In vivo and in vitro radiotherapy increased dentin enzymatic activity

Objectives: The present study investigated the effects of in vitro and in-vivo radiotherapy on endogenous enzymatic activity in dentin using gelatin zymography and in-situ zymography. Methods: Gelatin zymographic assays were performed on protein extracts obtained from dentin powder of sound non-irradiated (NRT), in vitro irradiated (VTRT) and in vivo irradiated (VIRT) human teeth. Their proteolytic activities were quantified using band densitometric evaluation. For in-situ zymography, dentin specimens from NRT, VIRT and VTRT were covered with fluorescein-conjugated gelatin and examined with confocal laser-scanning microscopy. Fluorescence intensity emitted by the hydrolyzed fluorescein-conjugated gelatin was quantified and statistically analyzed. In-situ zymography data were statistically analyzed using Kruskal-Wallis ANOVA and Dunn's multiple comparison procedures (α = 0.05). Results: No difference between in vitro and in vivo radiotherapy treatment was found. Both VTRT and VIRT groups showed increase in MMP-9 expression when compared to NRT group. Significant increases (p < 0.05) in gelatinolytic activity (26 % for VTRT; 55 % for VIRT) were observed when compared to the NRT group. Conclusion: Radiotherapy increase endogenous enzymatic activity in non-restored dentin.