Division of Periodontology, Department of Oral Function, Kyushu Dental University, Kitakyushu, Japan.
Department of Life and Environment Engineering, The University of Kitakyushu, Kitakyushu, Japan.
J Periodontal Res. 2018 Oct;53(5):870-882. doi: 10.1111/jre.12577. Epub 2018 Jun 14.
Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo.
Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids.
The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate "stemness" were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation.
Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.
人牙周膜间充质干细胞（hPDLMSCs）被认为是维持牙周组织稳态和再生的关键。尽管 hPDLMSCs 通常在单层中培养，但已有研究表明，单层培养不如球体等 3 维培养，球体是由自组装形成的细胞球形簇。本研究旨在比较 hPDLMSCs 球体与单层培养的 hPDLMSC 的成骨表型，分别在体外和体内进行研究。
使用标记有聚乙二醇的微井芯片形成球体。通过流式细胞仪检测 hPDLMSC 球体中的间充质干细胞（MSC）标志物。通过实时聚合酶链反应分析检测干性标志物和骨向相关基因在 hPDLMSC 球体和单层培养物中的表达。通过免疫荧光分析确认 PDLMSC 球体中干性标志物的蛋白表达。进行结节形成测定、碱性磷酸酶（ALP）活性测定和在小鼠颅骨缺损模型中的移植测定，以确认 hPDLMSC 球体的成骨潜力。为了阐明在成骨诱导培养基（OIM）中球体培养增强 hPDLMSCs 成骨的机制，使用针对分泌卷曲相关蛋白 3（SFRP3）的小干扰 RNA（siRNA）测定进行研究。通过实时聚合酶链反应和 Western 印迹分析测量 OIM 中单层和球体培养的 hPDLMSCs 中 SFRP3 的表达水平。在 SFRP3 缺陷的 hPDLMSC 球体中检测 ALP 基因表达和 ALP 活性。
hPDLMSC 球体表达 MSC 标志物，与在单层培养物中生长的 hPDLMSCs 相似。有趣的是，与在单层培养物中生长的 hPDLMSCs 相比，调节“干性”的转录因子的蛋白和 mRNA 表达显著增加。与单层培养的 hPDLMSCs 相比，在 OIM 中培养的 hPDLMSC 球体的结节形成显著增加。与单层培养物相比，hPDLMSC 球体中的 ALP 活性和骨向相关基因的表达也显著增强。在小鼠颅骨缺损模型中，与单层培养的 hPDLMSCs 相比，hPDLMSC 球体的治疗显著增强了新骨形成。最后，为了阐明在 OIM 中培养球体增强 hPDLMSCs 中 ALP 激活的机制，使用 siRNA 测定来操纵 Wnt 信号通路拮抗剂分泌卷曲相关蛋白 3（SFRP3）的表达。SFRP3 的敲低抑制了 OIM 中生长的 hPDLMSCs 中的 ALP 基因表达；进一步抑制了球体培养物中的 ALP 活性。这些数据表明，hPDLMSC 球体中成骨潜能的增强是通过 SFRP3 介导的 ALP 激活来调节的。
hPDLMSCs 球体培养可能是再生医学中的一种新颖而有用的工具。
