明，AREG 促进了 hDPSCs 的成牙本质分化，并促进了 hDPSCs 的再生和矿化过程。
