Odontoblastic differentiation and mineralized matrix deposition by dental pulp stem cells seeded on nanofibrous scaffolds associated to bioactive substances

Abstract

In this study, the odontoblastic differentiation of dental pulp stem cells (DPSCs) and their potential to deposit mineralized matrix after being seeded on nanofibrous scaffolds treated or not with bioactive molecules will be assessed. For this purpose, Poli (L-latic acid) nanofibrous scaffolds (NF-PLLA) and nanofibrous hollow microspheres from star-shaped PLLA (HNF-SSPLLA) will be fabricated and placed at the bottom of 24-well plates. Then, DPSCs from human teeth will be seeded onto the scaffolds (1,000.000 cells), and the cell/scaffold construct will be incubated for 24 hours at 37oC and 5% CO2 in complete culture medium (alpha-MEM). After this period, the culture medium will be replaced by complete ±-MEM osteogenic medium (with arcorbic acid and betha-glycerophosphate), containing 10-8 M dexametasone and 10-6 M simvastatin, alone or in association. Osteogenic ±-MEM without bioactive substances will be used as control group. The cells will be incubated up to 4 weeks in the different experimental conditions, and the cell proliferation (DNA content) and morphology (SEM) will be assessed every week. Also, after 1 or 2 weeks, the alkaline phosphatase activity (p-nitrophenyl phosphate assay) will be assessed, and the calcium content (calcium assay kit) will be analyzed after 4 weeks. Gene expression of odontoblastic markers (OCN, Col I and DSPP - Real Time PCR) will be assessed at 1, 2 and 4 weeks. Histological analysis of cell migration into the scaffolds (H&E), collagen deposition (Masson's trichrome) and calcium deposits (Von Kossa), as well as immunohistochemical analysis of DSP expression will be performed in the cell/scaffolds constructs after 4 weeks in culture. Data will be subjected to specific statistics tests. (AU)