Highlights
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This study evaluated the reversibility of the inactivation of dentin proteases by crosslinkers.
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The results confirm the longterm inhibitory effect of crosslinkers on dentin matrix.
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The study indicated that the longterm effect is both crosslinker and dose-dependent.
Abstract
Objective
Inactivation of dentin proteases by crosslinkers has been suggested as a way to prevent the degradation of dentin collagen in the hybrid layer. However, it is not known if the inhibition is reversible. The aim of this study was to evaluate the inactivation effect of various crosslinkers on dentin protease activity over a period of 6 months.
Methods
Demineralized dentin beams (1 × 2 × 6 mm, n = 10/group) were treated with (1) 1% glutaraldehyde (GA1), (2) 5% glutaraldehyde (GA5), (3) 1% grape seed extract (GS1), (4) 5% grape seed extract (GS5), (5) 10% sumac berry extract (S), (6) 20 μM curcumin (CR20), and (7) 200 μM curcumin (CR200) for 5 min. Untreated beams served as control. The beams were incubated up to 6 months and incubation media were used to analyze solubilized telopeptide (ICTP and CTX) fragments as indicators of MMP- and cathepsin K-mediated degradation after 1, 3 and 6 months of incubation. The relative MMP activity of dentin beams was tested using a generic MMP assay. Data were analyzed using repeated-measures ANOVA, α = 0.05.
Results
All treated groups showed significant decrease in CTX release (32.2–469.5 pg/mg dentin) and ICTP (1.8–47.6 ng/mg dentin) fragments during the first month of incubation compared to control (1159 pg/mg and 72.9 ng/mg dentin, respectively). GA5, GS5 and CR200 maintained their inhibitory effect during 6-month incubation. The results were confirmed by dry mass loss and relative MMP activity following 6 months.
Significance
The results of this study indicate that the long-term effect is both crosslinker and dose dependent.
1
Introduction
The activity of host-derived dentin proteases has been shown to be responsible for the enzymatic degradation of improperly impregnated dentin collagen at the resin/dentin interface. Among these enzymes, both matrix metalloproteinases (MMPs) and cysteine cathepsins have an important role considering their presence and activity in dentin matrices . Although endogenous matrix proteases are inactive and immobile in mineralized dentin, the application of etch-and-rinse or self-etch adhesives can activate matrix proteases in dentin, resulting in the degradation of the exposed collagen network from the hybrid layer over time .
The inhibition or inactivation of dentin proteases or increasing the biomechanical resistance of collagen structure are the main strategies for preventing the degradation of dentin collagen matrix . Recent studies have indicated that both synthetic (glutaraldehyde, carbodiimides etc.) and natural crosslinkers (grape seed extract, berries etc.) can improve the mechanical properties of dentin collagen matrix , resulting in more stable bonding properties over time . Moreover, the use of collagen crosslinkers was shown to inactivate exposed matrix-bound MMPs , increasing bond durability over time . A previous report, evaluated the effect of pretreatment of completely demineralized dentin matrix with synthetic or natural crosslinking agents on the loss of dry mass, and the liberation of ICTP and CTX telopeptide fragments during up to 14 days of incubation . However, no information is available on whether the inactivation effect of the crosslinkers is durable over time. In the current study, a similar model was used to evaluate the durability of the inactivation effect over 6-month incubation.
Thus, the two null hypotheses tested in the current study were that the inactivation of dentinal MMPs and dentinal cathepsin-K activity by the crosslinking agents is reversible over time, and that crosslinking agents do not reduce the loss of dry mass or total enzymatic activity of demineralized matrix over time.
2
Materials and methods
2.1
Specimen preparation
Eighty extracted non-carious human third molars from 18- to 21 year-olds were obtained under a protocol approved by the Dental College of Georgia, at Augusta University. The teeth were stored at 4 °C in 0.9% NaCl containing 0.02% NaN 3 to prevent microbial growth, and were used within one month after extraction.
Dentin beams with 6 mm × 2 mm × 1 mm were prepared from the mid-coronal dentin using a low-speed saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling. Samples were stirred in 10 wt% H 3 PO 4 for 24 h at 20 °C to completely demineralize dentin which was confirmed with digital radiography. Demineralized dentin samples were rinsed in distilled water for 1 h at 4 °C. Beams were divided into 8 groups (n = 10/group) so that the mean dry weight of each group was not statistically different from one another.
The beams in each group were treated with the respective crosslinker; glutaraldehyde, grape seed extract, sumac berry extract or curcumin at different concentrations ( Table 1 ), for five minutes. Following the crosslinker treatment, each beam was blot-dried and then placed in individually labeled polypropylene tubes containing 1 mL calcium and zinc-containing media (CM) at 37 °C for 1-, 3-, or 6-months in a shaking-water bath (60 cycles/min). The CM contained 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.02 mM ZnCl 2 , and 0.3 mM NaN 3 (pH 7.2). Untreated demineralized dentin beams served as control.
| Group | Composition | Description | Manufacturer |
|---|---|---|---|
| GA1 | 1% glutaraldehyde | Protein crosslinker OHC(CH 2 ) 3 CHO | Merck, Finland |
| GA5 | 5% glutaraldehyde | ||
| GS1 | 1% grape seed extract | Vitis vinifera, natural proanthocyanidin | Meganatural CA, USA |
| GS5 | 5% grape seed extract | ||
| S | 10% sumac (w/v) | Natural proanthocyanidin and gallotannin source | Collected natural seed |
| CR20 | 20 μM curcumin | (1E,6E)-1,7-Bis(4-hydroxy 3-methoxyphenyl)1,6-heptadiene-3,5dione | LKT Lab, USA |
| CR200 | 200 μM curcumin |
2.2
Loss of dry mass
The loss of demineralized dry dentin mass as a result of matrix degradation was used as an indirect indicator of total protease activity. After each incubation, demineralized dentin beams were rinsed in 1 mL distilled water at 4 °C. Subsequently, rinsed beams were blot-dried and placed in a sealed desiccator containing dry silica beads for 72 h. After drying, the dry mass of dentin beams was measured using an analytical balance (XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA). The loss of dry mass after each incubation period was calculated as percentage of dry mass loss according to the initial dry mass of each sample. Dehydrated beams were dipped into distilled water for rehydration at 4 °C prior to further incubation.
2.3
Solubilized telopeptides of collagen
C-terminal telopeptides of type I collagen are degraded by MMPs at neutral pH, resulting in the release of ICTP fragments, whereas cathepsin-K degrades CTX telopeptide fragments of C-terminal of type I collagen . Although the optimum pH for cathepsin K activity is about 5.0 , it does have low but significant catalytic activity at pH 7.4 . The amount of liberated telopeptide fragments due to MMP activity was measured using an ICTP ELISA kit (UniQ EIA, Orion Diagnostica, Finland), while the liberated fragments due to cathepsin-K activity were measured using the Serum CrossLaps ELISA (Immunodiagnostic System, Farmington, UK). Aliquots of the incubation medium from dentin beams were collected after each incubation period and analyzed. Ten to fifty microliter of the incubation medium was used for ICTP and CTX assays. The amount of CTX/ICTP release was calculated with a standard curve using standards with known concentrations provided in the kits. The assays were performed in duplicates using 10 samples/incubation period.
2.4
Measurement of the direct inactivation effect of crosslinkers
To evaluate the changes in the activity of dentin MMPs, we tested experimental groups initially before crosslinker pretreatment and after 6-month incubation by using a generic MMP assay kit (Sensolyte, Fremont, CA, USA). The assay is used to detect total MMs activity from the same beams. The method is based on a substrate with a color-developing thiol-reactive agent which releases a sulfhydyl group when cleaved by the MMPs. Beams were dipped into 100 μL chromogenic substrates and 100 μL assay buffer of the kit in 96-well plate for 60 min at 25 °C. The final product of this reaction, 2-nitro-5-thiobenzoic acid (TNB), was detected at 412 nm using a colorimetric spectrometer (Synergy HT; BioTek Instruments, Winooski, VT, USA) for 60 min. The MMP activity of the groups was calculated as a percentage of the baseline measurement for each specimen individually to determine the relative inhibition or activation.
2.5
Statistical analyses
The percentage loss of dry mass and the amount of liberated ICTP (ng telopeptide/mg dry dentin) and CTX (pg telopeptide/mg dry dentin) fragments from all groups were compared for normality (Kolmogorov–Smirnov test) and homoscedasticity (modified Levene’s test). When the normality and equality variance assumptions of the data were valid, they were analyzed using repeated measures ANOVA. Post hoc multiple comparisons were performed with the Tukey’s test using IBM SPSS v.23 (NY, USA).
2
Materials and methods
2.1
Specimen preparation
Eighty extracted non-carious human third molars from 18- to 21 year-olds were obtained under a protocol approved by the Dental College of Georgia, at Augusta University. The teeth were stored at 4 °C in 0.9% NaCl containing 0.02% NaN 3 to prevent microbial growth, and were used within one month after extraction.
Dentin beams with 6 mm × 2 mm × 1 mm were prepared from the mid-coronal dentin using a low-speed saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling. Samples were stirred in 10 wt% H 3 PO 4 for 24 h at 20 °C to completely demineralize dentin which was confirmed with digital radiography. Demineralized dentin samples were rinsed in distilled water for 1 h at 4 °C. Beams were divided into 8 groups (n = 10/group) so that the mean dry weight of each group was not statistically different from one another.
The beams in each group were treated with the respective crosslinker; glutaraldehyde, grape seed extract, sumac berry extract or curcumin at different concentrations ( Table 1 ), for five minutes. Following the crosslinker treatment, each beam was blot-dried and then placed in individually labeled polypropylene tubes containing 1 mL calcium and zinc-containing media (CM) at 37 °C for 1-, 3-, or 6-months in a shaking-water bath (60 cycles/min). The CM contained 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.02 mM ZnCl 2 , and 0.3 mM NaN 3 (pH 7.2). Untreated demineralized dentin beams served as control.
| Group | Composition | Description | Manufacturer |
|---|---|---|---|
| GA1 | 1% glutaraldehyde | Protein crosslinker OHC(CH 2 ) 3 CHO | Merck, Finland |
| GA5 | 5% glutaraldehyde | ||
| GS1 | 1% grape seed extract | Vitis vinifera, natural proanthocyanidin | Meganatural CA, USA |
| GS5 | 5% grape seed extract | ||
| S | 10% sumac (w/v) | Natural proanthocyanidin and gallotannin source | Collected natural seed |
| CR20 | 20 μM curcumin | (1E,6E)-1,7-Bis(4-hydroxy 3-methoxyphenyl)1,6-heptadiene-3,5dione | LKT Lab, USA |
| CR200 | 200 μM curcumin |
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