Effect of Adjunctive Ozone Application Protocols on Dentin-Derived Growth Factor Release: An In Vitro Study

Background/Objectives: Regenerative endodontic treatment (RET) depends on the release of dentin-derived bioactive molecules, which is commonly promoted by ethylenediaminetetraacetic acid (EDTA)-based dentin conditioning. However, whether adjunctive ozone delivery protocols can modify the measurable release of dentin-derived transforming growth factor beta 1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) remains unclear. This study evaluated the effects of two adjunctive ozone application protocols used with chelation on dentin-derived TGF-β1 and IGF-1 release, without directly assessing the in situ activation or functional bioactivity of TGF-β1. Methods: Sixty-four freshly extracted human mandibular premolars were randomly assigned to four groups (n = 16). The experimental protocols were as follows: 17% EDTA alone (Group A), 17% EDTA followed by ozonated distilled water and ozone gas (Group B), ozonated 17% EDTA followed by ozone gas (Group C), and a negative control group. Root segments were standardized. In the experimental groups, all external surfaces were coated with nail varnish, leaving only the intracanal dentin surface exposed. In the negative control group, all surfaces were sealed. After ultrasonic activation, the specimens were incubated in phosphate-buffered saline (PBS) at 37 °C. PBS samples were collected on day 1 to evaluate early measurable growth factor release and on day 7 to assess short-term changes in detectable growth factor levels. TGF-β1 and IGF-1 levels were measured by ELISA and normalized to internal dentin surface area derived from micro-computed tomography (micro-CT) analysis. Results: No detectable growth factor values were observed in the negative control group. For TGF-β1, no significant intergroup difference was observed on day 1, whereas a significant difference was found on day 7 (p = 0.022). On day 7, the ozonated EDTA followed by ozone gas group showed approximately threefold higher surface-area-normalized TGF-β1 values than the EDTA followed by ozonated distilled water and ozone gas group (p = 0.018). TGF-β1 values increased from day 1 to day 7 in Groups A and C, whereas no significant temporal change was observed in Group B. IGF-1 values showed no significant intergroup or intragroup differences. Conclusions: Adjunctive ozone application showed a protocol-dependent effect on dentin-derived growth factor values, mainly for TGF-β1, while IGF-1 remained unaffected. The highest TGF-β1 values were observed when ozonated EDTA was followed by ozone gas. However, these in vitro findings indicate measurable growth factor release and should not be interpreted as direct evidence of TGF-β1 activation or clinical regenerative efficacy.