Engineering Research Center of Oral Translational Medicine, West China Hospital of Stomatology, Ministry of Education, Sichuan University, Chengdu, P.R. China.
National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, P.R. China.
Stem Cell Res Ther. 2024 Aug 26;15(1):270. doi: 10.1186/s13287-024-03882-2.
Periodontal tissue loss is the main reason for tooth mobility and loss caused by periodontal disease. Dental follicle stem cells (DFSCs) have significant therapeutic potential in periodontal regeneration, which maybe mainly depends on their potent immunomodulatory capacity. Consequently, this study aims to elucidate the impact of implanted xenogenous DFSCs on innate immune responses during early and late stages in the periodontal defect repair period.
To trace and investigate the immunomodulation mechanisms of DFSCs in vivo, DFSCs were engineered (E-DFSCs) using lentiviral vectors expressing CD63-enhanced green fluorescent protein (CD63-EGFP) and β-Actin-mCherry protein (ACTB-mCherry) to exhibit green and red fluorescence. The biological characteristics and functions of E-DFSCs were verified by proliferation, differentiation, and co-culture experiments in vitro. In vivo, the periodontal regeneration capacity of E-DFSCs was detected by implantation of murine periodontal defect model, and the response of innate immune cells was detected at the 1st, 3rd, and 5th days (early stage) and 4th week (late stage) after implantation.
In vitro assessments showed that E-DFSCs retain similar properties to their non-engineered counterparts but exhibit enhanced macrophage immunomodulation capability. In mice models, four-week micro-CT and histological evaluations indicated that E-DFSCs have equivalent efficiency to DFSCs in periodontal defect regeneration. At the early stage of repair in mice periodontal defect, fluorescence tracking showed that implanted E-DFSCs might primarily activate endogenous cells through direct contact and indirect actions, and most of these cells are myeloperoxidase-positive neutrophils. Additionally, compared with the control group, the neutrophilic infiltration and conversion of N2-type were significantly increased in the E-DFSC group. At the late stage of defect regeneration, more M2-type macrophages, fewer TRAP + osteoclasts, and an upregulated OPG/RANKL ratio were detected in the E-DFSC group compared to the control group, which indicated that immune balance tilts towards healing and bone formation.
The xenogenous implanted DFSCs can induce the N2 phenotype of neutrophils in the early stage, which can activate the innate immune mechanism of the host to promote periodontal tissue regeneration.
牙周组织丧失是牙周病导致牙齿松动和脱落的主要原因。牙囊干细胞（DFSCs）在牙周再生中有显著的治疗潜力，这可能主要取决于其强大的免疫调节能力。因此，本研究旨在阐明植入的异种 DFSCs 在牙周缺损修复期的早期和晚期对固有免疫反应的影响。
为了追踪和研究 DFSCs 在体内的免疫调节机制，我们使用表达 CD63-增强型绿色荧光蛋白（CD63-EGFP）和β-肌动蛋白-红色荧光蛋白（ACTB-mCherry）的慢病毒载体对 DFSCs 进行工程化改造（E-DFSCs），以显示绿色和红色荧光。通过体外增殖、分化和共培养实验验证 E-DFSCs 的生物学特性和功能。在体内，通过植入小鼠牙周缺损模型检测 E-DFSCs 的牙周再生能力，并在植入后第 1、3 和 5 天（早期）和第 4 周（晚期）检测固有免疫细胞的反应。
体外评估表明，E-DFSCs 保留了与其非工程化对应物相似的特性，但表现出增强的巨噬细胞免疫调节能力。在小鼠模型中，四周的微 CT 和组织学评估表明，E-DFSCs 在牙周缺损再生中的效率与 DFSCs 相当。在小鼠牙周缺损的修复早期，荧光追踪显示植入的 E-DFSCs 可能通过直接接触和间接作用主要激活内源性细胞，其中大多数细胞是髓过氧化物酶阳性的中性粒细胞。此外，与对照组相比，E-DFSC 组中性粒细胞浸润和 N2 型转化显著增加。在缺损再生的晚期阶段，E-DFSC 组中检测到更多的 M2 型巨噬细胞、更少的 TRAP+破骨细胞和上调的 OPG/RANKL 比值，表明免疫平衡向愈合和骨形成倾斜。
异种植入的 DFSCs 可在早期诱导中性粒细胞的 N2 表型，从而激活宿主的固有免疫机制，促进牙周组织再生。
