|Year : 2021 | Volume
| Issue : 3 | Page : 267-274
The effect of acid concentration and etch time on morphology and tensile bond strength of primary dentin: An in vitro study
Kirthana Satish1, Rashmi Nayak1, Kishore Ginjupalli2, Shwetha Balagopal1
1 Department of Pedodontics and Preventive Dentistry, Manipal Academy of Higher Education, Manipal College of Dental Sciences, Manipal, Karnataka, India
2 Department of Dental Materials, Manipal Academy of Higher Education, Manipal College of Dental Sciences, Manipal, Karnataka, India
|Date of Submission||10-Apr-2021|
|Date of Decision||04-Jul-2021|
|Date of Acceptance||03-Sep-2021|
|Date of Web Publication||22-Nov-2021|
Dr. Rashmi Nayak
Department of Pedodontics and Preventive Dentistry, Manipal Academy of Higher Education, Manipal College of Dental Sciences, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: There are conflicting results concerning the ideal time for etching primary dentin and its effect on the bond strength of adhesive restorative materials. Aim: To assess in vitro, the effect of varied acid concentration and etch time on the morphological features and tensile bond strength of primary dentin. Materials and Methods: Forty healthy primary molars were prepared by exposing the dentin of the occlusal surface, creating a smear layer. A 3 mm × 3 mm test indow was demarcated, and specimens were randomly allocated to four groups (n = 10): Group 1A 10% phosphoric acid, 7 s; Group 1B 10% phosphoric acid, 15 s; Group 2A 37% phosphoric acid, 7 s and Group 2B 37% phosphoric acid, 15 s. Surface analysis was done using atomic force microscope and scanning electron microscope. For tensile bond strength evaluation, 24 specimens prepared as mentioned were mounted in acrylic blocks and allocated to four groups according to the prescribed etching protocol. Resin rods were bonded and tested in tension after 24 h (n = 6). Data were analyzed statistically using unpaired t-test. Results: Etching of primary dentin using 37% phosphoric acid for 7 s produced the highest tensile bond strength of 9.51 ± 2.19 MPa. Conclusion: Etching time of 7 s may improve the adhesion of resin restorative materials with primary dentin.
Keywords: Acid etchant concentration, etching time, morphology, primary teeth, resin composite, tensile bond strength
|How to cite this article:|
Satish K, Nayak R, Ginjupalli K, Balagopal S. The effect of acid concentration and etch time on morphology and tensile bond strength of primary dentin: An in vitro study. J Indian Soc Pedod Prev Dent 2021;39:267-74
|How to cite this URL:|
Satish K, Nayak R, Ginjupalli K, Balagopal S. The effect of acid concentration and etch time on morphology and tensile bond strength of primary dentin: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2021 [cited 2021 Dec 5];39:267-74. Available from: https://www.jisppd.com/text.asp?2021/39/3/267/330703
| Introduction|| |
The introduction of acid etching has remarkably transformed the practice of dentistry. Total etching using 30%–40% orthophosphoric acid for 15 s is widely accepted as the protocol for conditioning the dentin of permanent teeth. The depth and extent of demineralisation caused by this process differ with etching time and acid concentration. Acid etching preferentially removes the mineral components of dentin and exposes the collagen meshwork. The subsequent application of a bonding agent helps in the creation of a long-lasting, acid-resistant intermixture of resin adhesive and dentin termed as the resin dentin interdiffusion zone.,
There are reasons why an extension of the total-etch protocol of permanent teeth to primary teeth is not acceptable. Primary dentin has unique morphological and histological features that contribute to a difference in bond strength compared with permanent teeth. They have higher tubular densityand hence, less intertubular dentin available for bonding. The dentinal tubules are smaller in diameter owing to thicker peritubular dentin. Besides, the low calcium and phosphate content of the peri and intertubular dentin in primary teeth renders it more reactive to acid etching.
The complex and highly hydrated nature of dentin makes it a challenging tissue for bonding. In vitro experiments have reported poor bonding of distinct adhesive systems particularly to primary dentin., A previous study conducted to study the thickness of hybrid layer in primary and permanent teeth with etch times of seven and 15 s observed a significantly thicker hybrid layer in primary dentin suggestive of its high reactivity to acid etching. Failure of adhesive resin to completely penetrate the thick hybrid layer was cited as the possible explanation for the lesser bond strength values of primary dentin.
Thus, the effects of etching and the resultant attributes of the dentin substrate influence the performance of composite resin restorations. Regardless of the peculiar structural, compositional, and morphological features and variations in bond strengths, an identical etching and bonding protocol is employed for etching and bonding to primary and permanent teeth. The differentiated resin-dentin interface in the two dentitions may have potentially deleterious consequences to the performance of the resin restoration. Decreasing the acid concentration and time of etching for primary dentin has been recommended to obtain sufficient bond strength values due to the development of a more uniform hybrid layer. Nonetheless, there is a lack of firmly established recommendations concerning the ideal time duration for etching the primary dentin to improve its bond strength with adhesive materials. Hence, this study aimed to evaluate the effect of acid concentration and etch time on surface morphology and tensile bond strength of primary dentin. The objectives of the study were:
- To evaluate the surface morphology of the primary dentin surface etched using 10% and 37% phosphoric acid for 7 s and 15 s using scanning electron microscopy (SEM)
- To evaluate and compare the surface roughness of primary dentin etched using 10% and 37% phosphoric acid for 7 s and 15 s using atomic force microscopy (AFM)
- To evaluate and compare the tensile bond strength of resin to primary dentin prepared using the above mentioned acid concentrations and etching times.
The null hypothesis tested was that there is no difference in the tensile bond strength of resin composite to primary dentin prepared using varied etch time or etchant concentration.
| Materials and Methods|| |
The study was commenced with the approval from the Institutional Ethics Committee (364/2014). Sixty-four noncarious primary molars, extracted due to preshedding mobility or for orthodontic reasons, were included in the study. Hypoplastic teeth, teeth with white spot lesions, and fractured teeth were excluded.
Phase I was carried out to study the changes in the morphology of primary dentin after the use of varied etchant concentrations and etch time.
Forty noncarious primary molars were selected. The occlusal surfaces were ground using a double-faced diamond disc with water coolant until the dentin was exposed. The surface was polished for 30 s by wet grinding sequentially using 400 and 600 grit silicon carbide paper to obtain a polished surface with a standardized smear layer [Figure 1]a and [Figure 1]b. A 3 mm × 3 mm adhesive tape was affixed on the prepared dentin surface, and nail varnish was applied on the remaining tooth structure to demarcate a window for etchant application [Figure 2]. The prepared teeth were randomly allocated to four groups with ten samples each as per the surface treatment protocol as follows:
|Figure 1: (a and b) Sound primary molar tooth sample prepared by grinding of the occlusal surface|
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|Figure 2: Adhesive tape of 3 mm × 3 mm applied on the prepared sample to create a window for bonding|
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- Group 1A – Treated with 10% phosphoric acid for 7 s
- Group 1B – Treated with 10% phosphoric acid for 15 s
- Group 2A – Treated with 37% phosphoric acid for 7 s
- Group 2B – Treated with 37% phosphoric acid for 15 s.
The dentin of the demarcated window was etched by gently agitating the gel with a bristle brush and washed using distilled water for 20 s. Time was recorded using a stop clock. Specimens were kept hydrated at room temperature in 0.9% saline solution until further evaluation to preserve the surface morphology of the demineralized layer.
Atomic force microscopy
The etched samples were subjected to AFM (Innova, Newport Vision Isostation, 28-12-18) to evaluate the surface roughness. Air-dried samples were mounted on the focusing platform [Figure 3], and the oscillating probe tip was adjusted to gently tap the surface. Tapping mode at 3V and 2-hz frequency over an area of 20 μm was used. The obtained two and three-dimensional images along the X, Y, and Z axes were used for the evaluation of surface roughness.
Scanning electron microscopy
After AFM evaluation, the samples were desiccated in a hot air oven for 10 min at 110 °C, gold-sputtered, and viewed under a SEM (JEOL-JSM-630 LA) at a magnification of 2000X. The surface was observed for the presence or absence of a smear layer and dentinal tubule opening. The diameter of the opened dentinal tubules was measured using a computerized scale.
Phase II was carried out to assess the tensile bond strength of resin composites to the conditioned dentin surface. Twenty-four samples were mounted on acrylic blocks of 2 × 2 inches [Figure 3] and prepared using the etching protocols mentioned earlier. The etched dentin surface was dried using a blotting paper to prevent excessive drying and collapse of the collagen network. Adper single bond (3M ESPE) was applied and cured for 20 s. A composite resin rod of 3 mm diameter and 2 mm height was built over the demarcated window. A 0.7 mm wire bent in a triangular shape [Figure 3] was incorporated into the composite to provide an attachment for the loop in the Universal testing machine (Instron 3366, USA). After 24 h in saline, tensile bond strength was measured by loading the samples at a cross-head speed of 1 mm/min until the composite debonded from the tooth surface [Figure 4]a and [Figure 4]b. The maximum load recorded during the test divided by the area of the specimen, in MPa, was considered as tensile bond strength.
|Figure 4: Universal testing machine being used for assessment of tensile bond strength (a) Experimental set up (b) Tensile bond strength Measurement|
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| Results|| |
Surface roughness with atomic force microscopy
[Graph 1] shows the mean surface roughness of the study groups as seen under AFM. [Figure 5], [Figure 6], [Figure 7], [Figure 8] show the graphical representation of surface roughness in a 20 μm × 20 μm window as seen under AFM after etching of the dentin. The samples treated with 37% phosphoric acid for 7 s recorded a significantly higher surface roughness ranging from 312 to 393 nm with a mean of 344.0 ± 28.72 nm.
|Figure 5: Atomic force microscopy images of a representative sample from Group 1A (10% phosphoric acid etchant at 7 s). (a) 20 μm × 20 μm surface area of the etched primary dentin showing depth of − 1.3 μm and height of 1.1 μm from the surface. (b-d) Image of the sample along the X, Y and Z axes respectively showing the partial opening of dentinal tubules and flat surface of the intertubular dentin|
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|Figure 6: Atomic force microscopy images of a representative sample from Group 1B (10% phosphoric acid etchant at 15 s). (a) 20 μm × 20 μm surface area of the etched primary dentin showing depth of − 1.5 μm and height of 1.0 μm from the surface. (b-d) Image of the sample along the X, Y and Z axes respectively showing the near complete opening of dentinal tubules with exposed odontoblastic process and flat surface of intertubular dentin|
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|Figure 7: Atomic force microscopy images of a representative sample from Group 2A (37% phosphoric acid etchant at 7 s). (a) 20 μm × 20 μm surface area of the etched primary dentin showing depth of -2.5μm and height of 1.8 μm from the surface. (b-d) Image of the sample along the X, Y and Z axes respectively showing the near complete removal of smear layer, opening of dentinal tubules with exposed odontoblastic process Image of the sample along the X,Y and Z axes respectively showing the near complete removal of smear layer, opening of dentinal tubules with exposed odontoblastic process and irregular surface of the intertubular dentin suggestive of increased surface roughness|
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|Figure 8: Atomic force microscopy images of a representative sample from Group 2B (37% phosphoric acid etchant at 15 s). (a) 20 μm × 20 μm surface area of the etched primary dentin showing depth of − 1.7 μm and height of 1.3 μm from the surface. (b-d) Image of the sample along the X, Y and Z axes respectively showing the complete removal of smear layer, opening of dentinal tubules with exposed odontoblastic process but no appreciable roughness of the intertubular dentin|
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Surface changes with scanning electron microscopy
[Figure 9] shows the qualitative changes in surface morphology of the dentin as seen in SEM analysis at ×2000 magnification following acid etching.
|Figure 9: Scanning electron micrographs of all four groups at ×2000 magnification. (a) Sample from Group 1A shows incomplete removal of smear layer and partially open dentinal tubules. (b) Sample from Group 1B shows partial smear layer removal with open dentinal tubules. (c) Sample from Group 2A shows near complete removal of smear layer and open dentinal tubules. (d) Sample from group 2B shows complete removal of smear layer, comparatively more number of open dentinal tubules and reduced intertubular dentin|
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Samples etched with 10% phosphoric acid for 7 s show a granular surface suggestive of partial removal of smear layer and incomplete opening of the dentinal tubules. The tubule diameter ranges from 1.5 μm to 3.12 μm [Figure 9]a. The effect of 15 s etching with 10% phosphoric acid is observed to be better with respect to smear layer removal. Few dentinal tubules are seen to be completely open with tubule diameters ranging between 2.26 μm and 3.63 μm. Additionally, the odontoblastic processes can be appreciated in the dentinal tubules [Figure 9]b.
In contrast, the samples of surfaces treated with 37% phosphoric acid for 7 s reveal complete smear layer removal with higher number of open dentinal tubules, the diameter ranging from 2.01 μm to 4.04 μm [Figure 9]c. Images of dentin treated with 37% phosphoric acid for 15 s show more number of open dentinal tubules per 10 μm of dentin surface with a tubule diameter ranging from 1.99 μm to 4.12 μm [Figure 9]d.
Peritubular dentin is visualised as rings around the dentinal tubules in all the groups suggesting that it was preserved irrespective of etchant concentration and etching time.
Tensile bond strength
A statistically significant difference was observed in the tensile bond strength of composite resin for samples etched with 37% phosphoric acid (P < 0.05) compared to samples etched using 10% phosphoric acid [Graph 2]. However, the highest value of 9.51 ± 2.19 MPa was obtained for samples treated with 37% phosphoric acid for 7 s, corroborating with the findings of AFM and SEM observations.
| Discussion|| |
This study was conducted to evaluate in vitro, the morphology of primary dentin treated with varying concentrations of phosphoric acid and etch times, and its effect on bond strength to composite resin.
Acid etching facilitates removal of the smear layer from the intertubular dentin and the tubule openings to aid in the establishment of the hybrid layer and a resultant strong and stable adhesion to dentin. The concentration of acid and its duration of contact with the tooth surface influence smear layer removal. Strong acids applied for long periods cause excessive demineralization of the peritubular and intertubular dentin resulting in a severe collapse of its collagen mesh-work. The action of hydrophilic primers may be insufficient to reconstitute it to the original level. Additionally, unnecessary deep demineralization due to long etch times can prevent the resin monomers from diffusing as deeply as the etchant agent, leaving behind nonimpregnated or poorly penetrated, unsupported sections at the base of the hybrid layer, making it prone to microleakage and nanoleakage over time.,,,,,,
A linear relationship has been observed between the etching time and the thickness of the hybrid layer. SEM studies demonstrate 25%–30% thicker hybrid layer in primary tooth dentin compared with that of permanent tooth dentin using identical etching time. However, there is no evidence of a proportional increase in tensile bond strength with the greater thickness of the hybrid layer. Hence, it is suggested that the goal of dentin bonding in primary teeth should be to achieve a narrow hybrid layer uniformly infused with resin and a differentiated protocol with a shorter time of acid etching can be conducive to this process.,,,, Sardella et al. reported that reducing the time and the acid concentration for etching primary dentin, did not compromise its bond strength with the restorative material. Nevertheless, no study has conclusively recommended an etching protocol for primary teeth. Therefore, this experimental study was planned to test if etching, using lower concentrations of acid and shorter application time was effective in increasing the strength of the bond between resin and primary dentin.
The action of the etchant is affected by the structure and composition of the dentin. Microbial action triggers a natural mechanism that alters the surface of the tooth which completely or partially obliterates the dentinal tubules making the dentin more resistant to the action of acid. Also, affected dentin presents lower hardness than sound or sclerotic dentine.,, Sound, noncarious primary teeth were selected for this study to avoid variations within the substrate and facilitate a more accurate evaluation of surface morphology and bond strength.
10% phosphoric acid was chosen based on studies by Gwinnet and Nör who reported that it sufficiently demineralizes the primary dentin surface and makes it receptive to adhesive restorations.
Etching times of seven and 15 s were tested in this study. Lowering the time to 5 s with 37% phosphoric acid has been evaluated, but the resultant bond strength proved to be very low in comparison to 15 s and 30 s etching time. Several studies have proven that 7 s etch time had higher bond strength in comparison to 10, 15, and 20 s on primary dentin.,,, Therefore the lower etch time of 7 s was considered to be evaluated in the present study in comparison to the currently accepted time of 15 s.
SEM provides a two-dimensional image of the etched surface. The qualitative analysis of surface details such as the presence or absence of smear layer, the diameter of the dentinal tubule opening, and the quantification of intertubular dentin is possible with SEM evaluation. AFM when used to study dentin demineralization, provides three-dimensional visualization of the roughness produced by acid etching which is an important criterion in assessing the bonding potential. Hence, a combination of both was employed in our study. AFM has the added advantage of minimal sample preparation, ease of observation of roughness, and the samples can be used for further evaluation since they are not destroyed. It can be used in an array of conditions like the presence of moisture or reduced temperatures. SEM on the other hand is more sensitive to environmental conditions. Sample desiccation and high vacuum are chief requirements for SEM evaluation. Hence, in the present study, AFM was followed by SEM of the same samples, to avoid sample loss and facilitate comparison of the observations.,,,
All samples in the study showed a change in surface roughness following acid etching when viewed under AFM owing to dentinal tubule opening. A preferential attack on peritubular dentin resulted in the funnel shape of the tubules with the narrow portion inwards. But the degree of roughness varied with the acid concentration and etch time. Significantly higher mean surface roughness was observed with 37% phosphoric acid used for 7 s. Much of this surface roughness is due to the increase in the intertubular roughness as observed in AFM images of this group. This change of surface topography of the dentin provides a higher total surface area for bonding. Similar AFM results are reported by Osorio et al. with 7 s of etching time with 35% phosphoric acid.
It is seen in the present study that smear layer removal is also influenced by the acid concentration and etch time. SEM observations showed lower efficacy of smear layer removal by 10% of phosphoric acid compared with 37% concentration. When the same acid concentration was used, the smear layer removal was more effective with prolonged etching time. Upon comparing the acid concentrations, a higher concentration resulted in better removal of the smear layer. These results are as per Goes et al. Contradictory to our results Nör reported that 10% of phosphoric acid used for 10 and 15 s, completely removed the smear layer.
Peritubular dentin could be visualized as rings around the dentinal tubules under the SEM, suggesting that peritubular dentin was adequately preserved during etching irrespective of etchant concentration and etch time. Smaller tubule diameter was noted for the group etched with 37% phosphoric acid for 7 s. This means that a greater amount of intertubular dentin was preserved to favor better bond strength. Similar results are reported by Pimenta et al.
Dentin has high water content. Most modern adhesive systems possess hydrophilic characteristics and require a moist dentin substrate for high-quality bonding. It is well documented that the bond strength of over dried dentin is lower than that of moist dentin. This is because the collagen fibre mesh collapses if dentin is excessively air-dried and the resin monomers cannot optimally penetrate to form the hybrid zone. Single bond adhesive system used in this study, contains water as a cosolvent, which does not allow collagen saturation by hydroxyethyl methacrylate (HEMA) due to its lower volatility compared to acetone-based adhesive systems. Therefore, if the substrate is excessively moist, the water present in the adhesive system may lead to water saturation and decrease the bonding efficiency. On the other hand, Single Bond also contains HEMA and polyalkenoic acid, which enhance adhesion to moist dentin. In our study, care was taken to prevent excess moisture by blotting the dentin using check the terminology once!!! paper before application of the adhesive.
In summary, the application of 37% phosphoric acid for 7 s causes sufficient conditioning of primary dentin to achieve adequate tensile bond strength. Similar findings are reported by Rontani et al., Sardella et al. Torres et al.and Osorio et al. More effective removal of the smear layer, the smaller diameter of the dentinal tubules of the primary dentin, and the resultant increased volume of intertubular dentin as evidenced by SEM can be credited for these results. AFM observations of increased surface roughness when 37% phosphoric acid was used for 7 s, further corroborates with the SEM observations, adding credibility to the findings.
Thus, the null hypothesis that there is no difference in the tensile bond strength of resin composite to primary dentin when prepared using varied etch time or etchant concentration is rejected.
| Conclusion|| |
Primary dentin conditioned with 37% phosphoric acid for 7 s results in superior etching and facilitates a strong bond with adhesive restorative materials.
We sincerely acknowledge and appreciate the technical support provided by Manipal Institute of Technology, Manipal in the conduct of this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]