HHS Public Access Author manuscript Author Manuscript
Dent Mater. Author manuscript; available in PMC 2017 February 01. Published in final edited form as: Dent Mater. 2016 February ; 32(2): 211–222. doi:10.1016/j.dental.2015.11.024.
EFFECT OF CARBODIIMIDE ON THE FATIGUE CRACK GROWTH RESISTANCE OF RESIN-DENTIN BONDS Zihou Zhang1, Dylan Beitzel1, Hessam Majd1, Mustafa Mutluay2, Arzu Tezvergil-Mutluay2, Franklin R. Tay3,4, David H. Pashley3, and Dwayne Arola5,6,♠
Author Manuscript
1Department
of Mechanical Engineering, University of Maryland Baltimore County Baltimore, MD, USA Dentistry Research Group, Department of Cariology, Institute of Dentistry, University of Turku, Turku, Finland 3Department of Oral Biology, College of Dental Medicine, Georgia Health Sciences University, Augusta, GA, USA 4Department of Endodontics, College of Dental Medicine, Georgia Health Sciences University, Augusta, GA, USA 5Department of Materials Science and Engineering, University of Washington Seattle, WA USA 6Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA USA 2Adhesive
Abstract
Author Manuscript
Recent studies have shown that ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) inactivates endogenous dentin proteases, thereby preventing collagen degradation and improving the durability of adhesive bonds to dentin. Bond durability is routinely assessed by monotonic microtensile testing, which does not consider the cyclic nature of mastication. Objective—to characterize the effect of an EDC pretreatment on the fatigue crack growth behavior of resin-dentin bonds. Methods—Bonded interface Compact Tension (CT) specimens were prepared using a three-step etch-and-rinse adhesive and hybrid resin-composite. Adhesive bonding of the treated groups included a 1 min application of an experimental EDC conditioner to the acid-etched dentin. The control groups did not receive EDC treatment. The fatigue crack growth resistance was examined after storage in artificial saliva for 0, 3 and 6 months.
Author Manuscript
Results—There was no significant difference in the immediate fatigue crack growth resistance of the EDC-treated and control groups at 0 months. However, after the 3 and 6 months storage periods the EDC-treated groups exhibited significantly greater (p≤0.05) fatigue crack growth resistance than the control specimens. Significance—Although the EDC treatment maintained the fatigue crack growth resistance of the dentin bonds through 6 months of storage, additional studies are needed to assess its effectiveness over longer periods and in relation to other cross-linking agents.
♠ Corresponding Author. Dwayne D. Arola, Ph.D., Department of Materials Science and Engineering, University of Washington, Roberts Hall, 333; Box 352120, Seattle, WA 98195-2120 USA, [email protected], (206) 685-8158 (v), (206) 543-3100 (f). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Zhang et al.
Page 2
Author Manuscript
Keywords collagen; cross-linker; dentin bonding agents; durability; EDC; endogenous proteinases; fatigue crack growth; fracture
INTRODUCTION Contemporary bonding procedures used in the placement of resin-composite restorations can cause exposure and activation of endogenous dentin proteases [1]. This process causes gradual destruction of poorly infiltrated collagen fibrils within the hybrid layers of adhesive bonds to dentin [2,3]. Degradation of collagen within the hybrid layer can compromise the durability of adhesive bonds and facilitate a reduction in bond strength over time.
Author Manuscript
Manufacturers have simplified etch-and-rinse adhesives from three-step (involving etchant, primer and adhesive) to two-step systems (etchant and adhesive) by combining hydrophilic primers with hydrophobic adhesives with various solvents. But with this change the durability of resin-dentin bonds has decreased [2]. More recently, phosphoric acid etchants have been replaced by incorporating acidic monomers into a solvated adhesive to create single-step, self-etching adhesives. Application of both etch-and-rinse and self-etch adhesives causes activation of matrix-metalloproteinases (MMP)s and cysteine cathepsins [e.g. 4–7]. These are host-derived proteolytic enzymes that are bound to the dentin collagen matrix. When uncovered by etching, the MMPs slowly solubilize the collagen fibrils [1] and remain active even after resin-infiltration. Apparently, the time-dependent degradation of the hybrid layer is most evident in the use of etch-and-rinse adhesives [8].
Author Manuscript
Several strategies are being explored for preventing enzymatic degradation of the dentin collagen and to address the concerns related to the poor durability of resin-bonded dentin interfaces. Tjäderhane et al. [9], Montagner et al. [10] and Sabatini and Pashley [11] have recently reviewed the current understanding of collagen degradation and have discussed the relative merits/drawbacks of the techniques under exploration. One of the foremost approaches for inactivation of the dentin proteases involves using cross-linking agents. Covalent cross-links produced with exogenous cross-linkers (e.g. glutaraldehyde, grape seed extract and carbodiimides) inactivate the active sites of dentin proteases by reducing the molecular mobility of the active site or by changing negatively charged ionized carboxyl groups into positively charged amides [9]. Of the current crosslinkers, carbodiimide, has some attractive qualities, including very low cytotoxicity, and an ability to preserve dentin bond strength within clinically acceptable treatment times [12, 13].
Author Manuscript
Mazzoni et al. [14] recently reported promising results on the use of an EDC conditioning treatment in stabilizing dentin bonds. They evaluated the microtensile strength of dentin bonds for two different etch-and-rinse systems (Optibond FL and Adper Scotch bond MultiPurpose) and assessed the degradation over a 12-month period. The EDC treatment consisted of exposing the etched dentin to a 0.3 M EDC solution for 1 min prior to bonding. In comparison to the control groups, the EDC treated samples exhibited between 25 and 35% higher bond strengths after 12 months storage. A related study by the group [15] using
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
Page 3
Author Manuscript
zymography showed that the EDC treatment was successful in inactivating dentin gelatinases, thereby preventing degradation of the collagen. While bond strength is an important metric of performance, resin-dentin bonded interfaces are subjected to cyclic loading and thus may undergo failure by fatigue and/or fatigue crack growth. Fatigue failures are considered of substantial importance to the success of restoratives [e.g. 16, 17]. Yet, studies in this area are scant and the contribution of fatigue to resin-dentin bond failures has received rather limited attention overall [18–26]. If flaws are located within either the resin adhesive or the hybrid layer, e.g. as a result of hydrolysis or incomplete resin infiltration [27], then the interface durability depends on its resistance to the “propagation” of these defects via cyclic crack extension. However, progressive failure of the resin-dentin bonded interface by either cyclic or slow crack growth has not been addressed by the dental materials community.
Author Manuscript
Soappman et al. [28] proposed an approach for evaluating the fatigue crack growth resistance of resin-dentin bonds, but it has not been applied to assess the effectiveness of an EDC treatment on dentin bond durability. Therefore, the primary objective of this study was to evaluate the effect of an experimental EDC conditioning treatment applied during dentin bonding, on the fatigue crack growth resistance of the adhesive interface. The null hypotheses to be tested were that an EDC treatment (consisting of 0.5 M and 1 min exposure) applied during dentin bonding (1) does not change the immediate fatigue crack growth resistance of the resin/dentin interface, and (2) has no effect on the fatigue crack growth resistance up to a 6 month period of storage.
MATERIALS AND METHODS Author Manuscript
The specimens utilized for this approach involve sections of coronal dentin that were obtained from caries-free human third molars and obtained with informed signed consent. The teeth were obtained from participating clinics in Maryland with record of age (18≤age≤30 yrs) according to an approved protocol (#Y04DA23151). Each tooth was sectioned using a slicer/grinder (Chevalier Smart-H818II, Chevalier Machinery, Santa Fe Springs, CA, USA) with diamond abrasive slicing wheels (#320 mesh abrasives) and copious water coolant. The sections were obtained from the mid coronal region (Fig. 1a) as necessary for the specimen geometry. The remaining materials used in the development of the specimens included a three-step etch-and-rinse adhesive (Scotch bond Multipurpose, SBMP, 3M ESPE) and compatible resin composite (Z100, 3M ESPE).
Author Manuscript
Bonded interface Compact Tension (CT) specimens were prepared from the dentin sections using a special molding technique that has been described in detail in previous studies [28, 29]. Briefly, the dentin sections represented half of the completed CT specimen geometry (Fig. 1b). Adhesive bonding was performed to the occlusal aspect of the dentin sections, with the pulp side facing away from the bonded interface. The occlusal edge was etched for 15 sec (SB 37% phosphoric etchant) and rinsed with water in preparation for bonding. Then the SBMP primer and adhesive were applied to the etched surface according to the manufacturer’s recommendations. Thereafter, these sections were placed in a specially designed mold that enabled incremental application of the resin composite as necessary to
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
Page 4
Author Manuscript
complete the CT geometry. A thin Mylar sheet was placed at one end of the interface to introduce a molded notch as evident in Figure 1b. The composite was cured on both sides for 40 sec using a quartz–tungsten–halogen light-curing unit (Demetron VCL 401, Demetron, CA, USA) with output intensity of 600 mW/cm^2 and with tip diameter wider than 10 mm. Power emission of the curing light was measured and validated using a PM10 thermopile (Coherent, Santa Clara, CA) attached to a Fieldmate meter (Coherent) calibrated to NIST standards. The bonded sections were released from the mold and two holes were introduced using a miniature milling machine to facilitate the opening mode loading. A detailed description of the procedures used for specimen preparation has been presented previously [28].
Author Manuscript Author Manuscript
The durability of the bonded interfaces was evaluated with and without an experimental treatment formulated to inactivate endogenous dentin proteases. For the treated specimens, the application of primer and adhesive was preceded by conditioning the demineralized collagen using an experimental solution of 0.5 M ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 60 sec. The specimens were then rinsed with water (15 sec) and then lightly blotted. The remainder of the specimen preparation process was identical to that for those specimens without EDC. Following the aforementioned procedures, the specimens were placed within a phosphate-buffered artificial saliva at 37°C until further evaluation. A total of 36 specimens were prepared overall and consisted of an equal number of non-crosslinked control and cross-linked specimens (that received EDC treatment). The fatigue crack growth resistance of the specimens was evaluated after a storage period of 0, 3 or 6 months, resulting in six specimens in each cell (treatment and time). Those specimens evaluated at 0 months (i.e. without storage) are considered to represent the “immediate” fatigue crack growth resistance and were tested after a period of at least 48 hours from the date of preparation.
Author Manuscript
The CT specimens were subjected to cyclic Mode I loading using a universal testing system (EnduraTEC Model ELF 3200, Minnetonka, MN, USA) with load capacity and sensitivity of 225 N and ±0.01 N, respectively. All experiments were performed within a bath of Hanks Balanced Salt Solution (HBSS) at room temperature (pH 7.4). The loading was applied under load control actuation, a frequency of 5 Hz and stress ratio (R) of 0.1. Measurement of the crack length was accomplished using an imaging system that consisted of a microscope (Optem zoom 70xl 391940, QIOPTIQ, Luxembourg) and CCD camera. Sequential measurements of the crack length were used to estimate the incremental crack extension (Δa) as a function of the loading cycles in the increment ΔN. In general, the increment of cyclic loading ranged from 5≤ΔN≤30k cycles and the increment of crack growth extended from 0.02≤Δa≤0.15mm. Cyclic loading was continued until the specimen underwent complete fracture. These procedures have been used previously for evaluating the resindentin bonded interface [28, 29] and the fatigue crack growth resistance of dentin [30, 31]. The fatigue crack growth experiments provided measurements of crack length as a function of the cyclic loading history for each of the specimens evaluated. The data was used to determine the incremental crack growth rate (Δa/ΔN) and the corresponding stress intensity range (ΔK) over the total length of crack extension achieved. For the resin/dentin inset CT specimens the stress intensity range was estimated as a function of crack length according to
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
Page 5
Author Manuscript
(1)
where ΔP is the opening load range (Pmax – Pmin), B is the thickness of the specimen, W is the distance between the center of the loading points and free boundary in front of the crack, and α = a/W is the ratio of the average crack length to the in-plane specimen width (Fig. 1b). The incremental crack growth rate was then plotted in terms of the stress intensity range for each specimen to assess the fatigue crack growth history and important parameters defining the resistance to cyclic crack growth.
Author Manuscript
According to the distribution of data, the threshold stress intensity range (ΔKth) was estimated at the onset of cyclic crack growth, which represents the minimum stress intensity necessary for cyclic crack extension. The steady-state portion of the fatigue crack growth responses was also identified and modeled using the Paris Law [32] according to (2)
where C and m are the fatigue crack growth coefficient and exponent, respectively. The average values of these two parameters were determined for each group of specimens. Significant differences in the fatigue crack growth parameters resulting from EDC treatment and storage time were identified using a one-way analysis of variance (ANOVA) with Tukey’s HSD; significance was identified by p≤0.05. The fatigue crack growth distributions were compared to establish significant differences between the groups using the Wilcoxon Rank Sum test with the critical value (alpha) set at 0.05.
Author Manuscript
Selected fractured specimens were evaluated via Scanning Electron Microscopy (SEM) in secondary electron imaging mode using a scanning electron microscopy (FEI Model Nova NanoSEM 450; Hillsboro, OR, USA). Prior to the SEM analysis, the specimens were dehydrated in an ascending ethanol series (70–100%), fixed in hexamethyldisilazane, and then sputtered with gold/palladium to enhance conductance of the hard tissue and resin adhesive. The fracture surfaces were inspected to identify any potential mechanisms of degradation or other factors contributing to the fatigue crack growth behavior.
Author Manuscript
The additional fractured specimens were prepared for analysis using Transmission Electron Microcopy (TEM) according to the protocol of Tay et al. [33]. All specimens were completely demineralized in 8 mole/L formic acid buffered with sodium formate to pH 2.5. After demineralization, the specimens were fixed in Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mole/L cacadylate buffer, pH 7.3) for a minimum of 1 hr and rinsed thoroughly with fresh 0.1 mole/L sodium cacodylate buffer. Post-fixation was performed with 1% osmium tetroxide in 0.1 mole/L sodium cacodylate buffer (pH 7.3) for 1 hr at room temperature. Then the specimens were dehydrated in an ascending ethanol series (30% to 100%), immersed in propylene oxide as a transition fluid, and embedded in epoxy resin (TAAB 812 resin, TAAB Laboratories, Aldermaston, UK) at 60°C for 48 hrs. After resin embedding two 2 × 2 mm blocks were trimmed and reembedded in epoxy resin to ensure proper orientation of the resin-dentin interface.
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
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Author Manuscript
Following initial screening of all semi-thin sections from each group, representative 1 × 1 mm ultrathin sections about 90 nm thick were prepared with an ultramicrotome (Reich Ultracut, Leica, Vienna, Austria) using a diamond knife (Diatome, Bienne, Switzerland) and collected on 100-mesh copper grids (TAAB Laboratories). Grids were double stained with 2% uranyl acetate for 10 min, followed by lead citrate for an additional 5 min. After drying, the stained sections were examined with a Phillips EM208S (Eindhoven, The Netherlands) at 80 KV.
RESULTS
Author Manuscript
A representative fatigue crack growth response resulting from cyclic crack extension within a bonded interface CT specimen is shown in Figure 2a; the results in this figure correspond to a specimen that received EDC treatment and was evaluated without storage in artificial saliva (i.e. at 0 months). The threshold stress intensity range (ΔKth) is highlighted at the onset of incremental crack growth, as well as the Paris Law exponent, which quantitates the slope of the steady-state response. The fatigue crack growth responses for all of the EDC treated specimens evaluated without storage are shown in Figure 2b, along with the responses obtained for the control group. According to the Wilcoxon Rank Sum test, there was no significant difference between the fatigue crack growth distributions of the control and EDC-treated specimens (Z = −0.20; p=0.85) that were evaluated without storage in artificial saliva.
Author Manuscript
The fatigue crack growth distributions obtained from the specimens evaluated without storage (at 0 months) were used in estimating the corresponding fatigue crack growth parameters. The mean and standard deviation of the values obtained for the control and EDC-treated specimens are listed in Table 1. Note that only the mean is presented for the crack growth coefficient (C) due to the comparatively large variation in this parameter. Considering the initiation of cyclic crack extension, there was no significant difference in the values obtained for the ΔKth between the control and EDC-treated groups. Similarly, there was no significant difference (p>0.05) in the Paris Law parameters (m, C) between the two groups.
Author Manuscript
A comparison of the fatigue crack growth resistance of the control and EDC treated specimens after storage in artificial saliva is shown in Figure 3. Specifically, the responses obtained after 3 and 6 months of storage are shown in Figures 3a and 3b, respectively. According to the Wilcoxon Rank Sum test, after 3 months storage the EDC-treated specimens exhibited significantly greater resistance to fatigue crack growth than the control group (Z = −2.44; p=0.02). The EDC-treated group also exhibited significantly greater resistance to fatigue crack growth than the control group after 6 months storage as well (Z = −3.95; p
Dent Mater. Author manuscript; available in PMC 2017 February 01. Published in final edited form as: Dent Mater. 2016 February ; 32(2): 211–222. doi:10.1016/j.dental.2015.11.024.
EFFECT OF CARBODIIMIDE ON THE FATIGUE CRACK GROWTH RESISTANCE OF RESIN-DENTIN BONDS Zihou Zhang1, Dylan Beitzel1, Hessam Majd1, Mustafa Mutluay2, Arzu Tezvergil-Mutluay2, Franklin R. Tay3,4, David H. Pashley3, and Dwayne Arola5,6,♠
Author Manuscript
1Department
of Mechanical Engineering, University of Maryland Baltimore County Baltimore, MD, USA Dentistry Research Group, Department of Cariology, Institute of Dentistry, University of Turku, Turku, Finland 3Department of Oral Biology, College of Dental Medicine, Georgia Health Sciences University, Augusta, GA, USA 4Department of Endodontics, College of Dental Medicine, Georgia Health Sciences University, Augusta, GA, USA 5Department of Materials Science and Engineering, University of Washington Seattle, WA USA 6Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA USA 2Adhesive
Abstract
Author Manuscript
Recent studies have shown that ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) inactivates endogenous dentin proteases, thereby preventing collagen degradation and improving the durability of adhesive bonds to dentin. Bond durability is routinely assessed by monotonic microtensile testing, which does not consider the cyclic nature of mastication. Objective—to characterize the effect of an EDC pretreatment on the fatigue crack growth behavior of resin-dentin bonds. Methods—Bonded interface Compact Tension (CT) specimens were prepared using a three-step etch-and-rinse adhesive and hybrid resin-composite. Adhesive bonding of the treated groups included a 1 min application of an experimental EDC conditioner to the acid-etched dentin. The control groups did not receive EDC treatment. The fatigue crack growth resistance was examined after storage in artificial saliva for 0, 3 and 6 months.
Author Manuscript
Results—There was no significant difference in the immediate fatigue crack growth resistance of the EDC-treated and control groups at 0 months. However, after the 3 and 6 months storage periods the EDC-treated groups exhibited significantly greater (p≤0.05) fatigue crack growth resistance than the control specimens. Significance—Although the EDC treatment maintained the fatigue crack growth resistance of the dentin bonds through 6 months of storage, additional studies are needed to assess its effectiveness over longer periods and in relation to other cross-linking agents.
♠ Corresponding Author. Dwayne D. Arola, Ph.D., Department of Materials Science and Engineering, University of Washington, Roberts Hall, 333; Box 352120, Seattle, WA 98195-2120 USA, [email protected], (206) 685-8158 (v), (206) 543-3100 (f). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Zhang et al.
Page 2
Author Manuscript
Keywords collagen; cross-linker; dentin bonding agents; durability; EDC; endogenous proteinases; fatigue crack growth; fracture
INTRODUCTION Contemporary bonding procedures used in the placement of resin-composite restorations can cause exposure and activation of endogenous dentin proteases [1]. This process causes gradual destruction of poorly infiltrated collagen fibrils within the hybrid layers of adhesive bonds to dentin [2,3]. Degradation of collagen within the hybrid layer can compromise the durability of adhesive bonds and facilitate a reduction in bond strength over time.
Author Manuscript
Manufacturers have simplified etch-and-rinse adhesives from three-step (involving etchant, primer and adhesive) to two-step systems (etchant and adhesive) by combining hydrophilic primers with hydrophobic adhesives with various solvents. But with this change the durability of resin-dentin bonds has decreased [2]. More recently, phosphoric acid etchants have been replaced by incorporating acidic monomers into a solvated adhesive to create single-step, self-etching adhesives. Application of both etch-and-rinse and self-etch adhesives causes activation of matrix-metalloproteinases (MMP)s and cysteine cathepsins [e.g. 4–7]. These are host-derived proteolytic enzymes that are bound to the dentin collagen matrix. When uncovered by etching, the MMPs slowly solubilize the collagen fibrils [1] and remain active even after resin-infiltration. Apparently, the time-dependent degradation of the hybrid layer is most evident in the use of etch-and-rinse adhesives [8].
Author Manuscript
Several strategies are being explored for preventing enzymatic degradation of the dentin collagen and to address the concerns related to the poor durability of resin-bonded dentin interfaces. Tjäderhane et al. [9], Montagner et al. [10] and Sabatini and Pashley [11] have recently reviewed the current understanding of collagen degradation and have discussed the relative merits/drawbacks of the techniques under exploration. One of the foremost approaches for inactivation of the dentin proteases involves using cross-linking agents. Covalent cross-links produced with exogenous cross-linkers (e.g. glutaraldehyde, grape seed extract and carbodiimides) inactivate the active sites of dentin proteases by reducing the molecular mobility of the active site or by changing negatively charged ionized carboxyl groups into positively charged amides [9]. Of the current crosslinkers, carbodiimide, has some attractive qualities, including very low cytotoxicity, and an ability to preserve dentin bond strength within clinically acceptable treatment times [12, 13].
Author Manuscript
Mazzoni et al. [14] recently reported promising results on the use of an EDC conditioning treatment in stabilizing dentin bonds. They evaluated the microtensile strength of dentin bonds for two different etch-and-rinse systems (Optibond FL and Adper Scotch bond MultiPurpose) and assessed the degradation over a 12-month period. The EDC treatment consisted of exposing the etched dentin to a 0.3 M EDC solution for 1 min prior to bonding. In comparison to the control groups, the EDC treated samples exhibited between 25 and 35% higher bond strengths after 12 months storage. A related study by the group [15] using
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
Page 3
Author Manuscript
zymography showed that the EDC treatment was successful in inactivating dentin gelatinases, thereby preventing degradation of the collagen. While bond strength is an important metric of performance, resin-dentin bonded interfaces are subjected to cyclic loading and thus may undergo failure by fatigue and/or fatigue crack growth. Fatigue failures are considered of substantial importance to the success of restoratives [e.g. 16, 17]. Yet, studies in this area are scant and the contribution of fatigue to resin-dentin bond failures has received rather limited attention overall [18–26]. If flaws are located within either the resin adhesive or the hybrid layer, e.g. as a result of hydrolysis or incomplete resin infiltration [27], then the interface durability depends on its resistance to the “propagation” of these defects via cyclic crack extension. However, progressive failure of the resin-dentin bonded interface by either cyclic or slow crack growth has not been addressed by the dental materials community.
Author Manuscript
Soappman et al. [28] proposed an approach for evaluating the fatigue crack growth resistance of resin-dentin bonds, but it has not been applied to assess the effectiveness of an EDC treatment on dentin bond durability. Therefore, the primary objective of this study was to evaluate the effect of an experimental EDC conditioning treatment applied during dentin bonding, on the fatigue crack growth resistance of the adhesive interface. The null hypotheses to be tested were that an EDC treatment (consisting of 0.5 M and 1 min exposure) applied during dentin bonding (1) does not change the immediate fatigue crack growth resistance of the resin/dentin interface, and (2) has no effect on the fatigue crack growth resistance up to a 6 month period of storage.
MATERIALS AND METHODS Author Manuscript
The specimens utilized for this approach involve sections of coronal dentin that were obtained from caries-free human third molars and obtained with informed signed consent. The teeth were obtained from participating clinics in Maryland with record of age (18≤age≤30 yrs) according to an approved protocol (#Y04DA23151). Each tooth was sectioned using a slicer/grinder (Chevalier Smart-H818II, Chevalier Machinery, Santa Fe Springs, CA, USA) with diamond abrasive slicing wheels (#320 mesh abrasives) and copious water coolant. The sections were obtained from the mid coronal region (Fig. 1a) as necessary for the specimen geometry. The remaining materials used in the development of the specimens included a three-step etch-and-rinse adhesive (Scotch bond Multipurpose, SBMP, 3M ESPE) and compatible resin composite (Z100, 3M ESPE).
Author Manuscript
Bonded interface Compact Tension (CT) specimens were prepared from the dentin sections using a special molding technique that has been described in detail in previous studies [28, 29]. Briefly, the dentin sections represented half of the completed CT specimen geometry (Fig. 1b). Adhesive bonding was performed to the occlusal aspect of the dentin sections, with the pulp side facing away from the bonded interface. The occlusal edge was etched for 15 sec (SB 37% phosphoric etchant) and rinsed with water in preparation for bonding. Then the SBMP primer and adhesive were applied to the etched surface according to the manufacturer’s recommendations. Thereafter, these sections were placed in a specially designed mold that enabled incremental application of the resin composite as necessary to
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
Page 4
Author Manuscript
complete the CT geometry. A thin Mylar sheet was placed at one end of the interface to introduce a molded notch as evident in Figure 1b. The composite was cured on both sides for 40 sec using a quartz–tungsten–halogen light-curing unit (Demetron VCL 401, Demetron, CA, USA) with output intensity of 600 mW/cm^2 and with tip diameter wider than 10 mm. Power emission of the curing light was measured and validated using a PM10 thermopile (Coherent, Santa Clara, CA) attached to a Fieldmate meter (Coherent) calibrated to NIST standards. The bonded sections were released from the mold and two holes were introduced using a miniature milling machine to facilitate the opening mode loading. A detailed description of the procedures used for specimen preparation has been presented previously [28].
Author Manuscript Author Manuscript
The durability of the bonded interfaces was evaluated with and without an experimental treatment formulated to inactivate endogenous dentin proteases. For the treated specimens, the application of primer and adhesive was preceded by conditioning the demineralized collagen using an experimental solution of 0.5 M ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 60 sec. The specimens were then rinsed with water (15 sec) and then lightly blotted. The remainder of the specimen preparation process was identical to that for those specimens without EDC. Following the aforementioned procedures, the specimens were placed within a phosphate-buffered artificial saliva at 37°C until further evaluation. A total of 36 specimens were prepared overall and consisted of an equal number of non-crosslinked control and cross-linked specimens (that received EDC treatment). The fatigue crack growth resistance of the specimens was evaluated after a storage period of 0, 3 or 6 months, resulting in six specimens in each cell (treatment and time). Those specimens evaluated at 0 months (i.e. without storage) are considered to represent the “immediate” fatigue crack growth resistance and were tested after a period of at least 48 hours from the date of preparation.
Author Manuscript
The CT specimens were subjected to cyclic Mode I loading using a universal testing system (EnduraTEC Model ELF 3200, Minnetonka, MN, USA) with load capacity and sensitivity of 225 N and ±0.01 N, respectively. All experiments were performed within a bath of Hanks Balanced Salt Solution (HBSS) at room temperature (pH 7.4). The loading was applied under load control actuation, a frequency of 5 Hz and stress ratio (R) of 0.1. Measurement of the crack length was accomplished using an imaging system that consisted of a microscope (Optem zoom 70xl 391940, QIOPTIQ, Luxembourg) and CCD camera. Sequential measurements of the crack length were used to estimate the incremental crack extension (Δa) as a function of the loading cycles in the increment ΔN. In general, the increment of cyclic loading ranged from 5≤ΔN≤30k cycles and the increment of crack growth extended from 0.02≤Δa≤0.15mm. Cyclic loading was continued until the specimen underwent complete fracture. These procedures have been used previously for evaluating the resindentin bonded interface [28, 29] and the fatigue crack growth resistance of dentin [30, 31]. The fatigue crack growth experiments provided measurements of crack length as a function of the cyclic loading history for each of the specimens evaluated. The data was used to determine the incremental crack growth rate (Δa/ΔN) and the corresponding stress intensity range (ΔK) over the total length of crack extension achieved. For the resin/dentin inset CT specimens the stress intensity range was estimated as a function of crack length according to
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
Page 5
Author Manuscript
(1)
where ΔP is the opening load range (Pmax – Pmin), B is the thickness of the specimen, W is the distance between the center of the loading points and free boundary in front of the crack, and α = a/W is the ratio of the average crack length to the in-plane specimen width (Fig. 1b). The incremental crack growth rate was then plotted in terms of the stress intensity range for each specimen to assess the fatigue crack growth history and important parameters defining the resistance to cyclic crack growth.
Author Manuscript
According to the distribution of data, the threshold stress intensity range (ΔKth) was estimated at the onset of cyclic crack growth, which represents the minimum stress intensity necessary for cyclic crack extension. The steady-state portion of the fatigue crack growth responses was also identified and modeled using the Paris Law [32] according to (2)
where C and m are the fatigue crack growth coefficient and exponent, respectively. The average values of these two parameters were determined for each group of specimens. Significant differences in the fatigue crack growth parameters resulting from EDC treatment and storage time were identified using a one-way analysis of variance (ANOVA) with Tukey’s HSD; significance was identified by p≤0.05. The fatigue crack growth distributions were compared to establish significant differences between the groups using the Wilcoxon Rank Sum test with the critical value (alpha) set at 0.05.
Author Manuscript
Selected fractured specimens were evaluated via Scanning Electron Microscopy (SEM) in secondary electron imaging mode using a scanning electron microscopy (FEI Model Nova NanoSEM 450; Hillsboro, OR, USA). Prior to the SEM analysis, the specimens were dehydrated in an ascending ethanol series (70–100%), fixed in hexamethyldisilazane, and then sputtered with gold/palladium to enhance conductance of the hard tissue and resin adhesive. The fracture surfaces were inspected to identify any potential mechanisms of degradation or other factors contributing to the fatigue crack growth behavior.
Author Manuscript
The additional fractured specimens were prepared for analysis using Transmission Electron Microcopy (TEM) according to the protocol of Tay et al. [33]. All specimens were completely demineralized in 8 mole/L formic acid buffered with sodium formate to pH 2.5. After demineralization, the specimens were fixed in Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mole/L cacadylate buffer, pH 7.3) for a minimum of 1 hr and rinsed thoroughly with fresh 0.1 mole/L sodium cacodylate buffer. Post-fixation was performed with 1% osmium tetroxide in 0.1 mole/L sodium cacodylate buffer (pH 7.3) for 1 hr at room temperature. Then the specimens were dehydrated in an ascending ethanol series (30% to 100%), immersed in propylene oxide as a transition fluid, and embedded in epoxy resin (TAAB 812 resin, TAAB Laboratories, Aldermaston, UK) at 60°C for 48 hrs. After resin embedding two 2 × 2 mm blocks were trimmed and reembedded in epoxy resin to ensure proper orientation of the resin-dentin interface.
Dent Mater. Author manuscript; available in PMC 2017 February 01.
Zhang et al.
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Following initial screening of all semi-thin sections from each group, representative 1 × 1 mm ultrathin sections about 90 nm thick were prepared with an ultramicrotome (Reich Ultracut, Leica, Vienna, Austria) using a diamond knife (Diatome, Bienne, Switzerland) and collected on 100-mesh copper grids (TAAB Laboratories). Grids were double stained with 2% uranyl acetate for 10 min, followed by lead citrate for an additional 5 min. After drying, the stained sections were examined with a Phillips EM208S (Eindhoven, The Netherlands) at 80 KV.
RESULTS
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A representative fatigue crack growth response resulting from cyclic crack extension within a bonded interface CT specimen is shown in Figure 2a; the results in this figure correspond to a specimen that received EDC treatment and was evaluated without storage in artificial saliva (i.e. at 0 months). The threshold stress intensity range (ΔKth) is highlighted at the onset of incremental crack growth, as well as the Paris Law exponent, which quantitates the slope of the steady-state response. The fatigue crack growth responses for all of the EDC treated specimens evaluated without storage are shown in Figure 2b, along with the responses obtained for the control group. According to the Wilcoxon Rank Sum test, there was no significant difference between the fatigue crack growth distributions of the control and EDC-treated specimens (Z = −0.20; p=0.85) that were evaluated without storage in artificial saliva.
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The fatigue crack growth distributions obtained from the specimens evaluated without storage (at 0 months) were used in estimating the corresponding fatigue crack growth parameters. The mean and standard deviation of the values obtained for the control and EDC-treated specimens are listed in Table 1. Note that only the mean is presented for the crack growth coefficient (C) due to the comparatively large variation in this parameter. Considering the initiation of cyclic crack extension, there was no significant difference in the values obtained for the ΔKth between the control and EDC-treated groups. Similarly, there was no significant difference (p>0.05) in the Paris Law parameters (m, C) between the two groups.
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A comparison of the fatigue crack growth resistance of the control and EDC treated specimens after storage in artificial saliva is shown in Figure 3. Specifically, the responses obtained after 3 and 6 months of storage are shown in Figures 3a and 3b, respectively. According to the Wilcoxon Rank Sum test, after 3 months storage the EDC-treated specimens exhibited significantly greater resistance to fatigue crack growth than the control group (Z = −2.44; p=0.02). The EDC-treated group also exhibited significantly greater resistance to fatigue crack growth than the control group after 6 months storage as well (Z = −3.95; p
