Abstract
Objective
Recent studies supported the use of protein cross-linking agents during bonding procedures to inactivate endogenous dentin proteases, preventing dentin collagen degradation thus improving bond durability. The aim of this study was to evaluate the effect of a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-containing conditioner on the stability of the adhesive interface created by two etch-and-rinse adhesives.
Methods
Human dentin was etched with 35% phosphoric acid, treated with 0.3 M EDC-containing conditioner followed by a three-step or a two-step etch-and-rinse adhesive. Adhesives were applied to control specimens without EDC pre-treatment. Specimens were subjected to microtensile bond strength test and pulled to failure after 24 h or 1 year of storage and interfacial nanoleakage expression was evaluated and quantified by light microscopy. Additionally, to investigate endogenous dentin matrix metalloproteinase activity a zymographic assay was performed on protein extracts obtained from phosphoric-acid-etched dentin powder with or without EDC treatment.
Results
The use of the EDC-containing conditioner did not affect immediate bond strength to dentin but contributed to preserve the bond strength after 1 year ( p < 0.05) for both tested adhesives. No difference was found in the interfacial nanoleakage expression that increased after aging irrespective from the treatment. EDC pre-treatment inhibited dentin endogenous MMPs as assayed with the zymography.
Significance
In conclusion, the results of the study provide proof that EDC can produce long-term inactivation of MMPs in acid-etched dentin matrices contributing to bond strength preservation over time. Future studies are needed to support the use of EDC in vivo .
1
Introduction
Consistent evidence supports the hypothesis that exposure and activation of dentin endogenous proteases occurs during dentin bonding procedures. The resultant collagenolytic/gelatynolitic activity is thought to be responsible for the in vitro and in vivo manifestation of thinning and disappearance of collagen fibrils from poorly infiltrated aged hybrid layers . The application of resin monomer constituents of adhesive blends of either etch-and-rinse or self-etch adhesives have been recently shown to activate these endogenous proteases .
Among these enzymes, matrix-metalloproteinases (MMPs) and cathepsins have been shown to be present in dentin , being responsible for the slow hydrolysis of the collagen fibrils in hybrid layers that anchor resin composites to the underlying mineralized dentin . This hydrolysis causes a loss of bond strength, allowing gaps to open up between resin composites and tooth structure .
Recently, several efforts have been made to inactivate these proteases during dentin bonding procedures with the attempt to create more durable resin-dentin bonds. The identification of these enzymes as well as the understanding of their functions has prompted innovative approaches to retain the hybrid layer integrity and increase the longevity of dentin bonding over time . The proposed approaches to prolong the durability of resin-dentin bonds include the use of synthetic MMP inhibitors , quaternary ammonium methacrylates or benzalkonium chloride , as well as other approaches that act indirectly by chemical chelation of calcium ion, collagen cross-linking, ethanol wet bonding, or remineralization to protect the hybrid layer from enzymatic degradation .
Among these strategies, the use of collagen cross-linking agents seems to be very promising . The potential of cross-linkers is related to the possibility to improve the mechanical strength of the collagen network, improve the resistance to enzymatic degradation, and to inactivate exposed MMPs bound to matrix collagen. Together this leads to a stable dentin matrix network which ultimately determines the formation of a stable hybrid layer . Recently, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) has been proposed to effectively improve the durability of resin-dentin bonds by increasing the mechanical properties of the collagen matrix , although the 1–4 h time required for the EDC application step makes it clinically unacceptable. To overcome this problem, a previous in vitro study evaluated the feasibility of reducing the EDC application time up to 1 min, revealing the effectiveness of the cross-linker agent to inactivate soluble rhMMP-9 and matrix-bound dentin proteinases . However, additional data are required to validate the use of EDC (for a clinical reliable time) on demineralized dentin to stabilize the bond inhibiting endogenous MMPs.
Thus, the aim of this study was to evaluate the ability of an EDC-containing conditioner to cross-link dentin collagen (within 1 min of contact time) in order to improve immediate bond strength and stabilize the adhesive interface over time. Since the activity of dentinal MMPs has been implicated in the degradation of resin-dentin bonds, the effect of EDC on the activity of dentin MMPs was also be investigated. The null hypotheses tested were that EDC applied for 1 min to acid-etched dentin before bonding (1) does not affect immediate bond strength and interfacial nanoleakage expression, (2) does not prevent adhesive interface degradation over time, and (3) does not inhibit endogenous dentin MMPs activity.
2
Materials and methods
Reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise specified.
2.1
Microtensile bond strength test (μTBS)
Forty freshly extracted, non-carious, human third molars were used. Teeth were collected after obtaining patients’ informed consent for research purposes under a protocol approved by the institutional review board of the Georgia Regents University (USA).
Tooth crowns were flattened using a low-speed diamond saw (Micromet, Remet, Bologna, Italy) under water irrigation and a standardized smear layer was created with 600-grit silicon-carbide (SiC) paper. Dentin surfaces were etched for 15 s with 35% phosphoric-acid gel (3 M ESPE, St. Paul, MN, USA) and rinsed with water.
Specimens were then randomly assigned to the following treatments.
-
Group 1 (G1): acid-etched dentin was pretreated with 0.3 M EDC water-solution for 1 min, air-dried, primed and bonded with Optibond FL (OFL; Kerr, Orange, CA, USA) following the manufacturer’s instructions.
-
Group 2 (G2): OFL was applied on etched dentin without EDC pre-treatment in accordance with manufacturer’s instructions.
-
Group 3 (G3): acid-etched dentin was pretreated with 0.3 M EDC as described for G1 then bonded with Scotchbond 1XT (SB1XT 3 M ESPE).
-
Group 4 (G4): SB1XT was applied to acid-etched without EDC pre-treatment.
Each bonded specimen was light-cured for 20 s using a quartz-halogen light unit (Curing Light 2500; 3 M ESPE). The irradiance level of the light was monitored periodically with a radiometer (3 M ESPE) to ensure that it remained ≥600 mW/cm 2 . Four 1-mm-thick layers of microhybrid resin composite (Filtek Z250; 3 M ESPE) were placed and polymerized individually for 20 s.
Specimens were serially sectioned to obtain approximately 1-mm-thick beams in accordance with the microtensile non-trimming technique. The dimension of each stick ( ca. 0.9 mm × 0.9 mm × 6 mm) was recorded using a digital caliper (±0.01 mm) and the bonded area was calculated for subsequent conversion of microtensile strength values into units of stress (MPa). Beams were stressed to failure after 24 h ( T 0 ) or 1 year ( T 12 ) of storage in artificial buffer at 37 °C, prepared in accordance with the protocol of Pashley et al. , using a simplified universal testing machine (Bisco, Inc., Schaumburg, IL, USA) at a crosshead speed of 1 mm/min.
The number of prematurely debonded sticks in each test group was recorded, but these values were not included in the statistical analysis because all premature failures occurred during the cutting procedure, and they did not exceed 3% of the total number of tested specimens and were similarly distributed within the groups. A single observer evaluated the failure modes under a stereomicroscope (Stemi 2000-C; Carl Zeiss Jena GmbH) at magnifications up to 50× and classified them as adhesive, cohesive in dentin, cohesive in composite, or mixed failures.
Because a Kolmogorov–Smirnov test determined that values were normally distributed, data were analyzed using Two-Way (variables: dentin bonding system and storage time) analysis of variance (ANOVA) and post hoc Tukey test. p values of 0.05 were considered to indicate statistical significance.
2.2
Interfacial nanoleakage evaluation
Sixteen additional teeth ( N = 4/group) were processed for interfacial nanoleakage evaluation. Middle/deep dentin was selected, acid-etched and bonded for one of the adhesives with or without the EDC-containing conditioner as previously described. A 1-mm-thick flowable composite (Filtek Flow; 3 M ESPE) was applied on the bonded disks and light-cured. Composite-dentin specimens were cut vertically into 1-mm-thick slabs to expose the bonded surfaces and stored for 24 h ( T 0 ) or 1 year ( T 12 ) in artificial buffer at 37 °C. Specimens were covered with nail varnish, leaving 1 mm exposed at the bonded interface, and processed for interfacial nanoleakage evaluation. Bonded interfaces were immersed in 50 wt% ammoniacal AgNO 3 solution in darkness for 24 h according to the protocol described by Tay et al. . After immersion in the tracer solution, specimens were rinsed in distilled water and immersed in photo-developing solution for 8 h under a fluorescent light to reduce silver ions into metallic silver grain within voids along the bonded interfaces. Nanoleakage analysis was performed under light microscopy (LM – Nikon E 800; Tokyo, Japan) and the degree of interfacial nanoleakage was scored on a scale of 0–4 by two observers as described by Saboia et al. . Interfacial nanoleakage was scored based on the percentage of the adhesive surface showing silver nitrate deposition: 0, no nanoleakage; 1, <25% nanoleakage; 2, 25 to ≤50% nanoleakage; 3, 50 to ≤75% nanoleakage; and 4, >75% nanoleakage.
Statistical differences among nanoleakage group scores ( i.e. percentage of specimens falling within each score category) were analyzed using the χ 2 test. All statistical testing was performed at a pre-set alpha of 0.05. Inter-observer agreement was measured using Cohen’s kappa test.
2.3
Zymographic analysis
Zymographic analysis was performed in accordance with Mazzoni et al. . In brief, mineralized dentin powder was obtained from eight human third molars by freezing the dentin in liquid nitrogen and triturating it using Retsch miller (Model MM400, Retsch GmbH, Haan, Germany). Aliquots of mineralized dentin powder were treated as follows: G1 – left mineralized (control); G2 – demineralized with 10 wt% phosphoric acid for 10 min to simulate the first step of the etch-and-rinse approach; G3 – demineralized as for G2 and treated with EDC 0.3 M for 30 min.
Dentin powder aliquots were re-suspended in extraction buffer (50 mM Tris–HCl pH 6, containing 5 mM CaCl 2 , 100 mM NaCl, 0.1% Triton X-100, 0.1% nonionic detergent P-40, 0.1 mM ZnCl 2 , 0.02% NaN 3 ) for 24 h at 4 °C, intermittently sonicated for 10 min ( ca. ≈30 pulses), centrifuged for 20 min at 4 °C (20,800 G), then the supernatant was removed and re-centrifuged. The protein content was further concentrated using Vivaspin centrifugal concentrator (10,000 kDa cut off; Vivaspin Sartorius Stedim Biotech, Goettingen, Germany) for 30 min at 4 °C (15,000 G for 3 times). Total protein concentration in the dentin extracts was determined by the Bradford assay. Dentin proteins aliquots (60 μg) were diluted in Laemmli sample buffer in a 4:1 ratio and electrophorezed under non-reducing conditions in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 1 mg/mL fluorescein-labeled gelatin. Prestained low-range molecular weight SDS-PAGE standards (Bio-Rad, Hercules, CA) were used as molecular-weight markers. After electrophoresis, the gels were washed for 1 h in 2% Triton X-100 and the gels were incubated in zymography activation buffer (50 mmol/L Tris–HCl, 5 mmol/L CaCl 2 , pH 7.4) for 48 h. Proteolytic activity was evaluated and registered under long-wave UV light scanner (ChemiDoc Universal Hood, Bio-Rad). Gelatinase activities in the samples were analyzed in duplicate by gelatin zymography.
2
Materials and methods
Reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise specified.
2.1
Microtensile bond strength test (μTBS)
Forty freshly extracted, non-carious, human third molars were used. Teeth were collected after obtaining patients’ informed consent for research purposes under a protocol approved by the institutional review board of the Georgia Regents University (USA).
Tooth crowns were flattened using a low-speed diamond saw (Micromet, Remet, Bologna, Italy) under water irrigation and a standardized smear layer was created with 600-grit silicon-carbide (SiC) paper. Dentin surfaces were etched for 15 s with 35% phosphoric-acid gel (3 M ESPE, St. Paul, MN, USA) and rinsed with water.
Specimens were then randomly assigned to the following treatments.
-
Group 1 (G1): acid-etched dentin was pretreated with 0.3 M EDC water-solution for 1 min, air-dried, primed and bonded with Optibond FL (OFL; Kerr, Orange, CA, USA) following the manufacturer’s instructions.
-
Group 2 (G2): OFL was applied on etched dentin without EDC pre-treatment in accordance with manufacturer’s instructions.
-
Group 3 (G3): acid-etched dentin was pretreated with 0.3 M EDC as described for G1 then bonded with Scotchbond 1XT (SB1XT 3 M ESPE).
-
Group 4 (G4): SB1XT was applied to acid-etched without EDC pre-treatment.
Each bonded specimen was light-cured for 20 s using a quartz-halogen light unit (Curing Light 2500; 3 M ESPE). The irradiance level of the light was monitored periodically with a radiometer (3 M ESPE) to ensure that it remained ≥600 mW/cm 2 . Four 1-mm-thick layers of microhybrid resin composite (Filtek Z250; 3 M ESPE) were placed and polymerized individually for 20 s.
Specimens were serially sectioned to obtain approximately 1-mm-thick beams in accordance with the microtensile non-trimming technique. The dimension of each stick ( ca. 0.9 mm × 0.9 mm × 6 mm) was recorded using a digital caliper (±0.01 mm) and the bonded area was calculated for subsequent conversion of microtensile strength values into units of stress (MPa). Beams were stressed to failure after 24 h ( T 0 ) or 1 year ( T 12 ) of storage in artificial buffer at 37 °C, prepared in accordance with the protocol of Pashley et al. , using a simplified universal testing machine (Bisco, Inc., Schaumburg, IL, USA) at a crosshead speed of 1 mm/min.
The number of prematurely debonded sticks in each test group was recorded, but these values were not included in the statistical analysis because all premature failures occurred during the cutting procedure, and they did not exceed 3% of the total number of tested specimens and were similarly distributed within the groups. A single observer evaluated the failure modes under a stereomicroscope (Stemi 2000-C; Carl Zeiss Jena GmbH) at magnifications up to 50× and classified them as adhesive, cohesive in dentin, cohesive in composite, or mixed failures.
Because a Kolmogorov–Smirnov test determined that values were normally distributed, data were analyzed using Two-Way (variables: dentin bonding system and storage time) analysis of variance (ANOVA) and post hoc Tukey test. p values of 0.05 were considered to indicate statistical significance.
2.2
Interfacial nanoleakage evaluation
Sixteen additional teeth ( N = 4/group) were processed for interfacial nanoleakage evaluation. Middle/deep dentin was selected, acid-etched and bonded for one of the adhesives with or without the EDC-containing conditioner as previously described. A 1-mm-thick flowable composite (Filtek Flow; 3 M ESPE) was applied on the bonded disks and light-cured. Composite-dentin specimens were cut vertically into 1-mm-thick slabs to expose the bonded surfaces and stored for 24 h ( T 0 ) or 1 year ( T 12 ) in artificial buffer at 37 °C. Specimens were covered with nail varnish, leaving 1 mm exposed at the bonded interface, and processed for interfacial nanoleakage evaluation. Bonded interfaces were immersed in 50 wt% ammoniacal AgNO 3 solution in darkness for 24 h according to the protocol described by Tay et al. . After immersion in the tracer solution, specimens were rinsed in distilled water and immersed in photo-developing solution for 8 h under a fluorescent light to reduce silver ions into metallic silver grain within voids along the bonded interfaces. Nanoleakage analysis was performed under light microscopy (LM – Nikon E 800; Tokyo, Japan) and the degree of interfacial nanoleakage was scored on a scale of 0–4 by two observers as described by Saboia et al. . Interfacial nanoleakage was scored based on the percentage of the adhesive surface showing silver nitrate deposition: 0, no nanoleakage; 1, <25% nanoleakage; 2, 25 to ≤50% nanoleakage; 3, 50 to ≤75% nanoleakage; and 4, >75% nanoleakage.
Statistical differences among nanoleakage group scores ( i.e. percentage of specimens falling within each score category) were analyzed using the χ 2 test. All statistical testing was performed at a pre-set alpha of 0.05. Inter-observer agreement was measured using Cohen’s kappa test.
2.3
Zymographic analysis
Zymographic analysis was performed in accordance with Mazzoni et al. . In brief, mineralized dentin powder was obtained from eight human third molars by freezing the dentin in liquid nitrogen and triturating it using Retsch miller (Model MM400, Retsch GmbH, Haan, Germany). Aliquots of mineralized dentin powder were treated as follows: G1 – left mineralized (control); G2 – demineralized with 10 wt% phosphoric acid for 10 min to simulate the first step of the etch-and-rinse approach; G3 – demineralized as for G2 and treated with EDC 0.3 M for 30 min.
Dentin powder aliquots were re-suspended in extraction buffer (50 mM Tris–HCl pH 6, containing 5 mM CaCl 2 , 100 mM NaCl, 0.1% Triton X-100, 0.1% nonionic detergent P-40, 0.1 mM ZnCl 2 , 0.02% NaN 3 ) for 24 h at 4 °C, intermittently sonicated for 10 min ( ca. ≈30 pulses), centrifuged for 20 min at 4 °C (20,800 G), then the supernatant was removed and re-centrifuged. The protein content was further concentrated using Vivaspin centrifugal concentrator (10,000 kDa cut off; Vivaspin Sartorius Stedim Biotech, Goettingen, Germany) for 30 min at 4 °C (15,000 G for 3 times). Total protein concentration in the dentin extracts was determined by the Bradford assay. Dentin proteins aliquots (60 μg) were diluted in Laemmli sample buffer in a 4:1 ratio and electrophorezed under non-reducing conditions in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 1 mg/mL fluorescein-labeled gelatin. Prestained low-range molecular weight SDS-PAGE standards (Bio-Rad, Hercules, CA) were used as molecular-weight markers. After electrophoresis, the gels were washed for 1 h in 2% Triton X-100 and the gels were incubated in zymography activation buffer (50 mmol/L Tris–HCl, 5 mmol/L CaCl 2 , pH 7.4) for 48 h. Proteolytic activity was evaluated and registered under long-wave UV light scanner (ChemiDoc Universal Hood, Bio-Rad). Gelatinase activities in the samples were analyzed in duplicate by gelatin zymography.
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