Yang Nan, Xia Yu, Gao Hui, Wang Chen, Jiang Ying, Song Wei, Yu Ji-Feng, Liang Li
Department of Stomatology, Eighth Medical Center of Chinese PLA General Hospital, Beijing, PR China.
Clinical Laboratory, Eighth Medical Center of Chinese PLA General Hospital, Beijing, PR China.
J Dent. 2025 Jul;158:105772. doi: 10.1016/j.jdent.2025.105772. Epub 2025 Apr 25.
This study aimed to elucidate the role of regulatory T cells (Tregs) in promoting the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to investigate the underlying mechanisms involving Notch signaling.
Tregs were isolated via fluorescence-activated cell sorting (FACS) and co-cultured with PDLSCs. Osteogenic differentiation was assessed through in vitro assays and in vivo transplantation experiments. Gene expression profiles were quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. A murine periodontitis model was used to validated therapeutic outcomes, with bone remodeling quantified by micro-CT and histology (H&E, Masson's staining). Immunophenotypic analysis of Jagged1 expression in Tregs and Notch2 receptor localization in PDLSCs were performed using flow cytometry and immunofluorescence microscopy, respectively. The role of immobilized Jagged1 in osteogenic differentiation was further evaluated, while Notch pathway inhibition was achieved via γ-secretase inhibitor DAPT in vitro.
Elevated levels of Th17 cells and Tregs were observed in peripheral blood samples from periodontitis patients, with a significantly increased Th17/Treg ratio (p < 0.01). In vitro, co-culturing Tregs with PDLSCs significantly enhanced PDLSC osteogenesis, as evidenced by increased ALP activity (p < 0.01), elevated expression of osteogenesis-related genes (Runx2 and Osterix; p < 0.01), and enhanced mineralization (Alizarin Red staining) (p < 0.01). In vivo, intravenous infusion of Tregs into a periodontitis mouse model reduced periodontal damage and promoted bone regeneration, as demonstrated by reduced CEJ-ABC distance and increased BV/TV ratio (p < 0.01). Mechanistically, Tregs expressed the Notch ligand Jagged1 and upregulated Notch2 receptor expression in PDLSCs, indicating activation of the Notch signaling pathway. Jagged1 promoted osteogenic differentiation of PDLSCs in a dose- and time-dependent manner. Inhibition of Notch signaling using DAPT reduced Tregs-mediated enhancement of PDLSC osteogenesis (p < 0.05).
These findings suggest that Tregs promote PDLSC osteogenic differentiation via Jagged1-Notch2 signaling, highlighting the therapeutic potential of modulating Tregs and Notch signaling for periodontal regeneration and bone tissue engineering.
This study provides new insights into the complex interplay between immune modulation and stem cell differentiation, laying the foundation for potential Tregs-based therapeutic strategies for periodontal and bone tissue regeneration.
本研究旨在阐明调节性T细胞（Tregs）在促进牙周膜干细胞（PDLSCs）成骨分化中的作用，并探究涉及Notch信号通路的潜在机制。
通过荧光激活细胞分选（FACS）分离Tregs，并与PDLSCs共培养。通过体外实验和体内移植实验评估成骨分化。使用定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹分析对基因表达谱进行定量。使用小鼠牙周炎模型验证治疗效果，通过显微CT和组织学（苏木精-伊红染色、Masson染色）对骨重塑进行定量。分别使用流式细胞术和免疫荧光显微镜对Tregs中Jagged1表达和PDLSCs中Notch2受体定位进行免疫表型分析。进一步评估固定化Jagged1在成骨分化中的作用，同时在体外通过γ-分泌酶抑制剂DAPT抑制Notch信号通路。
在牙周炎患者的外周血样本中观察到Th17细胞和Tregs水平升高，Th17/Treg比值显著增加（p < 0.01）。在体外，Tregs与PDLSCs共培养显著增强了PDLSCs的成骨能力，碱性磷酸酶（ALP）活性增加（p < 0.01）、成骨相关基因（Runx2和Osterix）表达升高（p < 0.01）以及矿化增强（茜素红染色）（p < 0.01）均证明了这一点。在体内，将Tregs静脉注射到牙周炎小鼠模型中可减少牙周损伤并促进骨再生，牙骨质-牙槽嵴顶距离减小和骨体积分数增加（p < 0.01）证明了这一点。从机制上讲，Tregs表达Notch配体Jagged1并上调PDLSCs中Notch2受体表达，表明Notch信号通路被激活。Jagged1以剂量和时间依赖性方式促进PDLSCs的成骨分化。使用DAPT抑制Notch信号可降低Tregs介导的PDLSCs成骨增强作用（p < 0.05）。
这些发现表明Tregs通过Jagged1-Notch2信号通路促进PDLSCs的成骨分化，突出了调节Tregs和Notch信号通路在牙周再生和骨组织工程中的治疗潜力。
本研究为免疫调节与干细胞分化之间的复杂相互作用提供了新见解，为基于Tregs的牙周和骨组织再生治疗策略奠定了基础。
