Study On The Effect Of Citrus Flavonoids On Dentin Bonding Durability

Objects:Degradation of dentin collagen in hybrid layer(HL)is believed to be the most important factor of resin-dentin bonding interface destruction.Protecting the denuded collagen fibrils in HL is an effective method to improve the stability of dentin interface.Researches have demonstrated that citrus flavonoids could inhibit the degradation of dentin collagen,and keep collagen stable,indicating the possibility of citrus flavonoids to be used to stable the interface of resin-dentin bonding.There are various kinds of citrus flavonoid and it is essential to study the relationship between structure and their collagen protective effect,analyzing the effect of methylation and glycosylation on collagen stability.This study aimed to detect the effect of three kinds of citrus flavonoids,including Hesperidin(Hsd),hesperetin(Hst)and naringenin(Nge),on soluble and matrix-bound Matrix Metalloproteinases(MMPs)activity,exploring the functional group in there anti-MMPs ability;What’s more,pre-treated the completely demineralized dentin collagen with citrus flavonoids and evaluated the change of biological and physicochemical property of collagen,analyzing the effect of citrus flavonoids on structure of dentin collagen and exploring the mechanism of their protective effect on collagen,in order to provide the new thoughts and methods to improve dentin bonding durability.Methods:1.A fluorometric assay was used to measure the inhibitory effects of citrus flavonoids(Hst,Hsd,Nge)at different concentrations on soluble collagenase.Analyzed the effect of methylation and glycosylation on their anti-MMPs ability,seeking for the functional group and possible mechanism in there anti-MMPs ability.2.Matrix-bound endogenous MMPs activity was evaluated via dry mass loss and hydroxyproline(HYP)release of citrus flavonoids pretreated demineralized human dentin after aging for 15 days.Ultrastructural change of demineralized collagen fibers was exhibited by Transmission Electron Microscopy(TEM).Dentin slabs were used for in situ zymography and evaluated under confocal microscopy in order to further investigate citrus flavonoids’ effect on matrix-bound MMPs activity.3.Completely demineralized dentin matrix were treated by 500 μg/mL citrus flavonoids(Hst,Hsd,Nge)for 60 min.Then elasticity modulus and swelling ratio of dentin matrix,amount of hydroxyproline(HYP)releasing from collagen under collagenases digestion were evaluated.The spectra of demineralized dentin collagen before and after citrus flavonoids treatment were obtained and evaluated by Attenuated total reflection–fourier transform infrared spectroscopy(ATR-FTIR).Dentin slabs acid-etched by phosphoric were pretreated with the three citrus flavonoids,then air-dried and the configuration of exposed dentin network were observed under Scanning Electron Microscopy(SEM).Results:1.Citrus flavonoids exhibited inactivation function on soluble MMPs and the extent of inactivation increased in a dose-dependent manner.The inactivation percent of citrus flavonoids reached above 90% at the concentration of 500 μg/mL and there were no statistical difference among three experimental groups.2.Compared with control group,citrus flavonoids pretreated demineralized dentin beams exhibited less dry mass loss,lower hydroxyproline release and more intact collagen architecture after 15 days storage.Dentin samples pretreated with citrus flavonoids showed lower enzymes activities in in situ zymography.These results indicated that Hst、Hsd、Nge could also inhibit matrix-bound MMPs activity and there were no statistical difference among three experimental groups.3.After pretreated with Hst,Hsd,Nge,elasticity modulus of dentin matrix increased,swelling ratio reduced,and the amount of HYP release reduced comparing with blank control group.There were no statistical difference among three experimental groups.This treatment did not alter the structure and integrity of collagen triple helix according to the results of ATR-FTIR.After air-dried,exposed dentin collagen did not collapse or fuse together,maintaining its native network structure.These findings indicated that citrus flavonoids could inhibit enzymatic degradation effect on collagen through their collagen biomodification and stable dentin matrix.To sum up,citrus flavonoids could inhibit activity of MMPs and biologically modify the dentin collagen,so the collagen could be protected from being degraded by enzymes.These results indicate that citrus flavonoids have the potential to increase resin-dentin bonding durability.