Universidade Estadual de Campinas - UNICAMP, Faculdade de Odontologia de Piracicaba, Piracicaba, SP, Brasil.
J Appl Oral Sci. 2024 Nov 22;32:e20240160. doi: 10.1590/1678-7757-2024-0160. eCollection 2024.
Periodontal dental ligament mesenchymal stem cells (PDLMSCs) play a major role in periodontal tissue regeneration by the neoformation of root cementum and alveolar bone. These cells are highly heterogeneous, and many present low potential to renovate the hard tissue damaged by periodontal disease. A previous study found that the low osteoblast/cementoblast (O/C) differentiation potential of PDLMSCs is related to high asporin (ASPN) expression, which was identified as a negative regulator of PDL cells differentiation and mineralization, suppressing BMP-2-induced O/C differentiation. This study aimed to investigate whether 1,25(OH)2D3 treatment could stimulate the O/C differentiation of periodontal ligament mesenchymal progenitor cells characterized as low osteoblast potential (LOP), by asporin and bone morphogenetic protein-2 alteration.
Three LOP cell populations were cultured in standard medium (CONTROL), osteogenic medium (OM), and osteogenic medium associated with 1 nM of 1,25(OH)2D3 (OM + VD). The following assays were performed: 1) MTT to evaluate metabolic activity; 2) gene expression for asporin (ASPN), bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and vitamin D receptor (VDR) using qRT-PCR; 3) BMP-2 extracellular expression; and 4) quantification of mineralized nodule deposition by Alizarin Red Staining. Data were subjected to two-way ANOVA and Tukey's test (P<0.05).
The results showed that the 1,25(OH)2D3 treatment did not affect the cell viability, as demonstrated by metabolic activity increase over the 10 days in culture. After 14 days of 1,25(OH)2D3 treatment, the mRNA levels for ASPN and VDR decreased (P<0.05), while BMP-2 transcripts and extracellular expression increased (P<0.05). In parallel, RUNX2, ALP, and OCN gene expression was upregulated by 1,25(OH)2D3 treatment, resulting in an increase of mineral nodule deposition in vitro (P<0.05).
These data show that 1,25(OH)2D3 improves osteoblast/cementoblast differentiation of low osteoblast potential accompanied by alterations in ASPN and BMP-2 expression.
牙周韧带间充质干细胞（PDLMSCs）通过新生牙根骨质和牙槽骨在牙周组织再生中起主要作用。这些细胞高度异质，许多细胞重建牙周病损伤的硬组织的潜力较低。先前的研究发现，PDLMSCs 的低成骨细胞/成牙骨质细胞（O/C）分化潜能与高 asporin（ASPN）表达有关，ASPN 被鉴定为 PDL 细胞分化和矿化的负调节剂，抑制 BMP-2 诱导的 O/C 分化。本研究旨在探讨 1,25(OH)2D3 治疗是否可以通过改变 asporin 和骨形态发生蛋白-2 来刺激具有低成骨潜力（LOP）的牙周韧带间充质祖细胞的 O/C 分化。
在标准培养基（CONTROL）、成骨培养基（OM）和与 1 nM 1,25(OH)2D3 相关的成骨培养基（OM+VD）中培养三个 LOP 细胞群。进行以下测定：1）MTT 评估代谢活性；2）qRT-PCR 检测 asporin（ASPN）、骨形态发生蛋白-2（BMP-2）、 runt 相关转录因子 2（RUNX2）、碱性磷酸酶（ALP）、骨钙素（OCN）和维生素 D 受体（VDR）的基因表达；3）BMP-2 细胞外表达；4）茜素红染色定量矿化结节沉积。数据采用双因素方差分析和 Tukey 检验（P<0.05）。
结果表明，1,25(OH)2D3 处理在培养的 10 天内并没有影响细胞活力，表现为代谢活性增加。1,25(OH)2D3 处理 14 天后，ASPN 和 VDR 的 mRNA 水平降低（P<0.05），而 BMP-2 转录物和细胞外表达增加（P<0.05）。同时，1,25(OH)2D3 处理上调 RUNX2、ALP 和 OCN 的基因表达，导致体外矿化结节沉积增加（P<0.05）。
这些数据表明，1,25(OH)2D3 通过改变 ASPN 和 BMP-2 的表达来改善低成骨潜能的成骨细胞/成牙骨质细胞分化。
