Institute of Dental Research, Osaka Dental University, Osaka, Japan.
Department of Nanomedicine (DNP), Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
J Periodontal Res. 2019 Aug;54(4):364-373. doi: 10.1111/jre.12637. Epub 2018 Dec 30.
The periodontal ligament (PDL) has important roles in maintaining homeostasis, wound healing, and regeneration of periodontal tissues by supplying stem/progenitor cells. Periodontal ligament stem cells (PDLSCs) have mesenchymal stem cell (MSC)-like characteristics and can be isolated from periodontal tissues. The aim of this study was to examine the effect of three-dimensional spheroid culture on the characteristics of PDLSCs.
Periodontal ligament stem cells were isolated and cultured from healthy teeth, and PDLSC spheroids were formed by pellet culture in polypropylene tubes. The proliferation of PDLSCs in spheroids and conventional two-dimensional (2D) cultures were examined by immunostaining for Ki67. Cell death and cell size were analyzed using flow cytometry. Gene expression changes were investigated by quantitative real time PCR.
Periodontal ligament stem cells spontaneously formed spheroid masses in pellet culture. The size of PDLSC spheroids was inversely proportional to the culture period. Fewer Ki67-positive cells were detected in PDLSC spheroids compared to those in 2D culture. Flow cytometry revealed an increase in dead cells and a decrease in cell size in PDLSC spheroids. The expression levels of genes related to anti-inflammation (TSG6, COX2, MnSOD) and angiogenesis (VEGF, bFGF, HGF) were drastically increased by spheroid culture compared to 2D culture. TSG6 gene expression was inhibited in PDLSC spheroids in the presence of the apoptosis signal inhibitor, Z-VAD-FMK. Additionally, PDLSC spheroid transplantation into rat periodontal defects did not induce the regeneration of periodontal tissues.
We found that spheroid culture of PDLSCs affected several characteristics of PDLSCs, including the expression of genes related to anti-inflammation and angiogenesis; apoptosis signaling may be involved in these changes. Our results revealed the characteristics of PDLSCs in spheroid culture and have provided new information to the field of stem cell research.
牙周韧带（PDL）通过提供干细胞/祖细胞在维持内稳态、伤口愈合和牙周组织再生方面发挥重要作用。牙周韧带干细胞（PDLSCs）具有间充质干细胞（MSC）样特征，可从牙周组织中分离出来。本研究旨在探讨三维球体培养对 PDLSCs 特性的影响。
从健康牙齿中分离和培养牙周韧带干细胞，并通过在聚丙烯管中形成微球培养来形成 PDLSC 球体。通过 Ki67 免疫染色检查 PDLSC 球体和传统二维（2D）培养中的细胞增殖。使用流式细胞术分析细胞死亡和细胞大小。通过定量实时 PCR 研究基因表达变化。
牙周韧带干细胞在微球培养中自发形成球体团块。PDLSC 球体的大小与培养时间成反比。与 2D 培养相比，PDLSC 球体中的 Ki67 阳性细胞较少。流式细胞术显示 PDLSC 球体中的死亡细胞增加，细胞大小减小。与 2D 培养相比，球体培养显著增加了与抗炎（TSG6、COX2、MnSOD）和血管生成（VEGF、bFGF、HGF）相关的基因表达水平。TSG6 基因表达在凋亡信号抑制剂 Z-VAD-FMK 的存在下被抑制。此外，PDLSC 球体移植到大鼠牙周缺损中不会诱导牙周组织再生。
我们发现 PDLSC 的球体培养影响了 PDLSCs 的几种特性，包括与抗炎和血管生成相关的基因表达；凋亡信号可能参与了这些变化。我们的结果揭示了 PDLSC 球体培养中的特性，并为干细胞研究领域提供了新的信息。
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