Anti-proteolytic Property And Bonding Durability Of Mussel Adhesive Protein-modified Dentine Adhesive Interface

Objectives Contemporary adhesive systems,etch-and-rinse and self-etch adhesive systems,both fail to completely infiltrate the demineralized dentin matrix.Collagen fibrils are exposed without any protection of hydrophobic resin coatings along the bottom of the hybrid layer.Thus,naked collagen fibrils are vulnerable to degradation by the host-derived enzymes and finally fail to achieve the strong and durable resin-dentin bond strength.Therefore,this study aimed to evaluate the influence of mussel adhesive protein(MAP)application on collagenase activity,dentin collagen degradation and microtensile dentin bond strength(μTBS).Methods The influence of MAP on collagenase activity was measured by generic colorimetric assay.The experimental group was 1mg/ml MAP and 100U/ml collagenase(Clostridiopeptidase-A,Type VII).The positive control group was GM6001collagenase-inhibitor and 100U/ml collagenase.The negative control group was distilled water(DW)and 100U/ml collagenase.Each group contained five sub-groups.All reagents were mixed well in each experimental well and added the colorimetric substrate solution.After pre-incubation,the absorbance was read at 412 nm.Thirty human dentin slabs were prepared and allocated into 3 groups(n=10)for application of MAP,GM6001 collagenase-inhibitor and DW.After incubation in collagenase solution for 7 days,degradation of dentin collagen matrix was evaluated by measuring hydroxyproline released from the slabs.Another 60 dentin slabs were pretreated by either MAP,GM 6001 collagenase-inhibitor or DW followed by etch-and-rinse adhesive to nano-hybrid composite resin(n=20 in each group).μTBS testing was performed before and after thermocycling(×2,500 at 5°C and 55°C)and collagenase challenge(3weeks at 37°C).The morphology of dentin/adhesive interfaces or the fractured interfaces were examined by scanning electron microscopy(SEM).Results The collagenase activities(nmol/min/mg)of the MAP,GM6001 and DW group were 20.22±0.93,29.11±1.17 and 31.39±0.52,respectively(p<0.01).Their hydroxyproline concentrations(μg/m L)were 2.70±0.53,4.00±1.19 and 5.40±1.00,respectively(p<0.01).While there was no significant difference in the μTBS of the MAP,GM6001 and DW group,their μTBSs(MPa)after thermocycling and collagenase challenge were 11.39±2.52,10.77±3.16 and 5.83±2.02,respectively(p<0.001).SEM observation showed consistent results.The bonding interfaces detected before the immediate μTBS test showed that collagen fibres in the MAP group were hardly identified,while a few fibres were observed in the GM6001 and DW group.After thermocycling and collagenase challenge,the integrity of the bonding interfaces was broken.The collagen fibres in the MAP and GM6001 group had slighter degradation than it in the DW group.Meanwhile,in the DW group,larger spaces existed between the resin tags and dentinal tubules,suggesting that more collagen fibres had degraded.SEM of the fractured dentin surface revealed that fractures in the MAP and GM6001 group frequently occurred at the top of the hybrid layer,leaving the underlying dentin covered by adhesives.However,in the DW group,failures occurred at the bottom of the hybrid layer with numerous resin tags tightly filling the dentinal tubules.Conclusion MAP inhibits collagenase activity and prevents dentin collagen degradation.It may retard the deterioration of the dentin bonding of composite restoration over time.