Osteogenesis-induced exosomes derived from human periodontal ligament stem cells promote osteogenic differentiation of human periodontal ligament stem cells in an inflammatory microenvironment

doi:10.12307/2025.024

Abstract

Abstract: BACKGROUND: The osteogenic differentiation ability of exosomes derived from osteogenic mesenchymal stem cells is well established. However, their impact on the osteogenic differentiation of human periodontal ligament stem cells in an inflammatory microenvironment remains unclear.
OBJECTIVE: To examine the impact of exosomes derived from osteogenesis-induced human periodontal ligament stem cells on the osteogenic differentiation of human periodontal ligament stem cells within an inflammatory microenvironment.
METHODS: Human periodontal ligament stem cells were isolated and cultured. After 3 days of osteogenic induction, exosomes were extracted. Human periodontal ligament stem cells were divided into four groups. Control group was treated with osteogenesis-induced medium. The exosome group was treated with osteogenesis-induced medium containing 5 μg/mL exosomes. Inflammatory model and inflammatory model + exosome groups were treated with 1 μg/mL lipopolysaccharide for 24 hours to construct a cellular inflammatory microenvironment. The inflammatory model group was treated with osteogenesis-induced medium after lipopolysaccharide intervention. The inflammatory model + exosome group was treated with osteogenesis-induced medium containing 5 μg/mL exosome. The osteogenic differentiation ability of human periodontal ligament stem cells was assessed using alkaline phosphatase staining and alizarin red staining. The expressions of Runt-related transcription factor 2, osteopontin, osteoblast-specific transcription factor Osterix (OSX) and wnt pathway-related protein β-catenin were detected by real-time fluorescence quantitative PCR and western blot assay.
RESULTS AND CONCLUSION: (1) Compared with the control group, the relative area stained by alkaline phosphatase, the relative area stained by mineralized nodules and the expression levels of Runx2, osteopontin, and OSX were significantly decreased in the inflammatory model group (P < 0.05). (2) Compared with the inflammatory model group, the expression of Runx2, osteopontin, and OSX in the inflammatory model + exosome group was significantly increased in the relative area of alkaline phosphatase staining, the relative area of mineralized nodules staining (P < 0.05). (3) Compared with the control group, the expression of wnt pathway-related protein β-catenin was significantly increased in the inflammatory model group (P < 0.05). Compared with the inflammatory model group, the expression of β-catenin in the inflammatory model + exosome group was significantly decreased (P < 0.05). These findings indicate that exosomes derived from human periodontal ligament stem cells induced by bone formation can enhance the osteogenic differentiation of human periodontal ligament stem cells within an inflammatory microenvironment, and the mechanism may be related to wnt/β-catenin signaling pathway.

Key words: human periodontal ligament stem cell, exosome, inflammatory microenvironment, osteogenic differentiation, osteoblast, signaling pathway

CLC Number:

Aikepaer · Aierken, Chen Xiaotao, Wufanbieke · Baheti. Osteogenesis-induced exosomes derived from human periodontal ligament stem cells promote osteogenic differentiation of human periodontal ligament stem cells in an inflammatory microenvironment[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(7): 1388-1394.

Figures/Tables 2

2.1 hPDLSCs的鉴定结果原代hPDLSCs贴壁生长，相互黏附，细胞形态不均一，呈多边形或长梭形，见图1A。成骨诱导后hPDLSCs由纤维状开始变为鳞片状，显微镜下可见hPDLSCs逐渐形成钙结节，经0.2%茜素红染液染色后钙结节被染成红色，见图1B。成脂诱导培养后PDLSCs形态由原本的纤维状变圆，成脂诱导分化8-10 d在显微镜下能够看到脂滴，油红O染色后可见脂滴颗粒均被染成红色，见图1C。流式结果表明hPDLSCs高表达CD90、CD146和CD105表面抗原，阳性表达率分别为98.3%，73.7%和86.0%，低表达CD34，阳性表达率为0.62%，见图2。 2.2 OB-EXOs的鉴定结果 OB-EXOs为圆形或椭圆形，膜结构完整，见图3A；Western blot 结果显示 CD9、CD81和TGS101呈阳性表达，见图3B，符合文献[14]报道的外泌体的相关特征，表明成功提取OB-EXOs。 2.3 hPDLSCs摄取OB-EXOs 为检测OB-EXOs能否被hPDLSCs摄取，首先用PKH26标记OB-EXOs，进一步与hPDLSCs共培养，发现PKH26标记的OB-EXOs位于hPDLSCs内，并聚集在细胞核附近，说明OB-EXOs能够直接作用于hPDLSCs，见图4。 2.4 各组hPDLSCs成骨分化能力与对照组相比，炎症模型组的矿化结节相对面积与碱性磷酸酶相对面积显著降低；与对照组相比，外泌体组细胞矿化结节相对面积与碱性磷酸酶相对面积显著增加；与炎症模型组相比，炎症模型+外泌体组细胞碱性磷酸酶相对面积与矿化结节相对面积显著增加，见图5-7。 2.5 各组hPDLSCs成骨相关因子mRNA的表达与对照组相比，炎症模型组Runt相关转录因子2、骨桥蛋白和OSX mRNA表达降低；与对照组相比，外泌体组Runt相关转录因子2、骨桥蛋白和OSX mRNA表达升高；与炎症模型组相比，炎症模型+外泌体组Runt相关转录因子2、骨桥蛋白和OSX mRNA表达升高，见图8。 2.6 各组hPDLSCs成骨相关因子的蛋白表达与对照组相比，炎症模型组Runt相关转录因子2、骨桥蛋白和OSX 蛋白表达量显著降低；与对照组相比，外泌体组Runt相关转录因子2、骨桥蛋白和OSX 蛋白表达量显著升高；与炎症模型组相比，炎症模型+外泌体组Runt相关转录因子2、骨桥蛋白和OSX 蛋白表达量显著升高，见图9。 2.7 各组hPDLSCs中β-catenin表达与对照组相比，炎症模型组wnt通路相关蛋白β-catenin表达量显著增加；外泌体组与对照组之间无显著性差异；与炎症模型组相比，炎症模型+外泌体组β-catenin蛋白和mRNA表达量显著降低，见图10。 "

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