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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. Objective 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. (PDLMSCs heterogeneous disease osteoblastcementoblast cementoblast OC O C (O/C (ASPN mineralization BMP2induced BMPinduced BMP induced 1,25OH2D3 125OH2D3 OHD 1,25 OH 2D3 25 1,25(OH)2D LOP, , (LOP) protein2 protein protein- alteration CONTROL, CONTROL (CONTROL) OM, OM (OM) VD. VD . VD) performed ASPN, BMP2, BMP2 (BMP-2) runtrelated runt RUNX2 RUNX (RUNX2) ALP (ALP) OCN, (OCN) (VDR qRTPCR qRT PCR qRT-PCR 3 BMP- 4 Staining twoway two way Tukeys Tukey s P<0.05. P005 P P<0.05 0 05 (P<0.05) viability culture P<0.05, parallel 25OH2D3 1,25OH2D 125OH2D 125 1,2 2D (LOP (CONTROL (BMP-2 (RUNX2 (ALP (OCN P00 P<0.0 (P<0.05 25OH2D 12 1, (BMP- (RUNX P0 P<0. (P<0.0 (BMP P<0 (P<0. P< (P<0 (P< (P