Abstract
Objectives
To evaluate the micro-tensile bond strength (μTBS) and interfacial characteristics of adhesive–dentin bonds produced after caries-removal with contemporary techniques.
Methods
Carious molars were cut at the base of the fissure, exposing ‘sound’ and ‘carious’ dentin at different spots. After caries-excavation, a composite was bonded using a 2-step self-etch adhesive. The μTBS was measured and the mode of fracture analyzed using a stereomicroscope and imaged by Feg-SEM, while additional non-fractured specimens were histologically analyzed after Masson’s trichrome staining in order to identify potentially incompletely resin-enveloped collagen.
Results
μTBS to residual caries-excavated dentin was lower than to sound dentin. The different caries-removing techniques had a significant effect on the μTBS. Er:YAG laser guided by a LIF-feedback system (Kavo) resulted in the lowest μTBS (26.8% lower than to ‘sound’ dentin) and a distinct layer of incompletely resin-enveloped collagen at the interface. Although different degrees of collagen exposure were seen for other caries-removing techniques, such as a thick layer for CeraBur (Komet-Brasseler), some unprotected collagen areas for Cariex (Kavo), or completely resin-enveloped collagen for a tungsten-carbide-bur (Komet), the μTBS appeared not directly affected (10%, 16.6%, and 15.3% lower than to ‘sound’ dentin, respectively). Carisolv (MediTeam) resulted in the highest μTBS (only 1% reduction compared to that to ‘sound’ dentin), followed by the tungsten-carbide-bur aided by Caries Detector (Kuraray) (4.8% reduction). Enzymatic caries excavation using the experimental SFC-VIII (3M-ESPE) aided by a disposable plastic instrument resulted in a 19.4% reduction in μTBS as compared to that to ‘sound’ dentin.
Significance
The dentin bonding receptiveness depends to a large extent on the caries-excavation method employed.
1
Introduction
The modern concept of ‘minimal-invasive dentistry’ calls for more conservative elimination of the highly infected and irreversibly demineralized carious tissue. Such selective caries excavation should prevent lesion progression, while maintaining the strength and stability of the remaining tooth structure in order to guarantee long-term mechanical resistance against intra-oral forces . However, defining the actual endpoint of caries excavation and thus the start-point of restoration is often clinically challenging. Since soft and wet dentin carious lesions harbor significantly more bacteria than hard and dry lesions , clinicians are commonly advised to remove carious dentin to the level where it is ‘firm’ .
The most conventional method of removing caries involves the use of steel or tungsten-carbide burs mounted in a low-speed contra-angle. Although very efficient in terms of time spent for caries removal , the decision to stop caries removal using these burs is very subjective, and basically depends on the operator’s background and clinical experience. The recently marketed CeraBur (Komet-Brasseler, Lemgo, Germany) is a self-limiting ceramic bur (alumina-based with stabilized zirconia) , which according to the manufacturer efficiently cuts infected, soft dentin, while hardly acts on hard, sound tissue.
Another potentially more conservative caries-removing technique involves the use of sono-abrasive technology. Diamond-coated oscillating tips coupled to an airscaler have previously been investigated for caries removal. They were however reported to mostly leave considerable amounts of residual carious dentin . Sono-abrasive technology using tungsten-carbide tips (Cariex TC tips, Kavo, Biberach, Germany) has also recently been marketed, but its effectiveness for caries removal has not been evaluated yet.
Besides rotary and oscillating caries-removal methods, chemical agents to selectively dissolve carious dentin can today also be applied. The Carisolv system (MediTeam, Göteborg, Sweden) makes use of a NaOCl-based gel and was reported to perform well in laboratory and clinical research. Moreover, the dentin substrate left after caries-removal with Carisolv was found to be very compatible with adhesive procedures . Recently, an experimental pepsin-based gel was developed (exp. SFC-VIII, 3M-ESPE, Seefeld, Germany), and consists of a moderately acidic buffered solution of pepsin that possesses the ability to cleave denatured collagen fibrils. These fibrils are exposed in the carious lesion after dissolution of the surrounding hydroxyapatite by organic acids, so that carious dentin is specifically targeted . Preliminary results have indicated comparable caries-removing properties of a previous experimental version of this product (exp. SFC-V, 3M-ESPE) with Carisolv .
Caries removal using an Er:YAG laser has been introduced as a “pain-free” and more tissue-preserving cavity-preparation technique . To overcome the difficulties in establishing the caries-removal endpoint with this technique, a laser-induced fluorescence (LIF) method was coupled to the laser equipment. This feedback system activates the Er:YAG laser to ablade dentin tissue only when the LIF of the tissue is higher than the chosen threshold . The caries-removal effectiveness of a feedback-equipped Er:YAG laser and the resulting bonding receptiveness of the remaining dentin substrate have not yet fully been investigated.
Except in case traumatic tooth injuries need to be restored or teeth need to be corrected esthetically, adhesive tooth restoration mostly involves bonding to caries-affected dentin. Today’s adhesives bond effectively to sound dentin through hybridization, but this bonding mechanism remains vulnerable in the long term. Incomplete resin-envelopment exposes collagen to oral fluid attack and enzymatic degradation processes that may eventually lead to caries recurrence . Bonding to caries-affected dentin is even less predictable and durable, not only because wider zones of unprotected collagen , but also more cracks and pores are present .
The aim of the present study was to determine the bonding effectiveness of a ‘gold-standard’ self-etch adhesive to residual caries-excavated dentin, as produced following seven different contemporary caries-removing techniques. The hypotheses tested were (1) that the μTBS to ‘residual caries-excavated’ dentin is similar as to ‘sound’ dentin, and (2) that different caries-removing techniques result in dentin substrates equally receptive to bonding.
2
Materials and methods
2.1
Selection of teeth and caries removal
From a bulk of extracted, non-restored molars stored in 0.5% aqueous chloramine, those teeth presenting caries lesions on the occlusal surface that presumably involve dentin, were selected. After removing plaque, calculus and other debris with an airscaler (Sonicflex 2000 equipped with a scaler tip #5: Kavo, Biberach, Germany), the teeth were mounted for ease of manipulation in gypsum, leaving the occlusal surface exposed.
Digital radiographs were obtained with the aid of a CCD detector (Vista Ray CCD System, Dürr Dental AG, Bietigheim-Bissingen, Germany) so that teeth without radiographically detectable dentin caries could be excluded. All remaining teeth ( n = 35) were then divided in 7 groups according to the different caries-removing techniques tested ( Table 1 ).
| Caries-removal technique | Manufacturer | Excavation procedure | Caries-removal endpoint |
|---|---|---|---|
| Tungsten-carbide round bur | Komet-Brasseler, Lemgo, Germany | Low-speed contra-angle, approximate speed ≅1500 rpm/min | Hard cavity floor with a blunt explorer |
| CeraBur (K1SM) | Komet-Brasseler | Low-speed contra-angle, approximate speed ≅1500 rpm/min | Self-limiting cutting ability of the instrument |
| Cariex TC tips | Kavo, Biberach, Germany | Airscaler (Soniflex 2003L), oscillations <6.5 kHz with water cooling | Hard cavity floor with a blunt explorer |
| Tungsten-carbide round bur aided by Caries Detector | Komet-Brasseler/Kuraray Medical, Okayama, Japan | Low-speed contra-angle, approximate speed ≅1500 rpm/min | Light-pink residual carious dentin staining |
| Carisolv | MediTeam, Göteborg, Sweden | After dispensing using the auto-mix syringe system, a drop of the solution was placed on the carious lesion. After 30 s, the Carisolv metal mace tips (n.2–5) were used to scrape off the carious tissue | Self-limiting caries-removing ability of the solution |
| Exp. SFC-VIII + plastic instrument (star v1.3) | 3M-ESPE, Seefeld, Germany | After mixing the two solutions according to the manufacturer’s instruction, a drop of the solution was placed on the carious lesion. After 30 s, a prototype plastic instrument (Star v1.3) was used to scrape off the carious tissue | Self-limiting caries-removing ability of the solution |
| Er:YAG laser (Kavo KEY Laser III) | Kavo | Output settings: 250 mJ/pulse with a pulse-repetition rate of 4 pulses/s. Ablation was performed with a non-contact handpiece 2060 under water cooling | Feedback system threshold 7 |
The occlusal enamel was removed by cutting each tooth crown through the deepest part of the occlusal fissure with the aid of a 0.3-mm thick diamond cut-off wheel (Struers, Ballerup, Denmark) mounted in an Isomet low-speed saw (Buehler, Lake Bluff, IL, USA). The carious lesion was then removed by one of the methods described in Table 1 , using the respective caries-removing endpoints indicated. To prepare a standardized smear layer in the sound dentin area and to produce sound and caries-affected dentin substrates at approximately the same depth, each tooth was further ground with a medium-grit (100 μm) diamond bur (842, Komet-Brasseler, Lemgo, Germany) in a water-cooled high-speed turbine, mounted in a Micro-Specimen Former (University of Iowa, Iowa City, IA, USA). Extra care was taken not to touch the bottom of the excavated caries lesion.
2.2
Bonding procedures and micro-specimen preparation
Each tooth containing both sound and caries-excavated dentin was next thoroughly washed with water-spray and gently air-dried. A ‘mild’ two-step self-etch adhesive (Clearfil SE Bond, Kuraray, Osaka, Japan) was applied following the manufacturer’s instructions. A composite crown was built using Filtek Z100 composite (3M-ESPE, Seefeld, Germany) and the location of the caries-excavated dentin area was marked at the top of the composite build-up. After 24-h storage in water at 36 °C, the teeth were cut into 1-mm 2 stick-shaped micro-specimens with the aid of the 0.3 mm diamond cut-off wheel (Struers) mounted in an Accutom-50 cutting machine (Struers).
2.3
Laser-induced fluorescence (LIF) measurements
Each micro-specimen was assigned to either the ‘sound’ or ‘residual caries-excavated’ dentin group, based in the first place on the previously marked area at the top of the composite build-up. LIF measurements at each of the four sides of each micro-specimen were performed using a Diagnodent Pen (2190, Kavo, Biberach, Germany). The micro-specimen was definitively assigned to the ‘residual caries-excavated’ dentin group, if it presented readings similar or above 10 on at least one of the 4 sides.
2.4
μTBS testing
For μTBS testing, the micro-specimens were fixed to a BIOMAT jig with the aid of a cyanoacrylate-based glue (Model Repair II Blue, Dentsply-Sankin, Ohtawara, Japan), and stressed at a crosshead speed of 1 mm/min until failure using a universal testing device (LRX, Lloyd Instruments, Hampshire, UK), equipped with a load cell of 100 N. The μTBS was expressed in MPa, as derived from dividing the imposed force (N) at the time of fracture by the bond area of the individual specimen (mm 2 ). The occurrence of failure prior to the actual testing was included in the calculation of the mean μTBS as 0 MPa, with an explicit note of the number of pre-testing failures (ptf). The mode of failure was determined at a magnification of up to 50× using a stereomicroscope (LM fracture analysis: Wild M5A, Wild-Heerbrugg, Heerbrugg, Switzerland), and recorded either as ‘interfacial’, ‘cohesive in dentin’, ‘cohesive in composite’ or ‘mixed failure’ (including failures within the adhesive layer and composite). Five teeth were tested for each caries-removing technique.
2.5
SEM fracture analysis
Following failure analysis in the stereomicroscope, representative samples were processed for field-emission-gun scanning electron microscopy using secondary electron detection (Feg-SEM; FEI Nova NanoLab 600, Eindhoven, The Netherlands) in order to more accurately interpret (or confirm) the previously analyzed fracture mode in each group. For this purpose, samples that failed according to the most representative failure mode and had a μTBS close to the mean of that particular group, were selected. Common procedures for SEM-specimen preparation were employed, as described previously .
2.6
Interfacial characterization using a Masson’s trichrome staining protocol
For each caries-removing technique, two micro-specimens (belonging to the ‘residual caries-excavated’ dentin group) that were not tested for μTBS, were prepared for histological analysis using a standard Masson’s trichrome staining technique . The stick-shaped specimens were first individually embedded in epoxy resin (Epofix Kit, Struers, Ballerup, Denmark) and sectioned along the longest axis with the aid of the 0.3 mm thick diamond cut-off wheel mounted in the Accutom-50 cutting machine (Struers). The half-sectioned specimens were glued with cyanoacrylate (Sekunder-kleber, Renfert, Hilzingen, Germany) to a glass plate and ground to a thickness of 20–50 μm (Exakt AW 110, Exakt Technologies, Oklahoma City, OK, USA). Finally, the samples were stained according to the standard Masson’s trichrome technique , and imaged under transmitted light. Using this technique, fully resin-enveloped collagen stained ‘green’, unprotected collagen ‘red’, composite material ‘beige’ and adhesive ‘yellow’ due to a reaction with the fixative.
2.7
Statistical analysis
For the statistical analysis, every caries-excavated dentin specimen was randomly matched to one of the sound dentin (control) specimens from the same tooth. Doing so, 246 matched pairs were formed, and analyzed by two-way ANOVA with repeated measurements and Tukey multiple-comparisons in order to identify significant differences in μTBS among the different caries-removing techniques. A chi-square test was used to compare the incidence of the different fracture modes among the caries-removing techniques. A significance level of 5% was employed for all analyses.
2
Materials and methods
2.1
Selection of teeth and caries removal
From a bulk of extracted, non-restored molars stored in 0.5% aqueous chloramine, those teeth presenting caries lesions on the occlusal surface that presumably involve dentin, were selected. After removing plaque, calculus and other debris with an airscaler (Sonicflex 2000 equipped with a scaler tip #5: Kavo, Biberach, Germany), the teeth were mounted for ease of manipulation in gypsum, leaving the occlusal surface exposed.
Digital radiographs were obtained with the aid of a CCD detector (Vista Ray CCD System, Dürr Dental AG, Bietigheim-Bissingen, Germany) so that teeth without radiographically detectable dentin caries could be excluded. All remaining teeth ( n = 35) were then divided in 7 groups according to the different caries-removing techniques tested ( Table 1 ).
| Caries-removal technique | Manufacturer | Excavation procedure | Caries-removal endpoint |
|---|---|---|---|
| Tungsten-carbide round bur | Komet-Brasseler, Lemgo, Germany | Low-speed contra-angle, approximate speed ≅1500 rpm/min | Hard cavity floor with a blunt explorer |
| CeraBur (K1SM) | Komet-Brasseler | Low-speed contra-angle, approximate speed ≅1500 rpm/min | Self-limiting cutting ability of the instrument |
| Cariex TC tips | Kavo, Biberach, Germany | Airscaler (Soniflex 2003L), oscillations <6.5 kHz with water cooling | Hard cavity floor with a blunt explorer |
| Tungsten-carbide round bur aided by Caries Detector | Komet-Brasseler/Kuraray Medical, Okayama, Japan | Low-speed contra-angle, approximate speed ≅1500 rpm/min | Light-pink residual carious dentin staining |
| Carisolv | MediTeam, Göteborg, Sweden | After dispensing using the auto-mix syringe system, a drop of the solution was placed on the carious lesion. After 30 s, the Carisolv metal mace tips (n.2–5) were used to scrape off the carious tissue | Self-limiting caries-removing ability of the solution |
| Exp. SFC-VIII + plastic instrument (star v1.3) | 3M-ESPE, Seefeld, Germany | After mixing the two solutions according to the manufacturer’s instruction, a drop of the solution was placed on the carious lesion. After 30 s, a prototype plastic instrument (Star v1.3) was used to scrape off the carious tissue | Self-limiting caries-removing ability of the solution |
| Er:YAG laser (Kavo KEY Laser III) | Kavo | Output settings: 250 mJ/pulse with a pulse-repetition rate of 4 pulses/s. Ablation was performed with a non-contact handpiece 2060 under water cooling | Feedback system threshold 7 |
The occlusal enamel was removed by cutting each tooth crown through the deepest part of the occlusal fissure with the aid of a 0.3-mm thick diamond cut-off wheel (Struers, Ballerup, Denmark) mounted in an Isomet low-speed saw (Buehler, Lake Bluff, IL, USA). The carious lesion was then removed by one of the methods described in Table 1 , using the respective caries-removing endpoints indicated. To prepare a standardized smear layer in the sound dentin area and to produce sound and caries-affected dentin substrates at approximately the same depth, each tooth was further ground with a medium-grit (100 μm) diamond bur (842, Komet-Brasseler, Lemgo, Germany) in a water-cooled high-speed turbine, mounted in a Micro-Specimen Former (University of Iowa, Iowa City, IA, USA). Extra care was taken not to touch the bottom of the excavated caries lesion.
2.2
Bonding procedures and micro-specimen preparation
Each tooth containing both sound and caries-excavated dentin was next thoroughly washed with water-spray and gently air-dried. A ‘mild’ two-step self-etch adhesive (Clearfil SE Bond, Kuraray, Osaka, Japan) was applied following the manufacturer’s instructions. A composite crown was built using Filtek Z100 composite (3M-ESPE, Seefeld, Germany) and the location of the caries-excavated dentin area was marked at the top of the composite build-up. After 24-h storage in water at 36 °C, the teeth were cut into 1-mm 2 stick-shaped micro-specimens with the aid of the 0.3 mm diamond cut-off wheel (Struers) mounted in an Accutom-50 cutting machine (Struers).
2.3
Laser-induced fluorescence (LIF) measurements
Each micro-specimen was assigned to either the ‘sound’ or ‘residual caries-excavated’ dentin group, based in the first place on the previously marked area at the top of the composite build-up. LIF measurements at each of the four sides of each micro-specimen were performed using a Diagnodent Pen (2190, Kavo, Biberach, Germany). The micro-specimen was definitively assigned to the ‘residual caries-excavated’ dentin group, if it presented readings similar or above 10 on at least one of the 4 sides.
2.4
μTBS testing
For μTBS testing, the micro-specimens were fixed to a BIOMAT jig with the aid of a cyanoacrylate-based glue (Model Repair II Blue, Dentsply-Sankin, Ohtawara, Japan), and stressed at a crosshead speed of 1 mm/min until failure using a universal testing device (LRX, Lloyd Instruments, Hampshire, UK), equipped with a load cell of 100 N. The μTBS was expressed in MPa, as derived from dividing the imposed force (N) at the time of fracture by the bond area of the individual specimen (mm 2 ). The occurrence of failure prior to the actual testing was included in the calculation of the mean μTBS as 0 MPa, with an explicit note of the number of pre-testing failures (ptf). The mode of failure was determined at a magnification of up to 50× using a stereomicroscope (LM fracture analysis: Wild M5A, Wild-Heerbrugg, Heerbrugg, Switzerland), and recorded either as ‘interfacial’, ‘cohesive in dentin’, ‘cohesive in composite’ or ‘mixed failure’ (including failures within the adhesive layer and composite). Five teeth were tested for each caries-removing technique.
2.5
SEM fracture analysis
Following failure analysis in the stereomicroscope, representative samples were processed for field-emission-gun scanning electron microscopy using secondary electron detection (Feg-SEM; FEI Nova NanoLab 600, Eindhoven, The Netherlands) in order to more accurately interpret (or confirm) the previously analyzed fracture mode in each group. For this purpose, samples that failed according to the most representative failure mode and had a μTBS close to the mean of that particular group, were selected. Common procedures for SEM-specimen preparation were employed, as described previously .
2.6
Interfacial characterization using a Masson’s trichrome staining protocol
For each caries-removing technique, two micro-specimens (belonging to the ‘residual caries-excavated’ dentin group) that were not tested for μTBS, were prepared for histological analysis using a standard Masson’s trichrome staining technique . The stick-shaped specimens were first individually embedded in epoxy resin (Epofix Kit, Struers, Ballerup, Denmark) and sectioned along the longest axis with the aid of the 0.3 mm thick diamond cut-off wheel mounted in the Accutom-50 cutting machine (Struers). The half-sectioned specimens were glued with cyanoacrylate (Sekunder-kleber, Renfert, Hilzingen, Germany) to a glass plate and ground to a thickness of 20–50 μm (Exakt AW 110, Exakt Technologies, Oklahoma City, OK, USA). Finally, the samples were stained according to the standard Masson’s trichrome technique , and imaged under transmitted light. Using this technique, fully resin-enveloped collagen stained ‘green’, unprotected collagen ‘red’, composite material ‘beige’ and adhesive ‘yellow’ due to a reaction with the fixative.
2.7
Statistical analysis
For the statistical analysis, every caries-excavated dentin specimen was randomly matched to one of the sound dentin (control) specimens from the same tooth. Doing so, 246 matched pairs were formed, and analyzed by two-way ANOVA with repeated measurements and Tukey multiple-comparisons in order to identify significant differences in μTBS among the different caries-removing techniques. A chi-square test was used to compare the incidence of the different fracture modes among the caries-removing techniques. A significance level of 5% was employed for all analyses.
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