Inhibition of endogenous human dentin MMPs by Gluma

Abstract

Objective

The objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde and 35% HEMA in water) can inhibit the endogenous MMPs of dentin matrices in 60 s and to evaluate its effect on dentin matrix stiffness and dry mass weight.

Methods

Dentin beams of 2 mm × 1 mm × 6 mm were obtained from extracted human third molars coronal dentin. To measure the influence of Gluma treatment time on total MMP activity of dentin, beams were dipped in 37% phosphoric acid (PA) for 15 s and rinsed in water. The acid-etched beams were then dipped in Gluma for 5, 15, 30 or 60 s, rinsed in water and incubated into SensoLyte generic MMP substrate (AnaSpec, Inc.) for 60 min. Controls were dipped in water for 60 s. Additional beams of 1 mm × 1 mm × 6 mm were completely demineralized in 37% PA for 18 h, rinsed and used to evaluate changes on the dry weight and modulus of elasticity ( E ) after 60 s of Gluma treatment followed by incubation in simulated body fluid buffer for 0, 1 or 4 weeks. E was measured by 3-pt flexure.

Results

Gluma treatment inhibited total MMP activity of acid-etched dentin by 44, 50, 84, 86% after 5, 15, 30 or 60 s of exposure, respectively. All completely demineralized dentin beams lost stiffness after 1 and 4 weeks, with no significant differences between the control and Gluma-treated dentin. Gluma treatment for 60 s yielded significantly less dry mass loss than the control after 4 weeks.

Significance

The use of Gluma may contribute to the preservation of adhesive interfaces by its cross-linking and inhibitory properties of endogenous dentin MMPs.

Introduction

Although resin–enamel bond strengths are known to be stable over time , resin–dentin bonds are not very stable, falling 30–40% in 6 months and 60–70% in 1 year in vitro . The premature degradation of hybrid layers is known to be the consequence of a myriad of factors, including incomplete resin monomer infiltration of demineralized dentin and elution of unpolymerized monomers, both of which yield areas of poorly infiltrated collagen fibrils at the bottom of the hybrid layer that are prone to the attack by endogenous proteases. Armstrong et al. was the first to demonstrate that collagen fibrils in hybrid layers disintegrated over time as bond strengths fell over 5 years. That led Pashley et al. to demonstrate that dentin contained endogenous proteases that could cleave collagen. The presence of matrix metalloproteinase-2 (MMP-2) in dentin matrix was reported in 2000 by Martin-De Las Heras et al. . This was confirmed by Mazzoni et al. and expanded to include the presence of MMP-9. Sulkala et al. reported that dentin matrix also contained MMP-8, a true collagenase. In recent years, substantial work has been conducted to evaluate the role of host-derived proteases in the degradation of incompletely infiltrated collagen fibrils, as well as the mechanisms to regulate enzyme-mediated collagenolysis, such as the use of MMP inhibitors or cross-linking agents in dentin. Gendron et al. reported that chlorhexidine (CHX), a potent antimicrobial agent, was a nonspecific inhibitor of MMP-2, 8 and 9. Scaffa et al. reported that CHX also inhibited dentin cathepsins. Breschi et al. showed that a specific MMP-inhibitor (galardin) could prolong the durability of resin–dentin bonds. Many other dentin MMP inhibitors have also been described . Unfortunately, CHX and other proposed inhibitors do not co-polymerize with dental resins and can slowly leach out of hybrid layers within 1–2 years allowing proteases to resume collagen cleavage. This may explain recent observations by Sadek et al. who showed preservation of the bonds with the use of CHX after 9 months but not after 18 months.

Efforts are currently being directed toward creating hybrid layers with sustained anti-proteolytic potential. A proposed strategy to reduce collagen degradation within hybrid layers is to increase the extent of cross-linking of the demineralized matrix prior to adhesive application. One of the main advantages of using cross-linking agents is their ability to cross-link both active and allosteric enzyme sites of MMPs, as well as collagen molecules, preventing thus collagenases from binding to the collagen binding sites. Moreover, cross-linking agents can cross-link all MMPs, cysteine cathepsins and any other enzymes in the matrix simultaneously, precluding the need to treat dentin with separate enzyme inhibitors. By covalently cross-linking proteases to matrix components, they can be permanently inactivated. Several agents, including carbodiimide, glutaraldehyde, proanthocyanidin are currently being investigated for their cross-linking and anti-proteoytic properties .

Gluma is the brand name of a primer containing 5.0% glutaraldehyde, 35% hydroxyethylmethacrylate (HEMA) and 60% water, that was used in a three-step adhesive system that is no longer marketed . It was shown that this primer could prevent post-operative sensitivity if applied to crown preparations before the castings were cemented . Similar results were obtained by Dondi dall’Orologia and Malferrai and Davidson and Suzuki . However, the mechanism responsible for the desensitization of dentin remained elusive until the study by Schüpbach et al. . In that study, buccal cusps of human molars scheduled for extraction were flattened into dentin in vivo and then treated with EDTA, 5.0% glutaraldehyde or 35% HEMA or the combination of 5.0% glutaraldehyde and 35% HEMA in water. The teeth were then extracted and stained with fluorescein dye and the dentin examined by confocal laser scanning microscopy. The authors observed multiple intratubular septa in the lumens of the tubules of exposed dentin treated with 5.0% glutaraldehyde and HEMA, but not in dentinal tubules treated with either 5.0% glutarladehyde alone or 35% HEMA alone. They suggested that glutaraldehyde reacts with plasma proteins in dentin to precipitate them. Later work showed that HEMA reacts with the precipitate to form insoluble polymeric plugs in the tubules . In addition to its desensitizing properties, glutaraldehyde is a potent antimicrobial and cross-linking agent known to improve the resistance of uncross-linked or mildly cross-linked collagen matrices to enzymatic degradation by collagenases . By improving dentin’s mechanical properties, it is expected that glutaraldehyde can also contribute to improving the resistance to degradation of the bonds.

Recently, a simple model has been proposed to evaluate the rate of degradation of demineralized dentin matrices free of resin . Beams of coronal dentin are completely demineralized in 10% phosphoric acid, treated with a given MMP inhibitor or cross-linking agent, and then incubated in complete media for 30-day period. Changes in the modulus of elasticity, as determined by 3-point flexure, and dry mass loss before and after 30 days of incubation in complete media at 37 °C can be measured. If the MMPs are active, they will slowly solubilize collagen peptides resulting in the loss of dentin matrix stiffness and dry mass, thus providing indirect evidence of proteolytic activity.

The purpose of this work was to determine if Gluma can inactivate endogenous MMPs in acid-etched human dentin within 60 s of topical treatment, and to evaluate the effect of Gluma on dentin matrix stiffness and dry mass weight of demineralized dentin beams. The null hypotheses were that the 5.0% glutaraldehyde in Gluma cannot inactivate MMPs in acid-etched human dentin in 60 s, and that Gluma treatment of demineralized dentin beams has no effect on the matrix stiffness or loss of dry weight.

Materials and methods

Thirty extracted non-carious human third molars were obtained from 18 to 21 year-old patients with informed consent under a protocol approved by the Georgia Regents University and stored frozen until required. After removal of the roots, mid-coronal dentin was exposed by removing enamel and superficial dentin using an Isomet saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. One 1 mm-thick dentin disk was obtained from each tooth. Then, 50 dentin beams of 2 mm × 1 mm × 6 mm and 20 beams of 1 mm × 1 mm × 6 mm were sectioned from the dentin disks.

Dentin MMP activity

Because soluble MMPs extracted from dentin matrices or purchased rhMMPs are more susceptible to inhibitors or inactivators than matrix-bound MMPs , we elected to study dentin MMP activity while they were matrix-bound. To determine the total MMP activity, 50 beams of 2 mm × 1 mm × 6 mm were etched with 37% phosphoric acid (PA) for 15 s and then rinsed in deionized water (DW) for another 15 s. The etched-beams were treated either with DW for 60 s or Gluma (Gluma Desensitizer Liquid, Heraeus Kulzer GmgH, Lot# 010205, Hanau, Germany) for 5, 15, 30 or 60 s. Each group contained 10 specimens. Then the beams were immediately placed in 300 μl of a generic MMP substrate (SensoLyte Generic MMP colorimetric assay kit – catalog No. 72095, AnaSpec Inc., Lot# 131-029, Fremont, CA, USA) for 1 h at 25 °C in a 96-well plate. After 1 h, the beams were removed from the wells and the total MMP activity was spectrophotometrically determined by measuring the absorbance of each of the wells at 412 nm in a plate reader (Synergy HT microplate reader, BioTek Instruments, Winooski, VT, USA) against blanks. Ten individual values were averaged to obtain a mean value for each subgroup. All chemicals were purchased from Sigma/Aldrich Chemical Co. and used as received.

The generic MMP assay uses a proprietary thiopeptide to assay MMP-1, 2, 3, 7, 8, 9, 12, 13 and 14. Thus, the kit measured the total endogenous MMP activity of dentin. A standard curve of absorbance of the substrate vs. rhMMP-9 activity (ng) was constructed to permit expression of total MMP activity in MMP-9 equivalents. The rhMMP-9 was activated using trypsin at a final concentration of 10 μg/ml, pH 7.4 at 37 °C for 2 h. Then the trypsin was inactivated by addition of trypsin inhibitor at a final concentration of 100 μg/ml. As the ability of Gluma to inactivate MMPs was the same, regardless of whether we used the 412 nm absorbance values or converted it to MMP-9 equivalents, we elected to publish the absorbance values.

Elastic modulus and mass loss

Twenty beams of 1 mm × 1 mm × 6 mm were completely demineralized in 10 wt% liquid PA (pH 1.0) for 18 h at 25 °C under constant stirring and rinsed with DW for 2 h at 4 °C. Before demineralization, the initial modulus of elasticity of each mineralized beam was determined, by three-point flexure, to be between 16 and 18,000 MPa (16–18 GPa). After complete demineralization, the modulus of elasticity fell to 2.0–2.8 MPa indicating complete demineralization. An aluminum testing jig with a 2.5 mm support span was fixed to the bottom of a Petri glass dish. Specimens were tested under compression, while immersed in distilled water, by means of a testing machine (Vitrodyne V1000, Liveco Inc., Burlington, VT, USA) with a 100 g load cell, at a cross-head speed of 1 mm/min. Load–displacement curves were converted to stress–strain curves, and the apparent modulus of elasticity was calculated at 10% strain. The beams were then allocated into two groups ( n = 10) with statistically similar mean initial elastic modulus. The following formula was used to calculate the elastic modulus:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='E=mL34bd3′>E=mL34bd3E=mL34bd3
E = m L 3 4 b d 3

where m = slope (N/mm); L = support span (mm); d = thickness of beam (mm); b = width of beam (mm). Because specimen displacement was estimated from cross-head displacement, and the specimens thickness was not one-sixteenth of the length (ASTM D-790-03 ) , the calculated elastic moduli were only approximate but very reproducible.

To determine initial dry mass, the beams were dried in a desiccator containing calcium sulfate for 48 h and weighed to a constant dry weight in a microanalytical balance (XP6 Microbalance, Mettler Toledo, Columbus, OH). That is, the specimens were weighed four times, initial wet weight at time zero, dry weight at 24 h, 36 h and 48 h. Consistency of the last two weights indicates a constant dry mass. The beams were rehydrated in DW for 1 h and treated with either Gluma Desensitizer Liquid or DW (Control) for 60 s and stored in 500 μl of simulated body fluid (SBF, composition in mM/L: KCl, 13; KSCN, 2: Na 2 SO 4 ·10H 2 O, 2.4; HEPES, 5; CaCl 2 ·2H 2 O, 1.5; NaHCO 3 , 7.5; ZnCl 2 , 0.02, 0.02% sodium azide) at 37 °C for up to 4 weeks. After weeks 1 and 4, the same procedures described above were repeated to re-determine the elastic modulus and dry mass of the beams.

Statistical analysis

A one-way ANOVA and post hoc Student–Newman Keuls test were used to evaluate the effect of Gluma application time on the percentage inhibition of total MMP activity since the data was normally distributed. Kruskal–Wallis test followed by Dunn’s multiple comparisons test were used for evaluation of the effect of Gluma application time on the absorbance values since these values were not normally distributed. Two-way repeated measures ANOVA and a pairwise multiple comparisons Holm–Sidak test were used to determine the effect of Gluma treatment and application time on the modulus of elasticity and dry mass loss. A significance level of p < 0.05 was used for all tests. All statistical analysis was performed with SigmaStat version v.3.11 (San Jose, CA).

Materials and methods

Thirty extracted non-carious human third molars were obtained from 18 to 21 year-old patients with informed consent under a protocol approved by the Georgia Regents University and stored frozen until required. After removal of the roots, mid-coronal dentin was exposed by removing enamel and superficial dentin using an Isomet saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. One 1 mm-thick dentin disk was obtained from each tooth. Then, 50 dentin beams of 2 mm × 1 mm × 6 mm and 20 beams of 1 mm × 1 mm × 6 mm were sectioned from the dentin disks.

Dentin MMP activity

Because soluble MMPs extracted from dentin matrices or purchased rhMMPs are more susceptible to inhibitors or inactivators than matrix-bound MMPs , we elected to study dentin MMP activity while they were matrix-bound. To determine the total MMP activity, 50 beams of 2 mm × 1 mm × 6 mm were etched with 37% phosphoric acid (PA) for 15 s and then rinsed in deionized water (DW) for another 15 s. The etched-beams were treated either with DW for 60 s or Gluma (Gluma Desensitizer Liquid, Heraeus Kulzer GmgH, Lot# 010205, Hanau, Germany) for 5, 15, 30 or 60 s. Each group contained 10 specimens. Then the beams were immediately placed in 300 μl of a generic MMP substrate (SensoLyte Generic MMP colorimetric assay kit – catalog No. 72095, AnaSpec Inc., Lot# 131-029, Fremont, CA, USA) for 1 h at 25 °C in a 96-well plate. After 1 h, the beams were removed from the wells and the total MMP activity was spectrophotometrically determined by measuring the absorbance of each of the wells at 412 nm in a plate reader (Synergy HT microplate reader, BioTek Instruments, Winooski, VT, USA) against blanks. Ten individual values were averaged to obtain a mean value for each subgroup. All chemicals were purchased from Sigma/Aldrich Chemical Co. and used as received.

The generic MMP assay uses a proprietary thiopeptide to assay MMP-1, 2, 3, 7, 8, 9, 12, 13 and 14. Thus, the kit measured the total endogenous MMP activity of dentin. A standard curve of absorbance of the substrate vs. rhMMP-9 activity (ng) was constructed to permit expression of total MMP activity in MMP-9 equivalents. The rhMMP-9 was activated using trypsin at a final concentration of 10 μg/ml, pH 7.4 at 37 °C for 2 h. Then the trypsin was inactivated by addition of trypsin inhibitor at a final concentration of 100 μg/ml. As the ability of Gluma to inactivate MMPs was the same, regardless of whether we used the 412 nm absorbance values or converted it to MMP-9 equivalents, we elected to publish the absorbance values.

Elastic modulus and mass loss

Twenty beams of 1 mm × 1 mm × 6 mm were completely demineralized in 10 wt% liquid PA (pH 1.0) for 18 h at 25 °C under constant stirring and rinsed with DW for 2 h at 4 °C. Before demineralization, the initial modulus of elasticity of each mineralized beam was determined, by three-point flexure, to be between 16 and 18,000 MPa (16–18 GPa). After complete demineralization, the modulus of elasticity fell to 2.0–2.8 MPa indicating complete demineralization. An aluminum testing jig with a 2.5 mm support span was fixed to the bottom of a Petri glass dish. Specimens were tested under compression, while immersed in distilled water, by means of a testing machine (Vitrodyne V1000, Liveco Inc., Burlington, VT, USA) with a 100 g load cell, at a cross-head speed of 1 mm/min. Load–displacement curves were converted to stress–strain curves, and the apparent modulus of elasticity was calculated at 10% strain. The beams were then allocated into two groups ( n = 10) with statistically similar mean initial elastic modulus. The following formula was used to calculate the elastic modulus:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='E=mL34bd3′>E=mL34bd3E=mL34bd3
E = m L 3 4 b d 3
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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Inhibition of endogenous human dentin MMPs by Gluma
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