Effects of EDC crosslinking on the stiffness of dentin hybrid layers evaluated by nanoDMA over time

Abstract

Application of collagen cross-linkers to demineralized dentin improves bond durability. While the benefits of cross-linking treatments to bond strength and fatigue resistance have been explored, changes in hybrid layer stiffness with aging have not been examined.

Objective

To examine the influence of a cross-linking treatment using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) on hybrid layer stiffness of resin-dentin adhesive bonds, using spatially-resolved nanoscopic Dynamic Mechanical Analysis (nanoDMA).

Methods

Bonded interface specimens were prepared using a two-step (SB) or three-step (SBMP) etch-and-rinse adhesive. Adhesive bonding of the treated groups was preceded by a 1 min application of an experimental EDC conditioner to the acid-etched dentin. Control specimens did not receive EDC treatment. The bonded interfaces were evaluated using nanoDMA to determine the dynamic mechanical properties after storage in artificial saliva at 37 °C for 0, 3 and 6 months.

Results

The EDC treatment had no influence on the dynamic mechanical properties of the hybrid layer immediately after bonding. There was also no reduction in the hybrid layer stiffness after 3 and 6 months aging as defined by the complex modulus and storage modulus. However, there was a significant reduction in the loss modulus and tan δ components (i.e. viscous behavior) of the hybrid layers with aging. Degradation occurred to both adhesive systems with storage, but was greatest for SB. Without EDC treatment, the reduction in tan δ of the hybrid layer prepared with SB exceeded 80% in 6 months.

Significance

The application of EDC to acid-etched dentin helps maintain the viscoelasticity of hybrid layers.

Introduction

The placement of resin-composite restorations using contemporary bonding procedures exposes endogenous proteases in preparations that extend into dentin . Although dormant in mineralized dentin, acid-etching activates these proteases and enables slow enzymatic degradation of collagen fibrils within the hybrid layer . The degree of degradation increases with aging, and is largely concentrated at the bottom of the hybrid layer, where the collagen is poorly infiltrated . Mazzoni et al. demonstrated that this area has very high enzyme activity, which facilitates the initiation and progression of degradation. Because collagen fibrils are essential for anchoring composite restorations to dentin, this process is detrimental to bond durability. Indeed, degradation of the collagen fibrils within the hybrid layer has been identified as one of the principal contributors to the failure of resin-adhesive bonds to dentin .

Several strategies are being pursued to reduce enzymatic degradation of dentin collagen and maintain the integrity of resin-dentin adhesive bonds. One approach involves the use of 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 . These endogenous proteases within dentin matrices can be inactivated by application of crosslinkers for as little as 1 min .

Many crosslinkers are being considered, and of the present candidates carbodiimide (EDC) has low cytotoxicity and an ability to preserve dentin bonds within clinically acceptable treatment times . According to the work of Mazzoni et al. treatment of demineralized collagen with 0.3 M solution of carbodiimide for 1 min helped maintain dentin bond strength for over a year of aging in artificial saliva, which appears to result from inactivation of the endogenous proteases . Zhang et al. found treatment of the demineralized collagen with 0.5 M EDC for one minute also increases the resistance to fatigue. Dentin bonds treated with EDC exhibited significantly greater fatigue strength and fatigue crack growth resistance 6 months after bonding.

Apart from benefits to bond strength, the effects of cross-linking on the hybrid layer stiffness remains an important question. Cross-linking treatments increase the immediate stiffness of demineralized dentin matrices , but changes in stiffness of hybrid layers with aging has not been explored. Therefore, the primary objectives of this study were to evaluate the effect of a cross-linking treatment applied to the demineralized dentin matrix on the dynamic moduli of the hybrid layer, and to assess its stability during aging in artificial saliva. 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) has no effect on the immediate stiffness of the hybrid layer, and (2) has no effect on the stiffness of the hybrid layer after a 6 months aging regimen.

Materials and methods

Human third molars were obtained from participating clinics in Maryland with record of age (18 ≤ age ≤ 30 yrs) from anonymous donors according to an approved protocol (#Y04DA23151). Each tooth was evaluated to confirm that it was caries-free, and then 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. Sections were obtained from the mid-coronal region as necessary for the required specimen geometry. The remaining materials used in the development of the specimens included a three-step etch-and-rinse adhesive (Scotchbond Multipurpose (SBMP), 3M ESPE, Saint Paul, MN USA), a comparable two-step adhesive (Scotchbond (SB), 3M ESPE, Saint Paul, MN USA) and a compatible resin composite (Z100, 3M ESPE, Saint Paul, MN USA).

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 . Briefly, the dentin sections from the coronal region represented half of the CT specimen geometry. The edge oriented farthest from the pulp (outer dentin) was etched for 15 s (SB 37% phosphoric acid etchant) and rinsed with water in preparation for bonding. Then the primer and adhesive were applied (as appropriate for the resin adhesive system) 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 complete the CT geometry. The completed specimens were cured on both sides for 40 s 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.

The dynamic mechanical behavior of the bonded interfaces was evaluated with and without a crosslinking treatment 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 s. The specimens were then rinsed with water for 15 s 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. The solution contained 0.2% sodium azide to prevent microbial growth. A total of 36 specimens were prepared overall and consisted of an equal number of non-cross-linked (control) and cross-linked specimens (that received EDC treatment). The dynamic mechanical behavior of the bonded interfaces was evaluated after a storage period of 0, 3 or 6 months, resulting in six specimens in each cell (treatment and time) for each of the two adhesive systems. Those specimens evaluated at 0 months (i.e. without storage) are considered to represent the “immediate” response and were tested after a period of at least 48 h from the date of preparation.

The dynamic mechanical behavior of the bonded interfaces was evaluated according to the procedures described in previous investigations . Briefly, after completion of the aging period, the bonded interface specimens were cold-mounted in Epofix epoxy resin (Struers, Cleveland, OH, USA) taking care to prevent epoxy resin from touching the exposed surface. The specimens were arranged to fully expose the bonded interface and then were prepared for evaluation by nanoDMA, which required polishing. Preliminary polishing was performed with SiC abrasive paper from mesh numbers of #800–#4000. Final polishing was performed with abrasive cloth and diamond particle suspensions (Buehler) of sizes 9, 3, and 0.04 μm to produce a highly polished surface with a roughness of less than 50 nm RMS; the roughness was confirmed from scanning analysis of the topography with the nanoindenter. Nanoscale Dynamic Mechanical Analysis (nanoDMA) was performed with a Triboindenter (Model TI 900, Hysitron, Minneapolis, MN, USA) equipped with a Berkovich diamond indenter with a 100-nm tip radius. Scanning mode dynamic loading was conducted over scan areas of approximately 30 μm × 30 μm with 4 μN contact load, 2 μN dynamic loading amplitude and dynamic loading frequency of 100 Hz. The contact load and displacement signals were used to calculate the phase angle and to generate maps of the dynamic mechanical property distribution for the interface including mineralized dentin, hybrid layer, resin adhesive and resin composite. Scanning was performed on hydrated specimens using a layer of ethylene glycol over the specimen surface to prevent water evaporation.

The results of each scan were evaluated in terms of the complex modulus, storage modulus, loss modulus and phase map (i.e. tan δ ). Briefly, the storage and complex moduli are measures of the stored elastic energy and viscous components of the indentation response, respectively. The complex modulus represents a mathematic measure involving the sum of those two components, and the tan δ provides a measure of the ratio of the viscous and non-viscous components of the deformation. More complete descriptions of these components in reference to hybrid layers are presented by Ryou et al. . Subsets of the total scan area were selected within the center of the hybrid layer (equidistant from the mineralized dentin and resin adhesive) to characterize the properties in this region. Each of these subsets was roughly 15 × 15 pixels and slightly less than 2 μm 2 . Five subsets were chosen from each scan to describe the average properties of the hybrid layer.

Collected data from the scans obtained from the nanoDMA of the hybrid layers was analyzed using commercial statistical software (IBM SPSS, Endicott, New York, USA, version 20.0). For each of the dynamic properties including the moduli and tan δ values, a two-way ANOVA was performed to compare that property between the three time periods and between the two treatment conditions (i.e. without and with EDC). The data was checked for homoscedasticity and homogeneity of variance. All the ANOVA analyses included a Tukey’s post-hoc analysis and were performed with p ≤ 0.05 indicating a significant difference.

In addition, line scans of the complex modulus and tan δ distributions were obtained across the hybrid layer to distinguish if there were gradients in these properties that developed during the increments of storage. Details regarding application of nanoDMA and evaluating the resin-dentin interface using this approach have been previously described .

Materials and methods

Human third molars were obtained from participating clinics in Maryland with record of age (18 ≤ age ≤ 30 yrs) from anonymous donors according to an approved protocol (#Y04DA23151). Each tooth was evaluated to confirm that it was caries-free, and then 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. Sections were obtained from the mid-coronal region as necessary for the required specimen geometry. The remaining materials used in the development of the specimens included a three-step etch-and-rinse adhesive (Scotchbond Multipurpose (SBMP), 3M ESPE, Saint Paul, MN USA), a comparable two-step adhesive (Scotchbond (SB), 3M ESPE, Saint Paul, MN USA) and a compatible resin composite (Z100, 3M ESPE, Saint Paul, MN USA).

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 . Briefly, the dentin sections from the coronal region represented half of the CT specimen geometry. The edge oriented farthest from the pulp (outer dentin) was etched for 15 s (SB 37% phosphoric acid etchant) and rinsed with water in preparation for bonding. Then the primer and adhesive were applied (as appropriate for the resin adhesive system) 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 complete the CT geometry. The completed specimens were cured on both sides for 40 s 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.

The dynamic mechanical behavior of the bonded interfaces was evaluated with and without a crosslinking treatment 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 s. The specimens were then rinsed with water for 15 s 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. The solution contained 0.2% sodium azide to prevent microbial growth. A total of 36 specimens were prepared overall and consisted of an equal number of non-cross-linked (control) and cross-linked specimens (that received EDC treatment). The dynamic mechanical behavior of the bonded interfaces was evaluated after a storage period of 0, 3 or 6 months, resulting in six specimens in each cell (treatment and time) for each of the two adhesive systems. Those specimens evaluated at 0 months (i.e. without storage) are considered to represent the “immediate” response and were tested after a period of at least 48 h from the date of preparation.

The dynamic mechanical behavior of the bonded interfaces was evaluated according to the procedures described in previous investigations . Briefly, after completion of the aging period, the bonded interface specimens were cold-mounted in Epofix epoxy resin (Struers, Cleveland, OH, USA) taking care to prevent epoxy resin from touching the exposed surface. The specimens were arranged to fully expose the bonded interface and then were prepared for evaluation by nanoDMA, which required polishing. Preliminary polishing was performed with SiC abrasive paper from mesh numbers of #800–#4000. Final polishing was performed with abrasive cloth and diamond particle suspensions (Buehler) of sizes 9, 3, and 0.04 μm to produce a highly polished surface with a roughness of less than 50 nm RMS; the roughness was confirmed from scanning analysis of the topography with the nanoindenter. Nanoscale Dynamic Mechanical Analysis (nanoDMA) was performed with a Triboindenter (Model TI 900, Hysitron, Minneapolis, MN, USA) equipped with a Berkovich diamond indenter with a 100-nm tip radius. Scanning mode dynamic loading was conducted over scan areas of approximately 30 μm × 30 μm with 4 μN contact load, 2 μN dynamic loading amplitude and dynamic loading frequency of 100 Hz. The contact load and displacement signals were used to calculate the phase angle and to generate maps of the dynamic mechanical property distribution for the interface including mineralized dentin, hybrid layer, resin adhesive and resin composite. Scanning was performed on hydrated specimens using a layer of ethylene glycol over the specimen surface to prevent water evaporation.

The results of each scan were evaluated in terms of the complex modulus, storage modulus, loss modulus and phase map (i.e. tan δ ). Briefly, the storage and complex moduli are measures of the stored elastic energy and viscous components of the indentation response, respectively. The complex modulus represents a mathematic measure involving the sum of those two components, and the tan δ provides a measure of the ratio of the viscous and non-viscous components of the deformation. More complete descriptions of these components in reference to hybrid layers are presented by Ryou et al. . Subsets of the total scan area were selected within the center of the hybrid layer (equidistant from the mineralized dentin and resin adhesive) to characterize the properties in this region. Each of these subsets was roughly 15 × 15 pixels and slightly less than 2 μm 2 . Five subsets were chosen from each scan to describe the average properties of the hybrid layer.

Collected data from the scans obtained from the nanoDMA of the hybrid layers was analyzed using commercial statistical software (IBM SPSS, Endicott, New York, USA, version 20.0). For each of the dynamic properties including the moduli and tan δ values, a two-way ANOVA was performed to compare that property between the three time periods and between the two treatment conditions (i.e. without and with EDC). The data was checked for homoscedasticity and homogeneity of variance. All the ANOVA analyses included a Tukey’s post-hoc analysis and were performed with p ≤ 0.05 indicating a significant difference.

In addition, line scans of the complex modulus and tan δ distributions were obtained across the hybrid layer to distinguish if there were gradients in these properties that developed during the increments of storage. Details regarding application of nanoDMA and evaluating the resin-dentin interface using this approach have been previously described .

Results

An electron micrograph for a representative bonded interface specimen prepared with SB adhesive is shown in Fig. 1 a. The corresponding distribution of the complex modulus for this specimen is shown in Fig. 1 b. Scanning measurements yielded property maps for the dynamic mechanical moduli over the area of scanning. The scan window for these maps is 32 μm × 32 μm and is the window size adopted for all of the scanning performed in the present study. Note that the location of the electron micrograph and complex modulus distribution shown in Fig. 1 are not the same, which is evident from a comparison of the morphological details. However, the bonded interface microstructure (including mineralized dentin, hybrid layer, adhesive and resin composite) is clearly evident from the complex modulus variations in Fig. 1 b. Evident from the values in these scans is that the complex modulus of the resin adhesive layer is quite similar to that of the underlying hybrid layer.

Fig. 1
Analyzing the dynamic mechanical behavior of the resin-dentin bonded interface using nanoDMA. (a) Interface of a representative specimen prepared with SB resin adhesive and evaluated at time zero. Scale bar indicates 10 μm. (b) Typical complex modulus map obtained from nanoDMA scanning of the interface from the specimen in (a). The window of evaluation in (b) is 32 μm × 32 μm and is not from exactly the same location as shown in (a). Important features of the bonded interface are clearly evident from the modulus variation including the mineralized dentin (M) and resin-filled tubules (T), hybrid layer (H), resin adhesive (A) and resin composite (C).

A complete set of property maps resulting from nanoDMA scanning is shown in Fig. 2 for a representative bonded interface specimen prepared with SB adhesive. This particular specimen was subjected to treatment with EDC for 60 s prior to application of the resin adhesive. This specimen was not subjected to aging. The complex modulus, loss modulus and storage modulus distributions for this specimen are shown in Fig. 2 a–c, respectively. The corresponding distribution of tan δ is shown in Fig. 2 d. As evident from these property maps, the mineralized dentin, hybrid layer, resin adhesive and resin composite are clearly evident. Even the individual filler particles of the resin composite are apparent in this scan, particularly within the complex modulus and the storage modulus maps, which results from the high stiffness of these particles in comparison to the resin matrix. Features of the nanoDMA scans obtained for the specimens prepared with SB adhesives were very similar with those prepared with SBMP (not shown).

Fig. 2
Results from nanoDMA scanning of a representative specimen prepared with SB adhesive and EDC pretreatment. Property maps of (a) complex modulus (GPa), (b) loss modulus (GPa), (c) storage modulus (GPa) and (d) phase angle, represented by tan ( δ ). The image window for all the property maps is 32 μm × 32μm.

The maps obtained for each specimen were examined for consistency and to confirm that there were no anomalies present such as voids, etc. Thereafter, locations within the hybrid layer were selected randomly to quantify the average “bulk” response of the hybrid layer for each condition (age and treatment). Fig. 3 provides a summary of results obtained from nanoDMA testing of the bonded interface specimens prepared with SB as a function of the storage time. In particular, the distribution of the complex modulus, loss modulus and storage modulus measurements are presented in Fig. 3 a–c, respectively. The corresponding results for the tan δ are shown in Fig. 3 d. A list of the dynamic mechanical properties of the hybrid layer for the SB adhesive is presented in Table 1 in terms of the average and standard deviation.

Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Effects of EDC crosslinking on the stiffness of dentin hybrid layers evaluated by nanoDMA over time
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