Chlorhexidine preserves the hybrid layer in vitro after 10-years aging

Objective: The present study investigated the ability of a chlorhexidine (CHX)-containing primer (0.2% aqueous solution) to inhibit dentinal enzymes, preserve the hybrid layer (HL) and remain within the HL, after 10 years of aging in artificial saliva at 37 °C. Methods: Non-carious extracted molars were assigned to two groups, cut into slabs exposing middle/deep dentin, etched and bonded with Adper Scotchbond 1XT (SB1XT) with or without 0.2% CHX aqueous solution pretreatment. Composite build-ups were made, and the specimens were cut in 1-mm thick bonded sticks. In situ zymography was performed on freshly prepared specimens (T0) and specimens aged for 10 years (T10-yr) at 37 °C in artificial saliva, to investigate endogenous gelatinolytic activity within the HL. At T10-yr, specimens were also decalcified and embedded in epoxy resin for TEM analysis. Micro-Raman spectroscopy was performed at T0 and T10-yr to evaluate the chemical profiles in intertubular dentin and the HL. Results: In situ zymography showed less pronounced enzymatic activity in the CHX-pretreated group (p < 0.05) regardless of aging, maintaining a similar level of fluorescence at T0 and T10-yr (p > 0.05). TEM results showed that 98% of the HL had been degraded in the control group, while 95% of the HL was intact in the experimental group. Moreover, all the Raman spectra peaks assigned to CHX could be identified only in the CHX-pretreated group (T0 and T10-yr). Significance: In vitro, CHX remains in the HL after 10 years with its inhibitory effect preserved. This may be the underlying factor for HL preservation after this long aging period.