Ultracal xs

To simulate RET, calcium hydroxide (Ultracal XS; Ultradent, South Jordan, UT, USA) was placed inside the root canal, sealing the access Cavity with a cotton pellet and a temporary cement (Cavit; 3M Espe St Paul, MN, USA). Teeth were stored at 37 °C in a 100% humidity environment for 4 weeks. After the storage period, the temporary filling was removed, and the root canal was washed with 10 mL of saline. Finally, the canal was left filled with saline. At 6 mm from the CEJ, 1 mm of Teflon was placed, and 4 mm of the hydraulic material under observation was placed over it, according to the distribution shown in Table 1. Then, a humid cotton pellet and temporary cement (Cavit) were placed over it, and the teeth were kept for 48 h in an environment of 100% humidity at 37 °C. After this time, the temporary filling was removed and the access Cavity was sealed with an A1 shade Luna composite resin (SDI, Victoria, Australia) preceded by 37% orthophosphoric acid etching and the application of a universal adhesive system (Zipbond Universal; SDI, Victoria, Australia). The teeth were preserved in individual Eppendorf tubes with a saline solution for the duration of the study, which was changed every month.

Root canals were rinsed with saline, dried, and calcium hydroxide paste (UltraCal^® XS, Ultradent Products, South Jordan, UT, USA) was injected. Teeth were incubated (100% humidity, 37 °C and, 5% CO₂) for 72 h. Calcium hydroxide was removed by irrigation with EDTA, and canals were dried. In analogy to the previous samplings, EDTA was injected, activated as described above, and 100 µL of solution was collected (third sampling).
The concentration of TGF-β1 was quantified for each sample by a solid-phase sandwich ELISA for TGF-β1 (Human TGF-beta 1 Quantikine ELISA Kit, R&D Systems™, Wiesbaden, Germany), which was chosen as a representative growth factor within the mixture of dentin matrix-derived proteins. A previously established approach with a double set of standards was used to accurately calculate growth factor concentrations [3 (link)]. Based on volume and concentration, the mass of TGF-β1 collected from each root canal was calculated. Data from 12 samples per group (n = 12) were analyzed to compute median values and 25–75% percentiles. Furthermore, the obtainable volume per tooth and the number of collections that were necessary to reach 100 µL were determined for all teeth, and median values were calculated (n = 36).