In situ detection of endogenous proteolytic activity and the effect of inhibitors on tooth root surface

Abstract

Objectives: The aim of this study was to clarify the distribution and activity of proteolytic enzymes and examine the inhibitory effects of various substances on this proteolytic activity on tooth root surfaces in situ.

Methods: Disk-shaped bovine tooth root samples were partly pretreated in acid solution (50 mM lactic acid buffer, pH 4.0) for 48 h. The fluorescence intensity of samples was detected with fluorescent substrate solution for collagenase and gelatinase using a stereoscopic fluorescence microscope for 60 min. The acid-pretreated and non-acid-pretreated root samples were treated with chlorhexidine (CHX), sodium fluoride (NaF), epigallocatechin gallate (EGCG), and calcium hydroxide (Ca(OH)₂) for 10 min, and silver diamine fluoride (SDF) for 10, 30, and 60 s. These samples were immersed in the fluorescence substrate solution at pH 7.0, and the fluorescence intensity of samples was detected. The fluorescence intensity was calculated using analysis software.

Results: Gelatinase activity was detected in root samples. Gelatinase activity of the acid-pretreated side was significantly higher than that of the non-acid-pretreated side (1.63 times) at 60 min. CHX, EGCG, Ca(OH)₂, and SDF decreased the gelatinase activity of root samples, while NaF had no effect.

Conclusions: Gelatinase activity was detected from the root in situ and it was increased by acid-pretreatment. CHX, EGCG, Ca(OH)₂, and SDF inhibited gelatinase activity.

Clinical significance: Substances that inhibit proteolytic activity found in this research method will be useful for root caries prevention.

Keywords: In situ; Inhibitor; Proteolytic activity; Root caries.