A Novel Prime-and-rinse Agent For Increasing The Dentin Bond Durability Of Self-etch Adhesives

Part 1:A novel prime-and-rinse approach for increasing the dentin short-and long-term dentin bond strengths of a self-etch adhesive using MDP and MMPs inhibitors.Objectives:This study investigated the effects of the novel prime-and-rinse approach for the short-and long-term dentin bond strengths of a self-etch adhesive and the micro-morphologies of resin-dentin interfaces using MDP (10-methacryloxydecyl dihydrogen phosphate) and matrix metalloproteinase (MMPs) inhibitors (benzalkonium chloride, BAC; polyvinylphosphonic acid, PVPA; proanthocyanidin, PA).Materials and Methods:Eight novel prime-and-rinse agents were prepared using MDP and MMPs inhibitors as following:5%MDP,5%MDP-BAC,5%MDP-PVPA, 5%MDP-PA,15%MDP,15%MDP-BAC,15%MDP-PVPA,15%MDP-PA(wt) (The concentrations of BAC, PVPA and PA were 1%wt, 1000μg/ml and 15%wt, respectively). Fifty-four human third molars were randomly assigned to 9groups (n=6 teeth). The mid-coronal dentine surfaces were grounded with 320-grit SiC paper under running water. The dentin surfaces of the teeth were applied with or without (control) one of the eight prime-and-rinse agents for 30 s, water-sprayed for 30 s and gently air-dried (prime-and-rinse approach). Afterwards, the dentin surfaces were treated with a self-etch adhesive (Clearfil S3 Bond, Kuraray Noritake, Tokyo, Japan) for 20 s, strong air-dried for 5 s and light-cured (Radii Plus, SDI, Australia, output 1500 mW/cm2) for 10 s. Subsequently, the composite-resin Majesty was placed on the adhesive-treated dentin surfaces in 1-mm increment each and light-cured for 20 s to create a 4-mm high resin crown. After storage in water for 24 h, all the specimens were perpendicularly sectioned through the resin-dentin interface with the low-speed saw under continuous water cooling. Afterwards, they were prepared into multiple beams of about 1mm×1 mm×8mm. After half beamseach tooth were stored inwater after 24 h or 1 y, the micro-tensile bond strengths (MTBS) tests were performed using Bisco Micro Tensile Tester. The failure mode of the fractured samples was determined by a stereo-microscopy at 50-fold magnifications. Eight beams each subgroup were prepared for SEM test. The resin-dentin interfaces of the specimens were analyzed by the SEM. Thirty-six human third molars were prepared as the above-mentioned for the TEM tests. The MTBS data were analyzed by factorial design ANOVA followed by Post-hoc LSD for multiple comparisons (SPSS 22.0).Results:Compared with the control group,5%MDP groups (5%MDP,5%MDP-PVPA, 5%MDP-PA) except for 5%MDP-BAC could not significantly increase the short-term dentin MTBS (P>0.05), while 15%MDP groups (15%MDP,15%MDP-BAC, 15%MDP-PVPA,15%MDP-PA) and 5%MDP-BAC could significantly increase the dentin MTBS after 24-hr water storage (P<0.05). Compared with the control, the prime-&-rinse approach using MDP and MMPs inhibitors could significantly increase the long-term dentin MTBS (P<0.05). Among them,15%MDP-BAC achieved similar dentin MTBS of 15MDP-PVPA (P>0.05), but significantly higher dentin MTBS than the others (P<0.05). The dentin MTBS of the control group significantly declined after 1-yr water storage (P<0.05), but the dentin MTBS did not when the prime-&-rinse approach used (P>0.05). The SEM findings revealed that the micro-gaps could be observed along the resin-dentin interface after 1-yr water storage in the control group, but not when the prime-&-rinse approach used. The TEM findings revealed that the dentin smear layer was completely removed after the application of prime-&-rinse approach. The TEM findings revealed that in the control group the micro-morphology of the dentin smear layer was not uniform with some micro-gaps after 1-yr water storage, however, the hybrid layers did not significantly change. The adhesive layer, structure-less and non-structural layer and the hybrid layers could be detected in the resin-dentin interfaces. The abundant hydroxyapatite crystallites were still in the hybrid layers.Conclusions:Using the novel prime-and-rinse agents of 5%MDP-BAC,15%MDP, 15%MDP-BAC,15%MDP-PVPA or 15%MDP-PA could greatly increase the short-term and long-term dentin bond strengths. The dentin bond strengths would not decrease at least for one year. The prime-&-rinse approach could be used as a novel supplement to the contemporary self-etch approach.Part 2:The effect of the novel prime-and-rinse agents on the in activation of the soluble MMPsObjectives:This study investigated the inhibitory abilities of the novel prime-and-rinse agents on the soluble matrix metalloproteinase (MMPs).Materials and Methods:The rhMMP-8 and-9 (BioLegend Inc. San Diego, CA. USA) were incubated in the 10μg/ml trypsin solutions for 2 hours. The assay was performed in four replicates in a 96-well plate for each prime-&-rinse agent and control group. Eight prime-&-rinse agents were prepared as above-mentioned (5%MDP, 5%MDP-BAC,5%MDP-PVPA,5%MDP-PA,15%MDP,15%MDP-BAC,15%MDP-PVPA and 15%MDP-PA(wt). Each test compound group consisted of 2μl of rhMMPs (19.6 ng/well), 10μl of one prime-and-rinse agent,38μl assay buffer and 50μL of the thiopeptolide substrate solution (The Sensolyte Generic MMP colorimetric assay kit supplied, AnaSpec Inc., Fremont, USA). The control groups included:a positive control group containing 2μl rhMMP; an inhibitor control group containing 2μl rhMMP and 10μl of the 20μM GM6001 (i.e. Galadin), a known MMP inhibitor supplied in the assay kit; a test compound control group containing 10μl of one prime-and-rinse agent; a substrate control group containing 50μl assay buffer. Additional assay buffer was added to bring the totalvolume of each control group to 50μl prior to adding 50μl of the thiopeptolide substrate solution to obtain 100μl/well. The reagents were mixed and measured the absorbance at 412 nm at 10 min for six times by means of a plate reader. The mean absorbances were used for the percentage of MMP inhibition. Percentage of MMP inhibition=1-(absorbance of test compound group-absorbance of test compound control group)/(absorbance of the positive control group-absorbance of substrate control group). The percentage inhibition was analyzed using one-way ANOVA on ranks and Tukey multiple comparison tests at α=0.05.Results:The inhibitory rate of 5%MDP of inactivating rhMMP-8,9 was significantly lower than the other seven prime-and-rinse agents and the GM6001 group (P<0.05). The rhMMP-9 inhibition of the other seven groups were not significantly different from the GM6001 group (P>0.05). The rhMMP-8 inhibition of 15%MDP-PVPA and 15%MDP-PA groups were significantly higher than the GM6001 inhibitor(P<0.05).Conclusions:All the novel prime-and-rinse agents containing the MMPs inhibitors possessed the significantly high inhibitory rate of MMPs.Part 3:The novel prime-and-rinse agents resisted the resin-dentin degradation by the collagenasesObjectives:This study investigated the effects of the novel prime-and-rinse agents (15%MDP,15%MDP-BAC,15%MDP-PVPA) on the dentin MTBS of the self-etch adhesives and the micro-morphologies of resin-dentin interfaces before and after storage in collagenase solutions.Materials and Methods:One hundred and eight human third molars were randomly divided into 12 group according to 3 different adhesives and 4 different treatments. They included 3 self-etch adhesives:AdperTM Easy One,3M ESPE, USA; i Bond, Heraeus Kulzer GmbH, Germany; G-Bond, GC, Japan and 3 prime-&-rinse agents (15%MDP,15%MDP-BAC,15%MDP-PVPA),The mid-coronal dentine surfaces were grounded with 320-grit SiC paper for the uniform smear layer. The dentin surfaces were applied with or without (control) one of three prime-&-rinse agents for 30 s, water-sprayed for 10s and gently air-dried (prime-and-rinse approach). Afterwards, the dentin surfaces were treated with the self-etch adhesives (AdperTM Easy One, i Bond, G-Bond) and placed with the respective composite resin (FiltekTM Z250; Solitaire 2; Gradia Direct)from the same manufacturers according to the manufacturer’s instructions. After 72 specimens were kept in the tap water for 24hr at 37℃, they were prepared into multiple beams of about 1mm×1mm×8mm. The beams each tooth were randomly divided into three subgroups:control, type Ⅰ, typeⅡ collagenase’s group. The dentin MTBS were performed after the specimens were stored in 2mg/ml type Ⅰ, Ⅱ collagenase solution or water at 37℃ for 24h, respectively, After another 36 specimens were prepared as above-mentiond, they were prepared for ultra-sections for TEM observations. The MTBS data were analyzed by factorial design ANOVA followed by Post-hoc LSD multiple comparison (SPSS 22.0)Results:The dentin MTBS of three self-etch adhesives in descending order were listed as follows:AdperTM Easy One>i Bond>G-Bond (P<0.01). Compared with the control, the prime-&-rinse approach using MDP, MDP-BAC or MDP-PVPA could significantly increase the dentin MTBS (P<0.05). The MTBS of the control groups in three self-etch adhesives all significantly declined after incubation with type I and II collagenase’s solutions (P<0.05), however, the experimental groups still possessed significantly higher MTBS than the control group after enzymolysis (P<0.005). The anti-degaradation by collagenases of three prime-and-rinse agents in descending order were listed as follows:MDP-BAC>MDP-PVPA>MDP. The TEM findings revealed that in the control group the smear layer was loose with some micro-gaps and some substrate dissociated, in the experimental groups the hybrid layers were relatively uniform and integrated without gaps, along with abundant hydroxyapatite.Conclusions:Prior to the application of the self-etch adhesives, using thenovel prime-and-rinse agents could greatly increase the resistant ability of degradation by the collagenases and the dentin bonding strengths, and the MDP-BAC had the best ability, whichprovided experimental basis for improving the durability of dentin bonding.