Saiyasilp Suthasinee, Vaseenon Savitri, Srisuwan Tanida, Chuveera Patchanee
Division of General Dentistry, Department of Family and Community Dentistry, Chiang Mai University, Faculty of Dentistry, Chiang Mai, Thailand.
Department of Restorative Dentistry and Periodontology, Chiang Mai University, Faculty of Dentistry, Chiang Mai, Thailand.
Eur Endod J. 2025 Mar;10(2):142-150. doi: 10.14744/eej.2024.15010.
This study aimed to investigate the effects of D-galactose (D-gal) on cellular senescence induction, cell proliferation, mineralization production, and odontogenic gene expression of isolated human dental pulp cells (HDPCs).
Isolated HDPCs were cultured and assigned to four groups: control, 1 g/L D-gal, 10 g/L D-gal, and 10 g/L D-gal with Biodentine (BD). Cell proliferation was evaluated at 24, 48, and 72 hours using Alamar Blue® assay. To evaluate cellular senescence at 48 hours, senescence-associated beta-galactosidase (SA-β-gal) activity and senescence-related genes (p16 and p21) were assessed with SA-β-gal staining assay and quantitative reverse-transcription polymerase chain reaction (qRT-PCR), respectively. To examine the mineralization potential under differentiating conditions, quantitative staining with Alizarin Red S and mineralization-related gene expression (dentine sialophosphoprotein, DSPP) were investigated at 14 days. One-way ANOVA was used for statistical analysis. The statistical significance level was set at 0.05.
1 g/L D-gal and 10 g/L of D-gal significantly decreased cell proliferation at 72 hours compared to the control group (p<0.05). SA-β-gal-positive cells were significantly more prevalent in both D-gal-treated groups than in the control group (p<0.05). The expressions of genes p16 and p21 were markedly increased in cells treated with 10 g/L D-gal compared to the control group (p<0.05). The addition of BD did not promote cell proliferation but significantly improved cellular senescence by reducing SA-β-gal activity, p16, and p21 expression (p<0.05) compared to the group without BD. For mineralization potential, the amount of mineralization was similar among groups under differentiating conditions. The reduction of DSPP gene expression was obvious only in the 10 g/L D-gal group (p<0.05). The addition of BD did not show a significant effect on mineralization.
Ten g/L of D-gal can effectively induce aging phenotypes and reduce DSPP gene expression in HDPCs. Co-incubation with BD extract reduced the expression of these aging phenotypes. Mineralization production was not altered in the presence of D-gal. The data support the development of in vitro model for aging dental pulp. (EEJ-2024-07-108).
本研究旨在探讨D-半乳糖（D-gal）对分离的人牙髓细胞（HDPCs）的细胞衰老诱导、细胞增殖、矿化生成及牙源性基因表达的影响。
将分离的HDPCs进行培养并分为四组：对照组、1 g/L D-gal组、10 g/L D-gal组和10 g/L D-gal与生物活性牙本质（BD）共同处理组。使用Alamar Blue®检测法在24、48和72小时评估细胞增殖情况。在48小时评估细胞衰老时，分别采用衰老相关β-半乳糖苷酶（SA-β-gal）染色检测法和定量逆转录聚合酶链反应（qRT-PCR）评估SA-β-gal活性和衰老相关基因（p16和p21）。在分化条件下，于14天采用茜素红S定量染色及矿化相关基因表达（牙本质涎磷蛋白，DSPP）检测来考察矿化潜能。采用单因素方差分析进行统计分析。统计学显著性水平设定为0.05。
与对照组相比，1 g/L D-gal和10 g/L D-gal在72小时时显著降低细胞增殖（p<0.05）。在两个D-gal处理组中，SA-β-gal阳性细胞均显著多于对照组（p<0.05）。与对照组相比，10 g/L D-gal处理的细胞中p16和p21基因的表达显著增加（p<0.05）。添加BD并未促进细胞增殖，但与未添加BD的组相比，通过降低SA-β-gal活性、p16和p21表达显著改善了细胞衰老（p<0.05）。对于矿化潜能，在分化条件下各组间矿化量相似。仅在10 g/L D-gal组中DSPP基因表达的降低明显（p<0.05）。添加BD对矿化未显示出显著影响。
10 g/L D-gal可有效诱导HDPCs出现衰老表型并降低DSPP基因表达。与BD提取物共同孵育可降低这些衰老表型的表达。在存在D-gal的情况下矿化生成未发生改变。这些数据支持了牙髓衰老体外模型的建立。（EEJ - 2024 - 07 - 108）
