The progressive degradation of resin–dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium.
This study evaluated the effect of different storage media on changes in matrix stiffness, loss of dry weight or solubilization of collagen from demineralized dentin beams incubated in vitro for up to 60 days.
Dentin beams (1 mm × 2 mm × 6 mm) were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass and elastic modulus ( E ) (3-point bending, 15% strain) the beams were divided into 5 groups ( n = 11/group) and incubated at 37 °C in either media containing both zinc and calcium designated as complete medium (CM), calcium-free medium, zinc-free medium, a doubled-zinc medium or water. Beams were retested at 3, 7, 14, 30, and 60 days of incubation. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HOP) as an index of solubilization of collagen by MMPs. Data were analyzed using repeated measures of ANOVA.
Both the storage medium and the storage time showed significant effects on E , mass loss and HOP release ( p < 0.05). The incubation in CM resulted in relatively rapid and significant ( p < 0.05) decreases in stiffness, and increasing amounts of mass loss. The HOP content of the experimental media also increased with incubation time but was significantly lower ( p < 0.05) than in the control CM medium, the recommended storage medium.
The storage solutions used to age resin–dentin bonds should be buffered solutions that contain both calcium and zinc . The common use of water as an aging medium may underestimate the hydrolytic activity of endogenous dentin MMPs.
When the durability of resin–dentin bonds are tested in vitro , the commonly used method is to soak the bonded teeth in 37 °C water for 24 h and then section the teeth in the x and y planes into 1 mm 2 beams. This preparation is considered a form of accelerated aging because water needs only to diffuse 0.5 mm from each cut surface to reach the center of the beam . Water also diffuses into the adhesive and causes water sorption, plasticization of the polymers and solubilizes unreacted monomers . The other degradative mechanism in resin–dentin bonds is caused by the acid-etching step in bonding that uncovers and activates endogenous dentin matrix metalloproteinases (MMPs) These enzymes are neutral proteases that add water across specific peptide linkages in collagen peptides . Metalloproteinases require calcium and zinc ions to maintain their proper tertiary structure and functional active sites, respectively . These ions, that are normally constituents of body fluids may be lost during acid-etching and water rinsing. If the resin–bonded dentin beams are incubated in water rather than calcium and zinc containing buffers, the influence of MMPs on matrix degradation in vitro may be underestimated. Another important variable in the durability of resin–dentin bonds is how well the adhesive monomers infiltrate the acid-etched matrix which is regarded as a technique-sensitive procedure . Recently, a simple model to study the rate of degradation of demineralized dentin matrices independent of resin, was developed by making 6 mm × 2 mm × 1 mm beams of coronal dentin, and then completely demineralizing them in 10% phosphoric acid . By using demineralized beams devoid of resin, the decrease in their modulus of elasticity by 3-point flexure before and after 30 days of incubation in experimental media at 37 °C can be measured. By not infiltrating them with resin, uncertainty about how well the collagen fibrils were encapsulated could be eliminated . When the beams are incubated in experimental media containing calcium and zinc at 37 °C for 30–60 days, if the MMPs are active, they will slowly solubilize collagen peptide fragments that will accumulate in the media and can be collected and quantitated for collagen release.
The purpose of this in vitro study was to test the null hypothesis that there are no differences in matrix stiffness, loss of dry weight or solubilization of collagen from demineralized dentin matrices when incubated in water, in complete media or media deficient in calcium or zinc. The activity of endogenous dentin matrix MMPs was assessed indirectly by measuring the modulus of elasticity of demineralized beams before and after incubation for up to 60 days at 37 °C. Solubilization of collagen was quantitated gravimetrically by measuring the loss of dry mass over time and by measuring the amount of the hydroxyproline that was released from the solubilized collagen into the incubation media.
Materials and methods
Preparation of dentin disks
Fifty-five extracted human third molars were obtained with patient’s informed consent under a protocol approved by the Human Assurance Committee of the Medical College of Georgia. The teeth were stored at 4 °C in 0.9% NaCl supplemented with 0.02% sodium azide for no more than 1 month before use. The enamel and superficial dentin of each tooth were removed from the crown using a diamond-encrusted copper disk (Isomet saw, Buehler Ltd., Lake Bluff, IL, USA), by a horizontal section 1 mm below the deepest central groove. This removes the high concentration of MMP-2 reported to reside at the dentinoenamel junction . One millimeter thick dentin disks of mid-coronal dentin were then prepared by moving the blade slightly more than 1 mm apical to the first section. This second section removed the deep dentin/predentin source of MMP-2 and yielded mid-coronal dentin that has been shown to have a relatively constant distribution of MMPs . Dentin beams 6 mm × 2 mm × 1 mm were sectioned from each dentin disk to obtain fifty-five beams.
For demineralization, the beams were submerged in 10 wt% phosphoric acid for 18 h at 25 °C. Digital radiography was used to document the absence of residual minerals. The initial modulus of elasticity of the beams was measured by 3-point flexure to 15% strain. Previous work showed no plastic deformation of the beams at 15% strain . The initial modulus values also confirmed complete demineralization, since mineralized beams were previously shown to have moduli of elasticity of 16–19 GPa. Beams accepted as demineralized had moduli of elasticity <5 MPa . After initial modulus of elasticity measurements, the beams were distributed to five balanced groups ( n = 11/group) so that the mean initial elastic modulus of each group was statistically similar in all groups.
Five incubation media were used: group (1) complete media (CM) containing 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.05 mM ZnCl 2 , and 0.3 mM NaN 3 , pH 7.4 ( Table 1 ); group (2) a calcium-free modified media (calcium-free) containing all the other compounds except for CaCl 2 ; group (3) a zinc-free modified media (zinc-free) containing all the other compounds except ZnCl 2 ; group (4) a double-zinc media (double-zinc) containing all the other compounds, except that the concentration of ZnCl 2 which was doubled (0.1 mM); group (5) distilled water containing 0.3 mM NaN 3 (water).
|Whole saliva a||Complete medium (simulated oral fluid) b||Plasma c|
|Bicarbonate||1.0||5 mM HEPES||25|
|Fluoride μg%||8–25 μg/100 mL||0||10–20|
|Iodide μg%||4–24 μg/100 mL||0||3–8|
|NaN 3 (antimicrobial)||0||0.3 mM||0|
Each beam was separately incubated in one of the five incubation media, in individually labeled propylene tubes with threaded caps containing 1 mL of incubation media. The tubes were placed in a shaking water bath (Reciprocal Shaking Bath, Thermo Scientific, Marietta, OH, USA) (60 cycles/min) at 37 °C for 3, 7, 14, 30, 60 days of incubation.
Indirect assessment of the activity of the endogenous MMP activity in demineralized matrices
Loss of modulus of elasticity
Demineralized dentin beams were placed on the 3-point flexure jig with a 2.5 mm separation between supports, immersed under water. Using a 1 N load cell (Transducer Techniques, Temecula, CA, USA) mounted on a Vitrodyne universal tester (Model V1000, John Chatillon & Sons, Greensboro, NC, USA), the beams were deformed to a 15% strain at a displacement rate of 0.5 mm min −1 . After maximum displacement, the load was returned immediately to 0, to prevent creep of the demineralized collagen matrix . After initial testing, the beams were arranged into five balanced groups so that their mean values were not statistically different. They were then incubated in the control or experimental media in designated polypropylene tubes, and then retested after each incubation period. At every time point, the beams were rinsed free of salts for 10 min with running distilled water, and then retested in 3-point flexure to the same degree of strain.
The modulus of elasticity ( E ; in MPa) of each specimen was calculated as the steepest slope of the linear portion of the stress–strain curve using the following formula:
where m is the steepest slope of the linear portion of the load-displacement curve (N/mm), L is the span length (2.5 mm), b is the width of the test specimen (2.0 ± 0.1 mm) and h is the beam thickness (1.0 ± 0.05 mm).
Loss of dry mass over time
After the measurements of the initial modulus of elasticity, the beams were transferred to individually labeled polypropylene tubes and placed in a sealed plastic box containing anhydrous calcium sulfate (Drierite, W.A. Hammond Drierite Company, Xenio, OH, USA). With the vial caps off, the beams were desiccated to a constant weight within 8 h. The initial dry mass was measured to the nearest 0.001 mg on an analytical balance (Mettler XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA). After dry mass measurement, dried dentin beams were reimmersed in water and placed back in its corresponding polypropylene tube containing the original incubation medium. After each incubation period, determination of the dry mass was repeated under the same conditions following the modulus of elasticity measurements.
Analysis of incubation media for solubilized collagen peptides
The collagen in demineralized dentin is insoluble type I collagen. Demineralization of the matrix uncovers the endogenous MMPs and activates them even though they remain bound to collagen . If the MMPs are incubated in media that contain calcium and zinc , the enzymes can slowly solubilize collagen peptide fragments that accumulate in the 1 mL of test media over the 30–60 days experiment . At the end of each incubation period, 400 μL of the media was collected from each vial to an individually labeled ampule, diluted with an equal volume of 12 N HCl to give a final concentration of 6 N HCl. Ampules were sealed using an ampule sealer (Ampulmatic, Biosciences Inc., PA, USA). The sealed ampules were hydrolyzed at 120 °C in an oil bath for 18 h. After hydrolysis, the ampules were opened and placed in large glass desiccators containing anhydrous calcium sulfate and trays of sodium hydroxide pellets used to trap the HCl vapor that was released as the hydrolyzate evaporated to dryness in a mild vacuum. When dry, the hydroxyproline content of the hydrolyzate was analyzed using the method of Jamall et al. in a spectrophotometer (Model UV-A180, Shimadzu, Tokyo, Japan) at 558 nm. The measured amount of hydroxyproline was used to estimate the percent of solubilized (i.e. degraded) collagen assuming that 90% of the dry mass of the demineralized dentin beams consisted of type I collagen and that the dentin collagen contains 9.6 mass% of hydroxyproline . For each specimen, the solubilized collagen from demineralized dentin beam was expressed as μg of HOP/mg of the dry mass of the demineralized dentin before the incubation for each incubation period.
Modulus of elasticity, dry mass loss and HOP data were analyzed separately. Repeated measures analysis with “storage media” as the group variable and “timepoint” (3, 7, 14 days, etc.) as the repeated factor was used to analyze the data. Repeated measures analysis of variance (ANOVA) was used to test for interaction between the group and timepoint factors and, if no significant interaction was found, to test the main effects for group and timepoint. If significant interaction was found, then the five groups were compared separately at each timepoint and timepoints were compared separately for each group. If either the normality or equal-variance assumptions underlying the traditional repeated measures ANOVA tests were violated, rank-based ANOVA tests were used. The Tukey–Kramer method of pair-wise comparisons for repeated measures designs was used to evaluate the significance of the difference between each pair of groups and between each pair of timepoints. A significance level of 0.05 was used for all other statistical tests. All analyses were carried out using SAS 9.2 for Windows (SAS Institute Inc., Cary, NC, 2008).