To evaluate the effect of mussel adhesive protein (MAP) on collagenase activity, dentin collagen degradation and microtensile dentin bond strength (μTBS).
Three groups were designed: 1. experimental group: treated with MAP; 2. positive control: treated with GM6001 (collagenase-inhibitor); 3. negative control: treated with distilled water (DW). For collagenase activity, Clostridiopeptidase-A was added to each group (n = 5), and collagenase activity was assessed by colorimetric assay. For dentin collagen degradation, thirty dentin slabs were allocated to the three above groups (n = 10). Dentin collagen degradation was evaluated by measuring released hydroxyproline by colorimetric assay after being incubated in Clostridiopeptidase-A for 7 days. For microtensile bond strength, sixty human third molars with flat dentin surfaces were etched by phosphoric acid and then assigned to the three above groups (n = 20). An etch-and-rinse adhesive system was applied to all three groups as stated in standard clinic protocol. The test of μTBS was performed before and after thermocycling and collagenase challenge.
The collagenase activities (nmol/min/mg) in the group of MAP was significantly less inactive compared to the group of GM6001 and DW (MAP < GM6000 < DW, p < 0.01). The hydroxyproline concentrations (μg/mL) was significantly less in the group of MAP compared to the group of GM6001 and DW (MAP < GM6000 < DW, p < 0.01). While there was no significant difference in the immediate μTBS (MPa) among three groups (p > 0.06), the value of μTBSs after thermocycling and collagenase challenge was significantly greater in the group of MAP and GM6001 compared to the group of DW (MAP, GM6000 > DW, p < 0.001).
MAP inhibits collagenase activity, prevents dentin collagen degradation, and delays the deterioration of the dentin bonding of composite restoration over time.
Resin composite is widely used in dental restoration due to its superior aesthetics and excellent immediate bond strength ; however, long-term success and durability of the resin-dentin bond strength is challenging due to the degradation of the hybrid layer . Hydrolysis of adhesive resin and disorganization of collagen fibrils are the main reasons for the deterioration of the hybrid layer . Many etch-and-rinse and self-etch adhesive systems fail to completely infiltrate the demineralized dentin matrix . Collagen fibrils are exposed with no hydrophobic resin coatings at the bottom of the hybrid layer. These exposed collagen fibrils are vulnerable to proteases, leading to the degradation of the hybrid layer and the failure of the restoration. The proteases include reactivated endogenous matrix metalloproteinases (MMPs) from dentin , and other collagenases from bacteria or saliva .
To maintain the durability of the bond strength, researchers have explored different methods to inhibit protease activity and/or stabilize the ultrastructure of dentin collagen. Protease-inhibitors, such as chlorhexidine , ethylenediaminetetraacetic acid (EDTA) and doxycycline , act by chelating calcium and zinc ions, which are the essential cations to activate MMPs and bacterial collagenases . Although chlorhexidine and EDTA prevent collagen degradation in the hybrid layer, both of them are water-soluble and easily dissolved in oral cavity , which limit their long-term effects. Doxycycline could discolor the adhesive materials , which affects the aesthetic outcome. Other protease-inhibitors, such as glutaraldehyde, riboflavin and proanthocyanidin , inactivate the active sites of proteases and act as cross-linking agents to increase the stiffness of collagen ; however, these proteases also have their limitations. For example, the use of glutaraldehyde has been limited due to its cytotoxicity , while the cytotoxicity of riboflavin is unclear up until now . The incorporation of proanthocyanidin into the adhesive was reported to affect the polymerization of resin .
Mussel adhesive protein (MAP), a proteinaceous adhesive secreted by marine mussels, is a key component in the firm attachment of marine mussels to wet surfaces. MAP provides the mussels’ resistance to mechanical stresses which arise from tough and adverse marine environments, such as persistent turbulence, tidal waves, and fluctuation of temperature . The extraordinary adhesive property of MAP is attributed to the 3,4-dihydroxy-phenylalanine (DOPA) residues. DOPA contains catechol groups that possess hydrogen bonding and metal-liganding capabilities . Scientists recently developed various DOPA-derivative adhesives and found exceptional adhesive abilities to dentin and bone ; however, no study has so far reported on the enzyme-inhibiting properties of MAP. The aim of this study is to evaluate the effects of MAP on collagenase activity, dentin collagen degradation and resin-dentin bond strength. We hypothesized that MAP can (i) inhibit the activity of collagenase, (ii) prevent dentin collagen degradation, and (iii) finally improve the durability of the resin–dentin adhesion.
Materials and methods
The MAP (recombined mussel adhesive protein, Abcam, Cambridge, UK) was prepared at a concentration of 1 mg/mL. It was adjusted to a typical marine environmental (pH of 8.5 with 0.1 M sodium hydroxide) . The collagenase was prepared by adding lyophilized powder of Clostridiopeptidase-A in an incubation medium containing zwitterionic organic chemical buffering agent to achieve a concentration of 100 U/mL. The Clostridiopeptidase-A was highly purified collagenase from Clostridium histolyticum (Type VII, Sigma-Aldrich, St. Louis, MO, USA), and the buffering agent contained 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.36 mM calcium chloride, and 0.3 mM sodium azide (pH 7.4). The collagenase inhibitor was a synthetic broad spectrum MMP inhibitor (GM6001, Abcam, Cambridge, UK), which was prepared at 20 μM concentration with distilled water .
Inhibition of collagenase activity
The Sensolyte Generic MMP colorimetric assay kit (AnaSpec Inc, Fremont, CA, USA) was used for high throughput screening of the MMP’s inhibitors. It contains thiopeptolide, which can be cleaved by MMPs or collagenases to release a sulfhydryl group. The sulfhydryl group reacts with 5, 5′-dithiobis (2-nitrobenzoic acid) to produce 2-nitro-5-thiobenzoic acid . This acid is a colored-reaction product which can be detected spectrophotometrically at 412 nm . This assay was performed in a 96-well plate with five wells replicated for each group. The first group was the experimental (MAP) group (10 μL of MAP and 40 μL of Clostridiopeptidase-A). The second group was the positive control (GM6001) group (10 μL of GM6001 and 40 μL of Clostridiopeptidase-A). The third group was a negative control (DW) group with distilled water (10 μL of distilled water and 40 μL of Clostridiopeptidase-A). All reagents were mixed in each experimental well and pre-incubated for 20 min to avoid the burst of collagenase activity that may occur when all reagents are mixed together simultaneously. After pre-incubation, 50 μL of the thiopeptolide (Sensolyte Generic MMP colorimetric assay kit) was added to reach a total of 100 μL in each well. The reagents were mixed thoroughly for 30s. The absorbance at 412 nm was read by a microplate spectrophotometer (μQuant, BioTek, Winooski, VT, USA) at 10 min intervals for 60 min. MAP without collagenase cannot produce the colored reaction products in this assay which was shown in preliminary study. Glutathione reference standard containing 0–200 μM of reduced glutathione was prepared. Reduced glutathione is a tripeptide (g-glutamylcysteinylglycine) with a free thiol group, which reacts with 5, 5′-dithiobis (2-nitrobenzoic acid) and produces a yellow color. Reduced glutathione can be used as a reference standard to calculate the concentration of sulfhydryl groups in the sample. The collagenase activity under the effect of collagenase inhibitor was calculated as:
C o l l a g e n a s e a c t i v i t y = μ M ( SH ) × r mg / mL ( Col ) × t
where μM(SH) represents the concentration of sulfhydryl groups, mg/ml(Col) represents the concentration of bacterial collagenase (mg/mL), r represents the dilution ratio of test sample, and t represents reaction time.
Inhibiting the degradation of demineralized dentin matrix
Dentin specimen preparation
Thirty intact human third molars were collected from patients requiring extraction with informed consent. The research protocol was approved by the local Independent Ethics Committee. The teeth were stored at 4 °C in 0.02% sodium azide to prevent bacterial growth and used within one month of extraction. Thirty dentin slabs (5 mm × 5 mm × 1.5 mm) were prepared and polished with a 600-grit silicon carbide paper under running water to create a smooth surface. The slabs were cleaned using detergent solution, acetone, ethanol and distilled water. They were etched with 37% phosphoric acid for 60 s and then rinsed thoroughly with distilled water. The etched slabs were incubated at 37 °C for 5 min in a polypropylene tube containing 100 μL of MAP, GM6001 collagenase-inhibitor, or distilled water. Clostridiopeptidase-A (400 μL) was were added into each polypropylene tube. The tubes were sealed and incubated in a shaker-water bath (60 cycles/min) at 37 °C for 7 days.
Determination of degraded collagen
The degradation of collagen was determined by measuring the quantity of hydroxyproline (HYP), an amino acid characteristic of collagen . After 7 days of incubation, 400 μL of the medium was collected from each tube and transferred to another labeled polypropylene tube. An equal volume of 6 M hydrochloric acid was added into each tube, mixed and hydrolysed at 98 °C for 24 h . After cooling, the hydrolysates were lyophilized and re-solubilized in 600 μL of distilled water. HYP for each sample was measured for three times and averaged. The measure method had slight modification according to Reddy and Enwemeka . Briefly, 0.056 M chloramine-T reagent was mixed with the above hydrolysate, and then oxidation was allowed to proceed at room temperature. After a 25-min reaction, 1 M Ehrlich’s reagent was added to react with the oxidation product, which generated the chromophore ultimately. The absorbance was measured by a spectrophotometer (μQuant, BioTek) at 550 nm. As MAP may contain the hydroxyproline, the absorbance of it was determined at the same time and subtracted from the MAP group. Meanwhile, a standard HYP solution containing 0–50 μg/mL of hydroxyproline was prepared. The standard curve had a coefficient of determination by a value of 0.98.
Microtensile bond strength test
Flat dentin surfaces of another 60 human third molars were prepared by sectioning off the occlusal one-third of the crown. They were etched with 37% phosphoric acid for 15 s and divided into one of the three group: MAP, GM6001 and DW group. In each group, the dentin surface was scrubbed with MAP, GM6001, or distilled water for 60 s. Then, all the dentin surface was conditioned with an ethanol-based, single-component adhesive resin (Gluma Comfort Bond, Heraeus Kulzer, Hanau, Germany) which was light-cured according to the manufacturer’s instructions. A universal nano-hybrid composite resin (Tetric EvoCeram, Ivoclar Vivadent, Schaan, Liechtenstein) of 6 mm was built up incrementally. The composite resin was polymerized by an increment of increment for 20 s using an LED device (Bluephase 20i, IvoclareVivadent, Schaan, Liechtenstein). The composite bonded tooth were stored in distilled water at 37 °C for 24 h. They were longitudinally sectioned into beams with a cross-sectional dimension of 0.8 × 0.8 mm 2 for microtensile bond strength (μTBS) test . Each group was further divided into two subgroups (n = 70). The test of μTBS was conducted immediately for the beams in the first subgroup. The beams in the second subgroup received thermocycling and collagenase challenge before the μTBS test. Thermocycling was performed for 2500 cycles by putting the beams between 5 °C and 55 °C with a dwell time of 15 s. The beams were stored in the collagenase solutions at 37 °C for 3 weeks after thermocycling. The collagenase solution was refreshed every week. The μTBS test was performed with a digital universal testing machine (WDS-20, Jinan Fangyuan Co., Shandong, China) at a crosshead speed of 1 mm/min until failure. The dimension of the fracture area was recorded using a digital calliper to the nearest 0.01 mm, and μTBS value (in MPa) was calculated.
Field emission scanning electron microscopy (FE-SEM; Sirion 200, FEI Co, Hillsboro, OR, USA) was used to study the morphology of the dentin/adhesive interfaces of the specimens before μTBS testing and the morphology of the fractured dentin interfaces of the fractured samples after μTBS testing.
Data of collagenase activity and HYP release were normally distributed (Kolmogorov–Smirnov Test) and homoscedastic (Levene Test), so one-way ANOVA was used to analyze the effect of collagenase inhibition and dentin collagen degradation. Post hoc multiple comparisons were performed with Tukey’s test. Since the homoscedasticity assumptions of the μTBS were violated, Kruskal–Wallis test with Dunn’s multiple comparison was used in between group comparison at each time point. Wilcoxon Rank Sum Test was used in within group comparison. The analyses were performed using IBM SPSS Version 20.0 software (IBM Corporation, Armonk, New York, USA). The cut-off level for significance was taken as 5% for all of the analyses.