1,25(OH)2D3 increase osteogenic potential of human periodontal ligament cells with low osteoblast potential

Abstract

Objective: 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.

Methodology: 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).

Results: 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).

Conclusions: 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.

Graphical abstract

Figure 1. (A) Effect of 1,25(OH)2D3 on metabolic activity. Three LOP cell populations (LOP 1, LOP 2, and LOP 3) were cultured in standard medium (CONTROL), osteogenic medium (OM), and OM + 1nM of 1,25(OH)2D3 (OM + VD). MTT assay to assess cell metabolism and viability was performed at one, three, seven and 10 days. The control represents 100% of viability. (B) VDR expression after 1,25(OH)2D3 treatment. Real-time PCR analysis after treatment confirmed that all three populations express mRNAs for VDR. Experiments were performed in triplicate three times, with comparable results obtained on each occasion. Bars represent mean ± standard deviation (SD), intergroup analysis statistical differences are indicated by different lowercase letters, and intragroup statistically significant differences are indicated by different uppercase letters. The letter “X” represents the difference in relation to day one, while the letter “Y” represents the difference in relation to day three and “Z'” to day seven (P<0.05).

Figure 2. Real-time PCR analysis indicated a downregulation of the mRNA levels for ASPN (A), and an increased level of transcripts for BMP-2 (B) after the treatment with 1nm of 1,25(OH)2D3. (C) Correlation between BMP-2 and ASPN on day 14. Experiments were performed in triplicate three times, with comparable results obtained on each occasion. Bars represent mean ± standard deviation (SD), intergroup analysis statistical differences are indicated by different lowercase letters, and intragroup statistical significant differences are indicated by different uppercase letters. The letter “Y” represents the difference in relation to day three, while “Z” represents a difference in relation to day seven. (P<0.05)

Figure 3. Osteoblast genes expression after 1,25(OH)2D3 treatment. Real-time PCR analysis after treatment indicated that 1,25(OH)2D3 upregulated the expression of mRNAs for RUNX2 (A), ALP (B), and OCN (C). Bars represent mean ± standard deviation (SD), intergroup analysis statistical differences are indicated by different lowercase letters, and intragroup statistical significant differences are indicated by different uppercase letters. The letter “Y” represents the difference in relation to day three, while “Z” represents a difference in relation to day seven. (P<0.05).

Figure 4. BMP-2 protein expression. Quantification of BMP-2 secreted in supernatant of LOP 1, LOP 2, and LOP 3 cells after three, seven, and 14 days of osteogenic induction associated with 1,25(OH)2D3. Bars represent mean ± standard deviation (SD), intergroup analysis statistical differences are indicated by different lowercase letters, and intragroup statistical significantly differences are indicated by different uppercase letters. The letter “Y” represents the difference in relation to day three, while “Z” represents a difference in relation to day seven. (P<0.05)

Figure 5. Effect of 1,25(OH)2D3 on mineral nodule deposition. (A-C) Microscopic of Alizarin Red Staining (AR-S) mineralization of LOP 1, LOP 2, and LOP 3, respectively. Scale bar = 40µm. (D-F) Quantification of AR-S at 28 days. Experiments were performed in triplicate three times, with comparable results obtained on each occasion. Bars represent mean ± standard deviation (SD) and intergroup analysis statistical differences are indicated by different lowercase letters (P<0.05).