Huang Xin, Wang Xiaoxuan, Ma Li, Wang Huiyi, Peng Yan, Liu Heyu, Xiao Junhong, Cao Zhengguo
The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST KLOS) & Key Laboratory of Oral Biomedicine Ministry of Education (KLOBME), School & Hospital of Stomatology, Wuhan University, Wuhan, China.
Department of Periodontology, School & Hospital of Stomatology, Wuhan University, Wuhan, China.
J Periodontol. 2023 Feb;94(2):290-300. doi: 10.1002/JPER.22-0048. Epub 2022 Nov 9.
Cementum regeneration was regarded as the critical goal for periodontal regeneration, and M2 macrophage-based therapy was expected to be a promising strategy. However, little is known about the effects of M2 macrophages on cementoblast mineralization and tropism, especially under inflammation. Here we investigated for the first time the crosstalk between M2 macrophages and Porphyromonas gingivalis (Pg)-stimulated cementoblasts.
M2 macrophages were induced with interleukin (IL)-4, and identified. CC-chemokine ligand 2 (CCL2) expression and secretion of inflammatory cementoblasts were detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), immunohistochemistry for apical periodontitis (AP) mice, and by enzyme-linked immunosorbent assay. Crystal violet staining was used to observe macrophage migration. Conditional medium (CM) and transwell coculture methods were applied to evaluate the effects of M2 macrophages on cementum mineralization with or without Pg, and to explore the mechanism. Mineralization-related markers and pathway-related proteins were measured by RT-qPCR and WB.
M2 macrophages were identified successfully. We found an increase of CCL2 in cementoblasts and their supernatant. Also, higher CCL2 in cementoblasts was observed in the AP model. Superior recruitment of M2 macrophages to supernatant from Pg-stimulated cementoblasts or CCL2-containing medium was verified. Moreover, CM2 and Trans-M2 showed better mineralization-accelerating and rescuing effects when compared to their controls, and application of p38 inhibitor partially blocked the promotion.
Our study demonstrated the inflammation-targeting and mineralization-promoting effects of M2 macrophages on cementoblasts, which may provide evidence for M2 macrophage-based cementum regeneration.
牙骨质再生被视为牙周组织再生的关键目标，基于M2巨噬细胞的治疗有望成为一种有前景的策略。然而，关于M2巨噬细胞对成牙骨质细胞矿化和归巢的影响，尤其是在炎症状态下，人们了解甚少。在此，我们首次研究了M2巨噬细胞与牙龈卟啉单胞菌（Pg）刺激的成牙骨质细胞之间的相互作用。
用白细胞介素（IL）-4诱导并鉴定M2巨噬细胞。通过逆转录定量实时聚合酶链反应（RT-qPCR）、蛋白质免疫印迹法（WB）、根尖周炎（AP）小鼠免疫组织化学以及酶联免疫吸附测定法检测炎性成牙骨质细胞中CC趋化因子配体2（CCL2）的表达和分泌。用结晶紫染色观察巨噬细胞迁移。采用条件培养基（CM）和Transwell共培养方法，评估M2巨噬细胞在有或无Pg情况下对牙骨质矿化的影响，并探讨其机制。通过RT-qPCR和WB检测矿化相关标志物和通路相关蛋白。
成功鉴定出M2巨噬细胞。我们发现成牙骨质细胞及其上清液中CCL2增加。此外，在AP模型中观察到成牙骨质细胞中CCL2水平更高。证实M2巨噬细胞对Pg刺激的成牙骨质细胞或含CCL2培养基的上清液具有更强的趋化作用。此外，与对照相比，CM2和Trans-M2显示出更好的矿化促进和挽救作用，并且应用p38抑制剂可部分阻断这种促进作用。
我们的研究证明了M2巨噬细胞对成牙骨质细胞具有靶向炎症和促进矿化的作用，这可能为基于M2巨噬细胞的牙骨质再生提供证据。
