Effect of conditioning solutions containing ferric chloride on dentin bond strength and collagen degradation

Abstract

Objective

To investigate the effects of conditioning solutions containing ferric chloride (FeCl 3 ) on resin–dentin bond strength; on protection of dentin collagen against enzymatic degradation and on cathepsin-K (CT-K) activity.

Methods

Conditioning solutions were prepared combining citric acid (CA) and anhydrous ferric chloride (FeCl 3 ) in different concentrations. The solutions were applied to etch flat dentin surfaces followed by bonding with adhesive resin. Phosphoric acid (PA) gel etchant was used as control. The microtensile bond strength (μTBS) was tested after 24 h of storage in water and after 9 months of storage in phosphate buffer saline. Dentin slabs were demineralized in 0.5 M EDTA, pre-treated or not with FeCl 3 and incubated with CT-K. The collagenase activity on dentin collagen matrix was examined and characterized by SEM. Additional demineralized dentin slabs were treated with the conditioning solutions, and the amount of Fe bound to collagen was determined by EDX. The activity of CT-K in the presence of FeCl 3 was monitored fluorimetrically. Data were analyzed by ANOVA followed by post-hoc tests as required ( α = 5%).

Results

Slightly higher bond strengths were obtained when dentin was conditioned with 5% CA/0.6% FeCl 3 and 5% CA–1.8%FeCl 3 regardless of storage time. Bond strengths reduced significantly for all tested conditioners after 9 months of storage. Treating dentin with 1.8% FeCl 3 was effective to preserve the structure of collagen against CT-K. EDX analysis revealed binding of Fe-ions to dentin collagen after 15 s immersion of demineralized dentin slabs into FeCl 3 solutions. FeCl 3 at concentration of 0.08% was able to suppress CT-K activity.

Significance

This study shows that FeCl 3 binds to collagen and offers protection against Cat-K degradation. Mixed solutions of CA and FeCl 3 may be used as alternative to PA to etch dentin in resin–dentin bonding with the benefits of preventing collagen degradation.

Introduction

In 1982, Nakabayashi et al. , proposed the used of 10-3 solution, containing 10% citric acid (CA) and 3% ferric chloride hydrate (FeCl 3 ·6H 2 O) to prepare dentin for infiltrating resin monomers. According to reports, FeCl 3 was useful to preserve the exposed collagen structure against denaturation, which was a concern associated with the use of acids on collagen at that time . Conversely, other authors suggested that FeCl 3 was important as an interfacial initiator for improved curing of the 4-META/MMA-TBB adhesive . Despite the claims above, the precise definition of the benefits, or the role of FeCl 3 in resin–dentin bonding has never been clarified. Additionally, no investigation has followed to investigate any possible interaction of FeCl 3 with enzymatic activities in resin–dentin bonding. That was probably because evidences of the role of host-derived enzymes in resin–dentin bonding were not available at that time; and also because current use of such dentin conditioner (i.e., 10-3 solution) is limited to few products available, such as the resin cement Super Bond C & B (Sun Medical, Japan), thus catching little attention from researchers.

The degradation of collagen fibrils and hydrophilic components of adhesive resins are considered the determining factors of destruction of the hybrid layer and consequent reduction of bond strength to dentin over time . The mineral removal by acid etching exposes collagen fibrils to degradation by two classes of proteolytic enzymes: metalloproteinases (MMPs) and cathepsins (CTs) . Several endogenous enzymes are present in dentin, and Cathepsin-K (CT-K) is one of them , CT-K deserves special attention for its ability to degrade collagen quickly . The collagenolytic activity of CT-K is directed to the cleavage of the non-helical telopeptides of collagen and cleavages within the helical region, being the activity higher at an acid pH . Thus, it is likely that CT-K could start the collagen degradation process once exposed by the etching procedure in a typical dentin bonding procedure. With time, both families of enzymes (MMPs and CTs) work together to degrade exposed collagen . In concert, they exert a significant role in the degradation of exposed collagen in incompletely infiltrated hybrid layers , and also participate in the progression of dentinal caries and erosion .

Despite of the successful performance in the 90s, no further attention has been given to 10-3 solution by researchers or manufacturers. While being almost forgotten in Dentistry, the Fe-ion gained recent prominence in archeological science, in studies investigating the preservation of type I collagen and other proteins in fossils . These studies suggest that the intriguing preservation of fossil soft tissues were a result of the action of ferric ions (likely from decaying red blood cells) interacting with collagen and making it resistant to the challenge of time. The researchers found that cross-linked type I collagen was present in fossil tissues of Tyrannosaurus rex in close association with iron nanocrystals .

It is known that various naturals and synthetic chemical products have the ability to increase the number of intramolecular cross-links in collagen , and increased cross-links in dentin collagen improve the mechanical properties and allows for potential protection against degradation . Assuming the preservation of the collagen fibrils in the hybrid layer is essential for the preservation of the resin–dentin interface, the archeological finding renewed interest in the study of agents based on iron and how it may play a role in preserving resin–dentin bonding against degradation. It has been observed, for instance, that iron showed inhibitory effect against MMP-2 and -9 activity . We speculate that iron in conditioning solutions can interact with dentin collagen and offers protection against enzymatic degradation. We also speculate that Fe may also present inhibitory effect on CT-K. In concert, these mechanisms could inhibit collagenase activity and preserve collagen structure, thus preserving the resin–dentin bonds against degradation.

Therefore, the aim of this study was to evaluate the effects of conditioning solutions containing CA and/or FeCl 3 in different concentrations on long-term dentin bond strength and dentin collagen degradation. This study investigated the hypotheses that; (1) the use of conditioning solutions with FeCl 3 will result in stable resin–dentin bond strength over time; (2) that FeCl 3 can inhibit CT-K activity; and (3) that dentin collagen exposed to FeCl 3 will bind Fe-ions and be more resistant to degradation against the collagenase CT-K.

Materials and methods

Teeth

A total of 80 human caries-free third molars were used in this study. The study was approved by the local Ethics Board (approval # H15-02264). Seventy eight teeth were used for microtensile bond strength (μTBS) test and 2 teeth were used for collagen degradation and Fe-ion binding examination.

μTBS test

Teeth preparation

After extraction, the teeth were frozen in tap water and kept at −4 °C for no longer than 3 months. After thawing to room temperature, they were scrapped to remove organic debris and pumiced with rubber cup (KG Sorensen, Barueri, SP, Brazil). The roots were sectioned 1 mm below the cementum/enamel junction and a parallel cut made to expose flat mid-coronal dentin surface using a water-cooled diamond saw (EXTEC Corporation, Enfield, CT, USA) coupled to a cutting machine (Isomet 1000, Buehler Ltd., Lake Bluff, Il, USA). The surrounding enamel was removed by grinding on wet 180-grit SiC paper and the teeth were kept in tap water until used.

Preparation of conditioning solutions

The CA and/or FeCl 3 powder (Sigma/Aldrich Corporation, St. Louis, Missouri, USA) were dissolved in w/w% ratios in distilled water according to different concentration ratios ( Table 1 ). The solutions were prepared from anhydrous FeCl 3 (Sigma/Aldrich Corporation, St. Louis, Missouri, USA). The concentration ratios of CA and FeCl 3 were chosen to be variants of similar solutions previously described in the literature as 10-3 and 1-1 . For instance, the 1.8% FeCl 3 used in this study equals in Fe amount to that of 3% hydrated FeCl 3 (FeCl 3 ·6H 2 O) used in previous studies (10-3 solution) . The conditioning solutions were labeled and kept refrigerated in sealed vials until used. A commercially available 35% phosphoric acid gel (Select HV Etch, Bisco Inc., Schaumburg, IL, USA) was also used in the study. All conditioning solutions had their pH measured with a pH-meter (model mPA-210p, MS Tecnopon Equipamentos Especiais LTDA, SP, Brazil) ( Table 1 ).

Table 1
Description of the experimental groups for the microtensile bond strength testing, and composition of conditioning solutions used in the study.
Experimental groups Conditioning solutions ratio w/w % Etching time pH
Citric acid Ferric chloride
10–1.8 10 1.8 15 0.57
10–0.6 10 0.6 15 0.84
10–0.0 10 0 15 1.42
5–1.8 5 1.8 15 0.65
5–0.6 5 0.6 15 0.94
5–0 5 0 15 1.87
1–1.8 1 1.8 15 0.85
1–0.6 1 0.6 15 0.98
1–0 1 0 15 1.96
0–1.8 0 1.8 15 1.58
0–0.6 0 0.6 15 1.80
PA5 35% phosphoric acid 5 0.1–0.4
Control PA15 35% phosphoric acid 15 0.1–0.4

Bonding procedures

The dentin surface was polished with 320-grit SiC paper under water-cooling for 10 s to standardize the smear layer. The surface was rinsed with water and air-dried to remove excess water without desiccating the surface. The crown segments were randomly allocated to 13 groups ( Table 1 ).

Twenty μL of each conditioning solution were dispensed to the dentin surface followed by spreading with disposable applicators (Vigodent, Rio de Janeiro, RJ, Brazil) for 15 s. The surface was then rinsed for 15 s with distilled water and blot/dried with tissue paper (Kimwipes, Kimberly-Clark Global Sales, Inc, GA, USA), keeping the surface visibly moist. This procedure was reproduced for all groups, except for the PA5 and PA15 groups, in which the phosphoric acid was applied for 5 and 15 s, respectively (see Table 1 ).

The Adper Scotchbond Multi-Purpose adhesive system (3M ESPE, St. Paul, MN, USA) was applied according to the manufacturer’s recommendations to all groups, and light/cured for 20 s at 1200 mW/cm 2 using Bluephase G2 (Ivoclar Vivadent, Schaan, Liechtenstein). After bonding, 4 mm composite (Aelite™ All/Purpose Body, Bisco Inc., Schaumburg, IL, USA) block was incrementally built-up. Each 2 mm increment was light-cured for 40 s using Bluephase G2.

Specimens preparation for μTBS test

The bonded teeth were stored in water at 37 °C for 24 h and sectioned perpendicular to the adhesive–dentin interface (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) with double/sided diamond saw (EXTEC Corporation, Enfield, CT, USA) under water cooling to obtain beams of approximately 1 mm 2 of cross-sectional area. Half of the beams obtained from each tooth were randomly selected and tested immediately after sectioning, while the remaining were kept in a sealed vial containing Phosphate Buffered Saline (PBS, Sigma Aldrich, St. Louis, MO, USA) at 37 °C for nine months. The PBS was renewed monthly and preservatives and/or antimicrobial agents were not added to the PBS in this study.

Bond strength test

Bonded beams were mounted on a microtensile testing jig (Odeme Dental Research, Luzerna, SC, Brazil) with cyanoacrylate glue (Super Glue Gel Control, Loctite, Mississauga, ON, Canada) and tested until failure at 1.0 mm/min. The load (N) at failure was divided by the cross-sectional area of the beam (mm 2 ) measured with a digital caliper to the nearest 0.01 mm (Fisher Scientific, Chicago, IL, USA) to calculate the microtensile bond strength that was expressed in MPa. Beams were carefully removed from the jig, and failure modes were evaluated under 40× using light microscope (Olympus, Tokyo, Japan).

Failure mode analysis

Fractures were classified as cohesive, adhesive or mixed. When it occurred exclusively in either dentin or composite, it was classified as cohesive in dentin (CD) or cohesive in composite resin (CC); when occurred at dentin/resin bonded interface as adhesive (AD), and mixed (M) when two modes of failures occurred simultaneously. To observe the ultrastructure of the failing sites, three selected beams of each failure mode were examined at 80× and 3000× under Scanning Electron Microscopy (SEM) (JSM/5600LV, JEOL, Tokyo, Japan) operating at 15 kV. For that, the beams were positioned side-by-side on metallic stub with carbon tape, dried overnight in a desiccator and sputter-coated with gold (MED 010 Baltec, Balzers, Leichtenstein).

Dentin slabs preparation for EDX and SEM analysis of collagen

The roots of two teeth were transversally cut 1 mm below the cementum-enamel junction and then coronally to expose mid-coronal dentin. The resultant tooth segment was fixed in an acrylic base with wax and sectioned perpendicularly in both X and Y directions to obtain rectangular dentin slabs (2 mm × 3 mm × 0.5 mm) using the same cutting machine under water cooling . The dentin slabs were then demineralized in 0.5 M ethylenediaminetetraacetic acid (EDTA) for 3 days followed by rinsing with distilled water for 2 h. A total of 42 demineralized dentin slabs were obtained and randomly divided into two groups, one for energy-dispersive X-ray spectroscopy ( EDX) analysi s of iron binding to collagen (n = 27) and another (n = 15) for SEM examination of dentin collagen surface features after exposure to CT-K ( Fig. 1 ).

Fig. 1
Schematic of specimen preparation for EDX and collagen degradation experiments. Two teeth were used to produce 42 dentin slabs.

EDX analysis of Fe-ion binding to collagen

Twenty seven dentin slabs were divided into nine groups. Three dentin slabs were used for each of the nine conditioning solutions: 10% CA (as negative control, no FeCl 3 ); 10% CA–1.8% FeCl 3 ; 10% CA–0.6% FeCl 3 ; 5% CA–1.8%FeCl 3 ; 5% CA–0.6% FeCl 3 ; 1% CA–1.8% FeCl 3 ; 1% CA–0.6% FeCl 3 ; 1.8% FeCl 3 ; and 0.6% FeCl 3 . The slabs were immersed for 15 s in the conditioning solution followed by immersion in distilled water for 15 s. This procedure was done to mimic the exposure time of conditioning and rinsing of a typical etch-and-rinse bonding approach. The specimens were then dried at 37 °C in a desiccator with silica gel for 24 h. Afterwards, the slabs were fixed on acrylic stubs with carbon tape and sputter-coated with carbon (MED 010 Baltec, Balzers, Liechtenstein) prior to EDX. Spectrometry analysis (Vantage, NORAN Instruments, Middleton, WI, USA) coupled to a SEM (JEOL, JSM/5600LV, Tokyo, Japan) was performed in order to identify the elements and quantify the Fe weight percent. Each specimen was examined at 400× and selected areas of approximately 490 μm 2 were scanned for elements. Each spectrum was acquired for 100 s (voltage 15 kV, working distance 20 mm). Quantitative data were obtained from 10 analyzed areas of each treatment group and averaged for analysis.

SEM analysis of collagen exposed to CT-K

Fifteen demineralized dentin slabs were divided into 5 groups. Three slabs per group were immersed for 15 s in either 0.06% FeCl 3 ; 0.08% FeCl 3 or 1.8% FeCl 3 ; and rinsed for 15 s with distilled water before incubation with CT-K. Two additional control groups were created. One was exposed to neither FeCl 3 solutions nor incubation with CT-K; the other was not exposed to FeCl 3 solutions, but was exposed to CT-K incubation. After exposure to the conditioning solutions, exposed and control specimens were dried at 37 °C with silica gel in a desiccator for 24 h before being prepared for CT-K incubation. We used recombinant, human CT-K that was prepared as previously described elsewhere .

The dried specimens were weighted and incubated at a ratio of 1 mg collagen/1 μM CT-K in 50 μl of activity buffer (100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM DTT (Dithiothreitol) and EDTA) for 20 h at 28 °C. The reaction was stopped by the addition of 10 μM E-64 (carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidino)). After centrifuging, the specimens were removed from solution, rinsed with water, fixed in 2.5% glutaraldehyde, rinsed with water five times, dehydrated in ascending ethanol and dried with HMDS (hexamethyl di-silizane) before taken to sputter-coating with gold (MED 010 Baltec, Balzers, Leichtenstein) prior to SEM (JEOL, JSM/5600LV, Tokyo, Japan) observation . Micrographs were taken (voltage 15 kV) from randomly selected areas of the collagen matrix surface to assess the morphological changes using magnifications of 2000×, 6000× and 15,000×.

Effect of FeCl 3 on CT-K activity

CT-K activity was monitored with fluorimetric analysis (Luminescence Spectrometer, PerkinElmer, UK) by the degradation of Z-FR-AMC (Carbobenzoxy-phenylalanine-arginine-7 amino-4-methylcoumarin; Weil am Rhein, Germany) substrate in the presence of FeCl 3 at 0.005%; 0.01%; 0.02%; 0.04%; 0.08% concentrations. Solutions were prepared by adding 5 μL of 2 mM Z-FR-MCA dissolved in DMSO (dimethyl sulfoxide) to 990 μL of acetate buffer (100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM DTT and EDTA) containing or not FeCl 3 at the above concentrations. The reaction was initiated by adding 5 μL of CT-K at a final concentration of 5 nM to the solution and the hydrolysis rate of the Z-FR-AMC substrate was monitored by measuring the rate of increase in fluorescence as a function of time (30 s). Control assays were performed without FeCl 3 and it was considered the total hydrolysis of fluorescent substrate (100% enzymatic activity) against which the tested solutions were compared. The percentage of substrate degradation was plotted against the different concentrations of FeCl 3 and analyzed by best-fit regression analysis. The experiments were conducted in triplicate.

Statistical analysis

Bond strength data were analyzed by 2-way ANOVA (conditioning solutions vs. storage time) followed by Holm/Sidak post hoc multiple comparisons. Significance level was pre-set to α = 5%. Standard error of the mean (SEM) was given by least-square means (LSM) analysis (SigmaPlot 11, Systat Software Inc.). For EDX data (Fe-ion weight%), one-way ANOVA followed by all pairwise multiple comparison procedures (Fisher) under grouping information using LSD (Least Significant Difference) method was used. Non-linear regression analysis was used to investigate the relationship between FeCl 3 concentration and CT-K activity. All statistical analyses were done at 95% of confidence.

Materials and methods

Teeth

A total of 80 human caries-free third molars were used in this study. The study was approved by the local Ethics Board (approval # H15-02264). Seventy eight teeth were used for microtensile bond strength (μTBS) test and 2 teeth were used for collagen degradation and Fe-ion binding examination.

μTBS test

Teeth preparation

After extraction, the teeth were frozen in tap water and kept at −4 °C for no longer than 3 months. After thawing to room temperature, they were scrapped to remove organic debris and pumiced with rubber cup (KG Sorensen, Barueri, SP, Brazil). The roots were sectioned 1 mm below the cementum/enamel junction and a parallel cut made to expose flat mid-coronal dentin surface using a water-cooled diamond saw (EXTEC Corporation, Enfield, CT, USA) coupled to a cutting machine (Isomet 1000, Buehler Ltd., Lake Bluff, Il, USA). The surrounding enamel was removed by grinding on wet 180-grit SiC paper and the teeth were kept in tap water until used.

Preparation of conditioning solutions

The CA and/or FeCl 3 powder (Sigma/Aldrich Corporation, St. Louis, Missouri, USA) were dissolved in w/w% ratios in distilled water according to different concentration ratios ( Table 1 ). The solutions were prepared from anhydrous FeCl 3 (Sigma/Aldrich Corporation, St. Louis, Missouri, USA). The concentration ratios of CA and FeCl 3 were chosen to be variants of similar solutions previously described in the literature as 10-3 and 1-1 . For instance, the 1.8% FeCl 3 used in this study equals in Fe amount to that of 3% hydrated FeCl 3 (FeCl 3 ·6H 2 O) used in previous studies (10-3 solution) . The conditioning solutions were labeled and kept refrigerated in sealed vials until used. A commercially available 35% phosphoric acid gel (Select HV Etch, Bisco Inc., Schaumburg, IL, USA) was also used in the study. All conditioning solutions had their pH measured with a pH-meter (model mPA-210p, MS Tecnopon Equipamentos Especiais LTDA, SP, Brazil) ( Table 1 ).

Table 1
Description of the experimental groups for the microtensile bond strength testing, and composition of conditioning solutions used in the study.
Experimental groups Conditioning solutions ratio w/w % Etching time pH
Citric acid Ferric chloride
10–1.8 10 1.8 15 0.57
10–0.6 10 0.6 15 0.84
10–0.0 10 0 15 1.42
5–1.8 5 1.8 15 0.65
5–0.6 5 0.6 15 0.94
5–0 5 0 15 1.87
1–1.8 1 1.8 15 0.85
1–0.6 1 0.6 15 0.98
1–0 1 0 15 1.96
0–1.8 0 1.8 15 1.58
0–0.6 0 0.6 15 1.80
PA5 35% phosphoric acid 5 0.1–0.4
Control PA15 35% phosphoric acid 15 0.1–0.4
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Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Effect of conditioning solutions containing ferric chloride on dentin bond strength and collagen degradation
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