Comparative histological and immunohistochemical analysis of new tissues after conditioning root canal dentin with different acids in regenarative endodontic treatment using dpsc seeding implanted in mice dorsum

The primary goal of the regenerative endodontic treatment is to regain vitality and function of pulp-dentin complex which lost due to trauma or microbial contamination. By this way, root development will continue and teeth will function as an organ in its lifespan. EDTA has been considered the gold standard in regenerative endodontic treatments. The aim of this study was to evaluate the effects of different dentin conditioning agents alternative to EDTA on released growth factors (TGF-β1, FGF , VEGF ve BMP-2) and investigate their effects on the newly formed tissues in vivo by histochemical and immunohistochemical methods. In the in vitro part of the study coronal sections of extracted human impacted third molar teeth were used. Crowns were seperated from roots and splitted from its half by cutting corono-apical direction and cut into a disc shape (approximately 1-mm thick) horizontally with isomet saw under sterile saline irrigation. First half of the dentin discs prepared from half of the crowns were immersed in the acids and grinded into dentin powder. Other half of the dentin discs prepared from other half of the crowns were conditioned with the same acids and medium containing human mesenchymal stem cells (MSCs) were seeded onto dentin discs for three days. After three days of incubation period, dentin discs were removed and grinded into dentin powder. Dentin powders were tested by ELISA kits for TGF-β1, FGF-2, VEGF and BMP-2 quantification. In the first half of the groups, highest TGF-β1 release was seen in citric acid group, highest VEGF release was seen in phoshoric acid group, highest BMP-2 and FGF-2 release was seen in the phytic acid group. In the second half of the groups highest TGF-β1, FGF-2 and BMP-2 release was seen in the phoshoric acid group while highest FGF-2 release was seen in the EDTA group. Three days of MSC seeding after dentin conditioning has made a dramatic increase in all of the growth factors. In the in vivo part of our study, root sections of extracted human incisor teeth were used. Crowns sections of extracted teeth were removed from roots under water cooling. Root canals were enlarged by endodontic files and approximately 3-4 mm length of root fragements were cut under water cooling. Root fragments were irrigated with the groups including control and acid solutions (distilled water, EDTA, citric acid, phytic acid, phosphoric acid). After irrigation narrow end of the canals were sealed with 1-2 mm thickness of MTA. In the first half of the groups only PLG scaffolds were inserted into the root fragments. In the second half of the groups, MSCs were seeded onto PLG scaffolds and then inserted into root fragments. Root fragments were implanted into the subcutaneous space of immunodeficient mice dorsum (2 teeth for each mice). Two months after implantation, mice were sacrificed and root fragments were evaluated by histochemical and immunohistochemical stainings. Pulp-like tissues were observed in the groups which include acid application and MSC seeding. The highest density stainings with odontoblast markers were observed in the phosphoric acid + MSC application group. We conclude that phosphoric acid could be an alternative conditioning agent to EDTA, which is considered as golden standard, in regenerative endodontic treatments.