Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
College of Stomatology, Southern Medical University, Guangzhou, China.
Int Endod J. 2020 Jan;53(1):72-83. doi: 10.1111/iej.13205. Epub 2019 Sep 18.
To comparatively evaluate changes in the proliferation and mineralization abilities of dental pulp stem cells (DPSCs) from juvenile and adult rats in a lipopolysaccharide (LPS)-induced inflammatory microenvironment to provide a theoretical basis for the age-related differences observed in DPSCs during repair of inflammatory injuries.
DPSCs were isolated from juvenile (JDPSCs) and adult rats (ADPSCs), and senescence-associated β-galactosidase staining was used to compare senescence between JDPSCs and ADPSCs. Effects of LPS on JDPSCs and ADPSCs proliferation were investigated by cell counting kit-8 assays and flow cytometry. Alizarin red staining, quantitative reverse transcription polymerase chain reaction and Western blot assay were used to examine the effects of LPS on mineralization-related genes and proteins in JDPSCs and ADPSCs. Immunohistochemistry was used to compare interleukin-1β (IL-1β) and osteocalcin (OCN) expression in the pulpitis model. Unpaired Student's t-tests and one-way anova were used for statistical analysis.
DPSCs were isolated from juvenile and adult rat dental pulp tissues. At low concentrations (0.1-1 μg mL ), LPS significantly promoted the proliferation of JDPSCs (P < 0.01) and ADPSCs (P < 0.01 or P < 0.05), with the effect being stronger in JDPSCs than in ADPSCs. In addition, mineralized nodules and the expression of mineralization-related genes (OCN, DSPP, ALP, BSP) increased significantly after stimulation with LPS (0.5 μg mL ) in JDPSCs and ADPSCs (P < 0.01 or P < 0.05), and JDPSCs displayed a more obvious increase than ADPSCs. Western blots revealed OCN and ALP expression levels in JDPSCs treated with LPS were significantly upregulated (P < 0.05); meanwhile, ALP expression in ADPSCs increased slightly but significantly (P < 0.05), and OCN expression was not affected. Finally, IL-1β expression was significantly higher (P < 0.05) and OCN expression was significantly lower (P < 0.05) in the inflamed dental pulp of adult rats than in juvenile rats.
A certain degree of inflammatory stimulation promoted the proliferation and mineralization of DPSCs; however, this effect declined with age. The DPSCs of adult donors in an inflammatory microenvironment have a weaker repair ability than that of juvenile donors, who are better candidates for tissues damage repair.
比较幼年和成年大鼠牙髓干细胞（DPSCs）在脂多糖（LPS）诱导的炎症微环境中增殖和矿化能力的变化，为 DPSCs 在修复炎症损伤过程中观察到的与年龄相关的差异提供理论依据。
从幼年（JDPSCs）和成年大鼠（ADPSCs）中分离 DPSCs，通过衰老相关β-半乳糖苷酶染色比较 JDPSCs 和 ADPSCs 之间的衰老情况。通过细胞计数试剂盒-8 检测和流式细胞术研究 LPS 对 JDPSCs 和 ADPSCs 增殖的影响。茜素红染色、定量逆转录聚合酶链反应和 Western blot 检测 LPS 对 JDPSCs 和 ADPSCs 矿化相关基因和蛋白的影响。免疫组织化学比较牙髓炎模型中白细胞介素 1β（IL-1β）和骨钙素（OCN）的表达。采用独立样本 t 检验和单因素方差分析进行统计学分析。
从幼年和成年大鼠牙髓组织中分离出 DPSCs。在低浓度（0.1-1μg/mL）时，LPS 显著促进 JDPSCs（P<0.01）和 ADPSCs（P<0.01 或 P<0.05）的增殖，其作用在 JDPSCs 中强于 ADPSCs。此外，在 LPS（0.5μg/mL）刺激下，JDPSCs 和 ADPSCs 的矿化结节和矿化相关基因（OCN、DSPP、ALP、BSP）的表达均显著增加（P<0.01 或 P<0.05），JDPSCs 的增加更为明显。Western blot 显示 LPS 处理后的 JDPSCs 的 OCN 和 ALP 表达水平显著上调（P<0.05）；同时，ADPSCs 中的 ALP 表达略有增加但显著增加（P<0.05），而 OCN 表达不受影响。最后，与幼年大鼠相比，成年大鼠炎症性牙髓中 IL-1β的表达明显升高（P<0.05），OCN 的表达明显降低（P<0.05）。
一定程度的炎症刺激促进了 DPSCs 的增殖和矿化，但随着年龄的增长，这种作用会减弱。在炎症微环境中，成年供体的 DPSCs 的修复能力比幼年供体弱，因此，成年供体更适合组织损伤修复。
