Inactivation of Matrix-bound MMPs by Cross-linking Agents in Acid Etched Dentin

Abstract

Objectives

Published TEM analysis of in vivo resin-dentin bonds shows that in 44 months almost 70% of collagen fibrils from the hybrid layer disappear. Matrix metalloproteinases (MMPs) play an important role in that process and are thought to be the main factor responsible for the solubitization of dentin collagen. Therefore, this study aimed to evaluate the inactivation of matrix-bound MMPs by carbodiimide (EDC) or proanthocyanidin (PA) both cross-linking agents, or the MMP-inhibitor, chlorhexidine (CHX), on acid-etched dentin using a simplified MMP assay method.

Methods

Dentin beams (1×1×6mm) were obtained from mid-coronal dentin of sound third molars and randomly divided into 6 groups (G) according to the dentin treatment: G1: Deionized water (control), G2: 0.1M EDC, G3: 0.5M EDC, G4: 0.5M EDC+35% HEMA, G5: 5% Proanthocyanidin (PA) and G6: 2% CHX. The beams were etched for 15s with 37% phosphoric acid, rinsed and then immersed for 60s in one of the treatment solutions. The total MMP activity of dentin was analyzed for 1 h by colorimetric assay (Sensolyte). Data were submitted to Wilcoxon non-parametric test and Mann-Whitney tests (p>0.05).

Results

All experimental cross-linking solutions significantly reduced MMP activity compared to control, except 0.1M EDC (53.6% ±16.1). No difference was observed between cross-linking agents and 2% CHX 0.5M EDC + 35% HEMA (92.3% ±8.0) was similar to 0.5M EDC (89.1% ±6.4), 5% PA (100.8% ±10.9) and 2% CHX (83.4% ±10.9).

Conclusion

Dentin treatment with cross-linking agents is effective to significantly reduce MMP activity. Mixing 0.5M EDC and 35% HEMA did not influence EDC inhibitor potential.

INTRODUCTION

Since the introduction of the total-etching concept by Fusayama in 1980,¹ the effects of acid-etching of dentin has been subject to many studies. Etching dentin with 32–37% phosphoric acid removes the mineral content of the top 10 um of dentin and exposes the collagen fibrils, thereby creating space for monomer infiltration to achieve micromechanical retention of adhesive resins.² Although acid etching of dentin provides satisfactory initial bond strength, those bond strengths fall over time, raising concerns about the long-term stability of adhesive restorations.³

Resin/dentin bond degradation is a complex process that is not completely understood, involving the hydrolysis of both the resin and the collagen component of hybrid layers. Demineralized dentin contains bound matrix metalloproteinases-2, -3, -8, -9 and -20 (MMPs) and cathepsins^4,5 in their active forms. These enzymes are exposed and activated by acid-etching and can slowly degrade collagen fibrils^6–9 within the hybrid layer, resulting in a significant bond strength loss of 36% to 70% between 12 to 14 months.^10,11

In order to reduce the activity of these proteases and preserve the long term integrity of adhesive interfaces, chlorhexidine (CHX) has been used as a nonspecific inhibitor of MMPs. ^{6–9,11–13} CHX is also effective inhibitor of cysteine cathepsins.¹⁴ However this substance is water-soluble and may undergo leaching from the hybrid layer, impairing its long-term anti-MMP effectiveness.¹³

A new alternative to the inhibition of proteases by inhibitors, is the treatment of demineralized dentin with cross-linking agents that can inactivate the catalytic site of these enzymes.¹⁵ The cross-linker 1-(3-dimethylaminopropyl) carbodiimide (EDC) is capable of forming covalent peptide bonds between proteins by activating the free carboxyl group of glutamic and aspartic acids present in protein molecules.^16–17 This results in the formation of a O-acylisourea intermediate that reacts with the epsilon amino group of lysine or hydroxylysine in an adjacent polypeptide chain to form a stable, covalent amide bond. The only byproduct of the reaction is urea.^18–19 Furthermore EDC shows no transdentinal cytotoxicity on odontoblast-like cells (Scheffel et al. unpublished data) and is able to increase the mechanical properties of the collagen matrix.²⁰

Other cross-linking agents, such as the proanthocyanidins (PA), are polyphenolic natural products composed of flavan-3-ol subunits linked mainly through C4– C8 (or –C6) bonds.²¹ This substance is widely present in fruits, vegetables, nuts, seeds, flowers and barks, shows numerous biological activities, such as antioxidant capacity,²² antimicrobial effects,²³ anti-inflammatory properties,²⁴ positive effects on cardiovascular diseases²⁵ and anti-allergic activity.²⁶

Thus, the purpose of this study was to evaluate the inactivation of matrix-bound MMPs by topical application of cross-linking agents on acid etched dentin. The null hypothesis was that cross-linker-treated and untreated dentin do not differ regarding MMP activity.

MATERIALS AND METHODS

Twenty extracted human third molars were obtained from 18–21 year-old patients with informed consent under a protocol approved by the Georgia Health Sciences University. The teeth were stored frozen until required. After thawing, the enamel and superficial dentin were removed using an Isomet saw (Buehler Ltd., Lake Bluff, IL) under water cooling. One 1 mm-thick dentin disks were produced from the mid-coronal dentin of each tooth. Then, sixty dentin beams (1×1×6 mm) were sectioned from the dentin disks. The beams were etched by dipping them into 37% phosphoric acid (pH -0.5) for 15 s and copiously rinsed with deionized water for 15 s. The beams were randomly divided into 6 groups (n=10) according to the dentin treatment. G1: Deionized water (positive control) (pH 6.73), G2: 0.1M 1-[3-dimethylaminopropyl) carbodiimide (EDC) (pH 6.07), G3: 0.5M EDC (pH 6.04), G4: 35 vol% hydroxyethylmethacrylate (HEMA) in water+0.5M EDC (pH 6.34), G5: 5% Proanthocyanidin (PA) (pH 4.85) and G6: 2% CHX digluconate (negative control) in water (pH 6.43). All beams were dipped in the treatment solutions for 60 s and rinsed with distilled water for 10 s, except for 2% CHX where the beams were only blot dried. After the treatment, each beam was placed in 200 uL of a generic MMP substrate (Sensolyte Generic MMP colorimetric assay kit - catalog No. 72095, AnaSpec Inc. Fremont, CA) for 60 min at 25°C in a 96-well plate. At the end of 60 min, the total MMP activity was determined by measuring the absorbance of the wells at 412 nm in a plate reader (Synergy HT microplate reader, BioTek Instruments, VT) against appropriate blanks. All chemicals were purchased from Sigma/Aldrich Chemical Co. 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.

Statistical Analysis

For determination of MMP activity, the absorbance data set was submitted to Wilcoxon non-parametric test and Mann-Whitney test at 5% level of significance. The percentage of MMP activity inhibition was calculated based on the water control group MMP activity.

RESULTS

When mineralized dentin beams were dipped in 37 wt% phosphoric acid for 15 s and then rinsed with water, the top 7–8 μm of the beams were completely demineralized (Fig. 1). When completely demineralized dentin beams were dipped in water (control) and then dropped into the generic MMP substrate, the absorbance at 412 nm gradually increased to 0.24±0.02 over 60 min. That value was considered as 100% of the total MMP activity in the etched dentin and it was used to calculate the percentage of MMP activity inhibition of the investigated cross-linking agents and CHX. All cross-linking agents, except EDC at 0.1M, significantly reduced MMP activity in acid etched dentin after 60 s of topical treatment (Table 1). Although 0.1M EDC inhibited 53.6% of total MMP activity, its effect was not significantly different when compared to the control. The percentage of MMP inhibition for the other EDC concentrations and CHX ranged from 83.4% for 2 wt% CHX to 100% for 5% PA (Table 1). There was no statistical difference in MMP activity when 0.5M EDC, 0.5M EDC+35% HEMA, 5% PA and 2% CHX were compared (Table 1). When 0.5M EDC was mixed with 35% HEMA to simulate the composition of an adhesive primer, the HEMA did not interfere with that cross-linker in inactivating the total MMP activity of acid-etched dentin.

Figure 1.

Confocal Laser Scanning Microscope (CLSM) images of the etched layer shown dyed green on the surface of a dentin beam. Dentin beams, 1.0×1.0×6.0±0.1mm, were etched in 37% phosphoric acid for 15s and than rinsed with deionized water for 60s. The beams were than cut in 0.3×1.0×1.6±0.1mm slabs and labelled for 5 h respectively with 1 %w/v Fluorescein isothiocyanate (FITC) in anhydrous dimethyl sulfoxide (DMSO) and 1 %w/v Xylenol Orange (XO) in water. The two fluorochromes selectively label collagen (FITC) and the mineralized matrix (XO), respectively. Prior to CLSM observation, the slabs were rapidly blotted with absorbent paper to remove the excess of fluorochrome, mounted on glass slides and promptly examined. Samples were scanned in two channels fluorescence mode with both 488nm excitation − 525nm emission (green channel) and 546nm excitation − 580nm emission (red channel), respectively, for FITC and XO labeling. (A) 10x projection of 53 images (final Z-stack thickness: 346μm), the sample was intentionally tilted to highlight the peripheral distribution of demineralized collagen. (B) 100x image (slice thickness: 6.54μm) of the border between demineralized surface collagen fibrils (etched layer) and underlying mineralized dentin matrix.

Table 1.

Absorbance and percent inactivation/inhibition of total matrix-bound MMP activity in dentin

	Acid-etched dentin	0.1M EDC	0.5M EDC	0.5M EDC+35% HEMA	5% Proanthocyanidin (PA)	2% Clorehexidine (CHX)
Absorbance	0.24 (±0.06) c	0.11 (±0.04) bc	0.03 (±0.02) a	0.02 (±0.02) a	0.00 (±0.03) a	0.04 (±0.02) ab
Percent inhibition (%)	0.0 (±27.4)	53.6 (±16.1)	89.1 (±6.4)	92.3 (±8.0)	100.8 (±11.2)	83.4 (±10.9)

Values are mean±SD (n=10) absorbance and % inhibition of total MMP activity of dentin measured by SensoLyte substrate (AnaSpec Inc. Fremont, CA). EDC=1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; HEMA=2-Hydroxyethyl methacrylate; GS=grape seed extract containing Proanthocyanidins; CHX=2wt% clorehexidine digluconate. Groups identified by different lower case letters are significantly different (p<0.05).

DISCUSSION

The conventional method to analyze the total bound MMP activity using Sensolyte Generic MMP colorimetric assay kit includes the complete demineralization of dentin beams for 18 h with 10% phosphoric acid.²⁷ The current study used a simplified MMP assay method in which the dentin was acid etched for 15 s with 37% phosphoric acid. That avoids the complete dentin demineralization and reproduces more closely the surface demineralization of dentin that is done during etch-and-rinse bonding procedures. The complete demineralization of the dentin beam creates a much deeper collagen area (1×1×6mm) to be infiltrated by the cross-linking solutions and adhesive resins. Under such extreme conditions, it is as if the hybrid layer is 500 μm deep from all exposed surfaces. Clinically, acid etching of dentin by 37 wt% phosphoric acid for 15 s only demineralizes dentin to a depth of 8–10 μm (Figure 1). Such relatively thin zones of demineralized dentin are easily saturated by test solutions within seconds. Nevertheless, this technique does not reproduce all in vivo conditions such as the presence of pulpal pressure and the outflow of dentinal fluid.

The hybrid layer is composed of 30 vol% collagen²⁸ (primarily type I), while the other 70% corresponds to resin and residual solvent. The collagen fibril network acts as an anchorage to resin, enabling the retention of adhesive restorations. However, TEM analyses revealed that almost 70% of collagen from the adhesive interface disappears after 44 months water storage.²⁹ Proteases such as metalloproteinases (MMPs) and cysteine cathepsins are thought to be responsible for collagen fibrils enzymatic degradation via hydrolysis.³⁰

Exogenous MMP inhibitors have been tested in order to reduce protease activity and prolong the durability of resin-dentin bonds. Chlorhexidine was the first MMP inhibitor proposed for such a purpose during bonding to dentin.³¹ It has been largely studied as a non-specific MMP¹² and cathepsin inhibitor.¹⁴ CHX adsorbs on dentin and decreases hybrid layer degradation in vitro^{7, 32–34} and in vivo.^6,8,11,13 However this inhibitor is soluble in water and can slowly leach from the adhesive interface over time,¹³ since no chemical bond is established between the CHX molecule and the collagen fibril.

One of the mechanisms proposed to explain how MMPs degrade collagen is that these proteases unwind collagen molecules when they bind to them. By doing that it allows the enzyme’s active site sufficient space to attack the specific glycine-isoleucine peptide bond in peptide chains.^35–37 Cross-linking agents stiffens collagen polypeptides so that they can not unwind, and they can also inactivate the catalytic site of proteases³⁸ by creating a new peptide bond across adjacent peptides. Hence, it is reasonable to expect that MMP inactivation by cross-linking agents should last much longer than the inhibition of proteases by matrix-bound CHX. Carbodiimides (EDC) and Proanthocyanidins (PA) were first used to increase the modulus of elasticity of collagen and make it more difficult to MMPs unwind the collagen triple-helix structure. However, EDC and PA are still not capable of increasing the stiffness of collagen in clinically relevant periods of times, such as 30 s and 60 s (Scheffel et al., unpublished data).

Despite the long application times that cross-linking agents require to increase collagen stiffness,²⁰ they are effective against MMPs in 60 s. The results of this study partially rejected the tested null hypothesis. All investigated solutions except 0.1M EDC significantly decreased MMP activity in acid etched dentin within 60 s. Both 0.5M EDC and 5% PA were able to inactivate more than 89% of the total active MMPs. EDC activates the free carboxylic acid groups of glutamic and aspartic acids without introducing additional methylene groups. MMPs-2 (EC 3.4.24.24), -8 (EC 3.4.24.34), -9 (EC 3.4.24.35) and -20 (EC 3.4.24) have glutamic acid in their active sites in position 404, 218, 402 and 227, respectively, allowing EDC to react to those sites. Additionally, the concentrations of EDC tested in this study did not produce any evidence of transdentinal cytotoxic effect on odontoblast-like cells in separate experiments (Scheffel et al., unpublished data), making EDC safe for in vivo application.

Proanthocyanidins (PA) is a natural plant cross-linking agent. The mechanism of cross-linking is not completely understood. There are four different theories to explain how PA interacts with proteins. They include covalent,³⁹ ionic,⁴⁰ hydrogen bonding,⁴¹ and hydrophobic interactions.⁴² This substance has been reported to increase the stiffness of demineralized dentin,⁴³ and to inhibit the progression of artificial root caries.^44,45 Additionally, scanning electron microscopy of demineralized dentin collagen treated with 15% PA for periods shorter than 120 s showed a homogeneous and regular collagen fibril arrangement, regardless of the surface moisture condition.³⁸ That result suggests that besides acting as MMP inhibitor cross-linking agents, it can stiffen demineralized dentin sufficiently to minimize the risk of collagen network collapse, resulted from air-drying. However, PA solution has a dark color, which stains the dentin despite rinsing. That could be a drawback for the clinical use of this cross-linker. Its rapid, complete inactivation of matrix-bound MMPs in dentin suggests that more research should be done to try to isolate an uncolored fraction of the PA.

When 0.5M EDC was solubilized in 35 vol% HEMA there was no reduction in its ability to inactivate all of the MMPs in dentin. That is, it was as effective as 0.5M EDC alone, 5% PA or 2% CHX. Since HEMA is an important component of adhesives, it may be possible to mix EDC with HEMA and other primer components in etch-and-rinse adhesive systems to inactivate MMPs during bonding. However it is not known whether EDC influences adhesive polymerization. Further studies are still needed to demonstrate the effects of short-time application of cross-linking agents over time in vitro and in vivo.

CONCLUSIONS

Dentin treatment with cross-linking agents is effective to significantly reduce MMP activity; 0.5M EDC and 5% PA showed the best results. Mixing 0.5M EDC and 35% HEMA did not influence EDC crosslinking of MMPs, indicating that EDC could be added to primers in adhesive systems.

Clinical significance statement.

Cross-linking agents used in clinically applicable periods of time are capable of inactivating matrix-bound MMP in demineralized dentin. Such treatment may render the hybrid layer less prone to degradation over time and produce long lasting resin-dentin bonds.