Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements

Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers’ instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic ﬁbroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6–8 h. Statistical analyses included Kruskal–Wallis, Mann–Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.


Introduction
Guided endodontic repair refers to regenerative therapies that have as their first priorities: periapical lesions healing, root development promotion, root canal walls thickening, and apical foramen maturation induction to maintain dental pulp vitality. These steps in the repair process are essential to ensure the repaired teeth durability and functionality.
Wound healing and repair depend on angiogenesis to promote neovascularization. 1 The angiogenic response is controlled by the cumulative effects of positive and negative regulatory factors. 2 In particular, a role for a number of polypeptide growth factors has been identified in the initiation of the angiogenic response and regulation of endothelial cell proliferation in wound healing. 3 These factors include basic fibroblast growth factor (FGF-2), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF; which is also designated as vascular permeability factor and fibroblast growth factor). VEGF is considered essential for the vascular system differentiation. 4 Similarly, FGF-2 stimulates new blood vessels growth and development (angiogenesis) that contribute to normal wound healing and tissue development 5 and plays a significant role in the neovascularization of damaged or traumatized tissue, 6 whereas PDGF functions in tissue regeneration and embryogenesis. VEGF production also provides important information regarding cells functionality. 7 Scientific literature indicates that these growth factors possibly participate in the angiogenic response of the dental pulp and periapical tissues; therefore, their role in regenerative or vital pulp therapies needs further exploration. In this respect, a key goal of relevant research should be to discern the interaction between bioactive endodontic materials and the growth factors released during regeneration and/or revascularization, as well as their effects on the angiogenic responses of adjacent tissues.
Guided endodontic repair has been conducted for many years in Dentistry using mineral trioxide aggregate (MTA) and other bioactive endodontic materials. 8,9 MTA has been recognized as the approved gold standard in guided endodontic repair therapies for many years because of its capacity to induce smooth hard tissue deposition with low pulpal inflammation 10 and for its biocompatibility on cells regarding its reparative, regenerative, and angiogenic effects. 11,12 However, novel tricalcium silicate-based cements, such as Biodentine (Septodont,. Saint-Maur-des-Fossés, France), have recently been introduced to overcome the somewhat intolerable drawbacks of MTA, such as its long setting time, 13 difficult handling properties, 13 and tooth discoloration. 14 Biodentine was produced using active biosilicate technology to serve as a bioactive dentin substitute. 13 The mixture is prepared in a powder-to-liquid form in a single-dose capsule, to be mixed with an amalgamator for 30 s. The cement is then applied to the cavity as a bulk dentin substitute without any requirement for adhesive technology. 15    The cells were analyzed by flow cytometry using a

MTS cell viability assays
Cell viability was measured on the 1 st , 3 rd , 7 th , 10 th , and 14 th days, using the 3-(4,5-dimethyl-thiazol-2-yl)-  The hDPSCs group showed the following Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements

ELISA
The hTGSC, hDPSC, and hPLSC groups showed no statistically significant differences in FGF-2, PDGF, and VEGF levels between the first, seventh, and fourteenth days when exposed to the test materials (p>0.05).
The ELISA results for FGF-2, PDGF, and VEGF are shown in Figure 3

Discussion
In this study, due to the known contributions of PDGF, FGF-2, and VEGF to angiogenesis, their release was evaluated from odontogenic stem cells exposed to the tricalcium silicate-based materials Well-Root ST, Biodentine, and ProRoot MTA. 6 Untreated cells were used as a negative control group, and Dycal served as a positive control group because Dycal was previously shown to be cytotoxic to hTGSCs. 20 The MTS results showed no statistically significant difference between the hTGSC groups on the first  The ELISA results also indicated no differences between the cell groups and the tested materials in terms of angiogenic-enhancing potential. A possible explanation for this effect could be differences in the test materials; that is, preparation of the cements in static conditions versus using extracts from set materials. Set materials in static conditions were used to recapitulate the long-term clinical conditions according to the manufacturers' instructions. 16,24,25 Consequently, the effect of the tested materials on cell behavior was similar and minor. Chung, et al. 23 (2016) found that VEGF levels were significantly higher in a ProRoot MTA group than in a control group, but no difference was found between the groups for