Abstract
Objective
Dentin matrices release ICTP and CTX fragments during collagen degradation. ICTP fragments are known to be produced by MMPs. CTX fragments are thought to come from cathepsin K activity. The purpose of this study was to determine if quaternary methacrylates (QAMs) can inhibit matrix MMPs and cathepsins.
Methods
Dentin beams were demineralizated, and dried to constant weight. Beams were incubated with rh-cathepsin B, K, L or S for 24 h at pH 7.4 to identify which cathepsins release CTX at neutral pH. Beams were dipped in ATA, an antimicrobial QAM to determine if it can inhibit dentin matrix proteases. Other beams were dipped in another QAM (MDPB) to determine if it produced similar inhibition of dentin proteases.
Results
Only beams incubated with cathepsin K lost more dry mass than the controls and released CTX. Dentin beams dipped in ATA and incubated for 1 week at pH 7.4, showed a concentration-dependent reduction in weight-loss. There was no change in ICTP release from control values, meaning that ATA did not inhibit MMPs. Media concentrations of CTX fell significantly at 15 wt% ATA indicating that ATA inhibits capthesins.
Beams dipped in increasing concentrations of MDPB lost progressively less mass, showing that MDPB is a protease-inhibitor. ICTP released from controls or beams exposed to low concentrations were the same, while 5 or 10% MDPB significantly lowered ICTP production. CTX levels were strongly inhibited by 2.5–10% MDPB, indicating that MDPB is a potent inhibitor of both MMPs and cathepsin K.
Significance
CTX seems to be released from dentin matrix only by cathepsin K. MMPs and cathepsin K and B may all contribute to matrix degradation.
1
Introduction
In order to bond tooth-colored resin composites to enamel and dentin, these hard tissues are acid-etched to increase their micro- and nanoporosity. After infiltrating resins into these porosities, the results is a new hard tissue that is called the hybrid layer .
Hybrid layers are formed when solvated comonomers are infiltrated into dentin surfaces that have been acid-etched with 37% phosphoric acid for 15 s. Acid-etching uncovers the collagen matrix and activates the proforms of endogenous dentin proteases . If resin does not replace all of the rinse water, portions of the hybrid layer will include water-filled, resin-poor, collagen fibrils containing activated proteases that can slowly destroy the very fibrils that anchor resin-composites to dental hard tissues. This causes a loss of retention of tooth colored restorations, requiring their replacement .
The cyclic loading of hybrid layers during mastication induces excessive strain in the low modulus of elasticity water-filled zones, causing accelerated degradation. This degradation is thought to occur at pH 7, in contrast to the cyclic changes in pH encountered in carious lesions where intralesion pHs swing from 7.4 to 5. As the optimum pH for cathepsin K is 5.0, its collagenolytic activity is very prominent in carious lesions . However, that does not mean that cathepsin K has no collagenase activity at pH 7.4. Kometoni et al. reported that rh cathepsin K enzyme activity in vitro at pH 5.5 was 91% of this maximal activity at pH 5.0; when the pH was adjusted to 6.5, the enzyme showed 85% of its residual activity. At pH 7.5, the enzyme still retained 11% of its activity.
Peripheral dentin is a cell-free, mineralized connective tissue that does not turn-over . Hybrid layers are sequestered from saliva by bonded resin composites, and from pulpal fluids by resin tags that occlude the tubules. Thus, there is no source of replacement proteases in resin bonded dentin. Any degradation that occurs in the absence of carious bacteria, probably occurs at pH 7.4.
Since both active cathepsins and MMPs have been identified in peripheral dentin , and since both CTX and ICTP telopeptide have been identified in the incubation medium of demineralized dentin beams at pH 7.4, we assume that both classes of proteases contribute to collagen degradation of hybrid layers.
Because hybrid layers are only 1–10 μm thick, they do not release enough telopeptides to be detected by specific ELISAs, even after prolonged incubation. Thus, many investigators use a macrohybrid layer model where dentin beams 0.3–1.0 mm thick are completely demineralized in 10–37% phosphoric acid for 12–16 h to uncover and activate the endogenous proteases of dentin. While this might seem extreme treatment that might inactivate endogenous proteases, Tezvergil-Mutluay et al. compared the ICTP and CTX telopeptide release from dentin beams that were completely demineralized in 0.5 M EDTA pH 7.4 (controls) to EDTA-demineralized beams that were then exposed to 1, 10 or 37% phosphoric acid for up to 15 min. After rinsing, they were incubated in pH 7.4 buffer for 3 days. There were no changes in either ICTP or CTX release from control or experimental beams, indicating that phosphoric acids at pH 0.4–1.0 for 15 min had no influence of telopeptidase activity. The CTX release was only one-tenth that of ICTP, presumably because cathepsin K was operating at pH 7.4 instead of its optimum pH of 5.0. These results suggest that collagen-bound proteases are resistant to inactivation or denaturation by acids used in adhesive dentistry and confirm that cathepsin K can continue to function, albeit more slowly at pH 7.4 that is 2.5 pH units away from its optimum pH.
The nonspecific, cationic protease inhibitor chlorhexidine has recently been shown to inhibit cysteine cathepsins , in addition to MMPs . We have recently shown that cationic quaternary ammonium methacrylates (QAMs) can also inhibit MMPs .
The purpose of this study was to determine if the most effective QAMs screened for their anti-MMP activity , can also inhibit cathepsin K at pH 7.4 by measuring the release of ICTP from demineralized dentin beams as an indirect measure of MMP activity, and the release of CTX as an indirect measure of cathepsin K activity. The test null hypotheses were that 1, 5, 10, 15 wt% QAMs have no inhibitory activity on either endogenous dentin matrix MMPs or cathepsin K, at pH 7.4, the pH of hybrid layers.
2
Materials and methods
2.1
Effects of rh cathepsins on mass loss (experiment 1)
Recombinant human cysteine cathepsins B, L and S (catalytic domain) were obtained from Athens Research & Technology (Athens, GA). Procathepsin K was obtained from EnzoLife Sciences (Plymouth Meeting, PA) and activated with 100 mM sodium acetate, pH 3.9, 10 mM DTT and 5 mM EDTA for 40 min at 25 °C. Cathepsins B, L and S were already active when purchased. Dentin beams in all groups (2 mm × 1 mm × 6 mm) were prepared from 18 to 21 year old mid-coronal dentin of human third molars using an Isomet saw (Buehler Ltd., Lake Bluff, IL). The beams were completely demineralized in 10 wt% phosphoric acid for 18 h at 25 °C with tumbling, and then rinsed free of acid in water. After obtaining constant dry masses, the individual beams ( n = 5 beams/group) were incubated in 10 μg of one of the specific cathepsins, dissolved in 0.5 mL of simulated artificial saliva (SBF) , containing KCl 13 mM, KSCN 2 mM, Na 2 SO 4 7.5 mM, HEPES 5 mM, NH 4 Cl 3 mM, CaCl 2 1.5 mM, NaHCO 3 7.5 mM, ZnCl 2 0.02, PO 4 3− 4, and NaN 3 0.02%, pH adjusted to pH 7.4. Another set of beams was incubated with 10 μg rh cathepsin K at pH 5.0 to demonstrate the maximum activity of the enzyme. After incubation in SBF (37 °C with shaking) for 24 h, the beams were removed, rinsed with water to remove buffer salts, and re-dried to a constant weight in sealed containers of dry calcium sulfate. Fifty microliters aliquots to media was analyzed for ICTP and CTX by ELISA kits (TSZ ELISA, Cat.#HU9655, Framingham, MA, and Serum CrossLaps ELISA, Immunodiagnotics Systems, Scottsdale, AZ, USA). The telopeptide results were expressed in ng ICTP or CTX/mg dry dentin/day and as ng/L. Loss of dry mass was expressed as % dry weight loss per 24 h. Statistical analyses were done using a one-way ANOVA and Tukey’s test at α = 0.05 for normally distributed data. The ICTP data were not normally distributed. They were analyzed using Kruskal–Wallis test, followed by Dunn’s multiple comparison with an α = 0.05.
2.2
Effects of QAMs on dry mass loss and ICTP/CTX production (experiments 2 and 3)
All demineralized beams were rinsed in water and dried to a constant weight to provide initial dry masses. Individual beams ( n = 10 separate beams/group) were dipped for 30 s in increasing concentrations of ATA in (1, 5, 10 or 15 wt% in experiment 2) or MDPB (0.1, 1.0, 2.5, 5.0 or 10 wt% in experiment 3) and then incubated separately in 500 μL of SBF (pH 7.4) in capped containers for 1 week at 37 °C with shaking (60 Hz). At the end of 1 week, the beams were removed from the media, rinsed in water to remove buffer salts, and dried to a constant weight in anhydrous calcium sulfate to obtain the post-incubation dry mass. Fifty microliters of media was analyzed ( n = 10) for ICTP/CTX telopeptides by specific ELISA kits mentioned above, and expressed in ng telopeptides/mg dry dentin mass/unit time. Statistical analyses were done using one-way ANOVA seeking significance differences of α = 0.05. Multiple comparisons were done with Tukey’s test at α = 0.05. When data were not normally distributed, they were analyzed using Kruskal–Wallis, followed by Dunn’s multiple comparison. Regression analysis was used to test the correlations between the ICTP or CTX release rates at different ATA or MDPB concentrations vs. loss of dry mass over 1 week incubation.
2
Materials and methods
2.1
Effects of rh cathepsins on mass loss (experiment 1)
Recombinant human cysteine cathepsins B, L and S (catalytic domain) were obtained from Athens Research & Technology (Athens, GA). Procathepsin K was obtained from EnzoLife Sciences (Plymouth Meeting, PA) and activated with 100 mM sodium acetate, pH 3.9, 10 mM DTT and 5 mM EDTA for 40 min at 25 °C. Cathepsins B, L and S were already active when purchased. Dentin beams in all groups (2 mm × 1 mm × 6 mm) were prepared from 18 to 21 year old mid-coronal dentin of human third molars using an Isomet saw (Buehler Ltd., Lake Bluff, IL). The beams were completely demineralized in 10 wt% phosphoric acid for 18 h at 25 °C with tumbling, and then rinsed free of acid in water. After obtaining constant dry masses, the individual beams ( n = 5 beams/group) were incubated in 10 μg of one of the specific cathepsins, dissolved in 0.5 mL of simulated artificial saliva (SBF) , containing KCl 13 mM, KSCN 2 mM, Na 2 SO 4 7.5 mM, HEPES 5 mM, NH 4 Cl 3 mM, CaCl 2 1.5 mM, NaHCO 3 7.5 mM, ZnCl 2 0.02, PO 4 3− 4, and NaN 3 0.02%, pH adjusted to pH 7.4. Another set of beams was incubated with 10 μg rh cathepsin K at pH 5.0 to demonstrate the maximum activity of the enzyme. After incubation in SBF (37 °C with shaking) for 24 h, the beams were removed, rinsed with water to remove buffer salts, and re-dried to a constant weight in sealed containers of dry calcium sulfate. Fifty microliters aliquots to media was analyzed for ICTP and CTX by ELISA kits (TSZ ELISA, Cat.#HU9655, Framingham, MA, and Serum CrossLaps ELISA, Immunodiagnotics Systems, Scottsdale, AZ, USA). The telopeptide results were expressed in ng ICTP or CTX/mg dry dentin/day and as ng/L. Loss of dry mass was expressed as % dry weight loss per 24 h. Statistical analyses were done using a one-way ANOVA and Tukey’s test at α = 0.05 for normally distributed data. The ICTP data were not normally distributed. They were analyzed using Kruskal–Wallis test, followed by Dunn’s multiple comparison with an α = 0.05.
2.2
Effects of QAMs on dry mass loss and ICTP/CTX production (experiments 2 and 3)
All demineralized beams were rinsed in water and dried to a constant weight to provide initial dry masses. Individual beams ( n = 10 separate beams/group) were dipped for 30 s in increasing concentrations of ATA in (1, 5, 10 or 15 wt% in experiment 2) or MDPB (0.1, 1.0, 2.5, 5.0 or 10 wt% in experiment 3) and then incubated separately in 500 μL of SBF (pH 7.4) in capped containers for 1 week at 37 °C with shaking (60 Hz). At the end of 1 week, the beams were removed from the media, rinsed in water to remove buffer salts, and dried to a constant weight in anhydrous calcium sulfate to obtain the post-incubation dry mass. Fifty microliters of media was analyzed ( n = 10) for ICTP/CTX telopeptides by specific ELISA kits mentioned above, and expressed in ng telopeptides/mg dry dentin mass/unit time. Statistical analyses were done using one-way ANOVA seeking significance differences of α = 0.05. Multiple comparisons were done with Tukey’s test at α = 0.05. When data were not normally distributed, they were analyzed using Kruskal–Wallis, followed by Dunn’s multiple comparison. Regression analysis was used to test the correlations between the ICTP or CTX release rates at different ATA or MDPB concentrations vs. loss of dry mass over 1 week incubation.
3
Results
3.1
Effects of exogenous cathepsins on dry mass loss and ICTP/CTX production (experiment 1)
Fig. 1 A shows the loss of dry mass (in %) of completely demineralized individual dentin beams incubated in SBF containing 10 μg of rh cathepsins B, K, L or S at pH 7.4 (and at pH 5 for cathepsin K only). Control beams incubated in SBF lost 6.11 ± 1.01% ( n = 4) per 24 h. Beams incubated in rh cathepsin B lost 5.37 ± 0.69% of dry mass/24 h which was not significantly different from controls. Beams incubated in rh cathepsin K lost 35.05 ± 5.63%/24 h ( p < 0.01) at pH 5.5 but only 8.1 ± 2.1% at pH 7.4 (cross-hatched region). Beams incubated in rh cathepsin L lost 5.28 ± 1.33% of dry mass/24 h, while beams incubated in rh cathepsin S lost 4.89 ± 0.88% of dry mass/24 h. Clearly, the only significant increase in mass loss of dry weight was due to cathepsin K.
When the media was analyzed for ICTP telopeptides, the control media released 20.27 ± 0.39 ng ICTP/mg dry dentin/24 h ( Fig. 1 B) (dotted line). The media from beams incubated with cathepsin B or S contained 24.7 ± 9.4 and 23.0 ± 0.21 ng ICTP/mg dry dentin/24 h, respectively, values that were not significantly different from controls. In contrast, the media from beams incubated with cathepsin K or L showed significantly less ( p < 0.01) ICTP; 1.4 ± 0.7 and 0.9 ± 0.2 ng ICTP/mg dry dentin/24 h, respectively.
When the incubation media was analyzed for CTX telopeptides ( Fig. 1 C), control media released 0.45 ± 0.12 ng CTX/mg dry dentin/24 h, while cathepsins B, L and S media contained 0.8 ± 0.3, 0.5 ± 0.1 and 0.6 ± 0.1 ng CTX/mg dry dentin/24 h. None of these values were significantly different. However, media from the beams incubated with cathepsin K contained 32.1 ± 9.8 ng CTX/mg dry dentin/24 h when incubated at pH 5. This value was significantly higher ( p < 0.01) compared to the others ( Fig. 1 C). When rh cathepsin K was incubated with demineralized beams at pH 7.4, the medium contained 6.1 ± 1.3 ng CTX/mg dry dentin/24 h indicating that cathepsin K activity at pH 7.4 was 6.1/32.1 × 100 = 19.0% as much as was seen at pH 5.0
3.2
Effects of increasing concentrations of ATA on dry mass loss or ICTP/CTX telopeptide production (experiment 2)
The results of experiment 2 are summarized in Table 1 . When completely demineralized dentin beams were incubated in control SBF for 1 week, their loss of dry mass was 12.8 ± 2.8% ( Fig. 2 A , dotted line). Experimental demineralized beams dipped in 1, 5, 10 or 15 wt% ATA made up in SBF (pH 7.4) for 30 s showed a loss of dry mass 11.9 ± 1.9, 10.0 ± 2.8, 8.2 ± 2.8 and 5.7 ± 1.8%. Only the 15 wt% ATA result was significantly different ( p < 0.05) from the rest. Was this loss of dry mass due to MMP or cathepsin activity or both?
