Effect of Infiltrating a Commercial Etch-and-Rinse Bonding Agent with Bioactive Nanoparticles on the Degree of Conversion of Adhesive and Flexural Strength of Dentin

doi:10.21203/rs.3.rs-4726867/v1

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Research Article

Effect of Infiltrating a Commercial Etch-and-Rinse Bonding Agent with Bioactive Nanoparticles on the Degree of Conversion of Adhesive and Flexural Strength of Dentin

https://doi.org/10.21203/rs.3.rs-4726867/v1

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Background: The aim of this study was to compare the degree of conversion of adhesive and the flexural strength of dentin after adding calcium silicate (CS) and amorphous calcium phosphate (ACP) nanoparticles (NP) to an etch-and-rinse bonding system and the application of these materials on dentin.

Methods: ACP and CS nanoparticles with an average size of 100 nm were synthesized and added to Tetric N-Bond two-step etch- and- rinse adhesive. To check the flexural strength, dentin samples (n=60) were divided into two groups based on immersion time (24 hours and 2 months), and each of these two groups was divided into 3 groups (n=10) including bonding agent containing ACP and CS nanoparticles and pure bonding agent.

In order to measure the degree of conversion (DC), bonding agent containing ACP and CS and pure adhesive were evaluated by Fourier Transform Infrared (FTIR) spectroscopy.

Results: Stepwise comparison of the bonding agent was carried out. The results revealed that the flexural strength in the group containing ACP is significantly higher than the pure bonding agent (P=0.02) regardless of the time, while the adhesive containing CS nanoparticles did not show a significant increase (P=0.74). The average flexural strength was lower in bonding agent containing ACP and CS nanoparticles two months after immersion compared to 24-hour period, but this difference was not significant in the pure type.

Conclusions: According to the results of the present study, the addition of ACP nanoparticles to the dentin bonding system increased the flexural strength of dentin, while the addition of CS nanoparticles did not lead to a significant increase in flexural strength. On the other hand, the degree of conversion of adhesive increased with the addition of nanoparticles.

Tooth Remineralization

Etch- and – rinse adhesive

Nanoparticle

Flexural strength

Degree of Conversion

Adhesives are bonded to enamel or dentin in dentistry, and tooth enamel is a suitable substrate for bonding because it is mainly composed of minerals. Dentin is a collagenous tissue that helps bond resin-based dental materials to tooth structure. However, progressive degradation of the resin-dentin bond remains a major concern in adhesive dentistry. This biodegradation leads to microleakage, which contributes to the development of secondary caries and leads to restoration failure (1). Dentin is a collagen-containing mineral tissue composed of mineral apatite crystals buried within an extracellular matrix. Type I collagen is the main component of the extracellular matrix of dentin, which constitutes up to 90% of organic materials (2). Demineralized dentin does not significantly induce the precipitation of calcium phosphate minerals in the remineralization solution because there are no residual crystals on its surface. This top-down mineralization method does not depend on the spontaneous nucleation of minerals on organic substrates, but on the growth of residual apatite nuclei (3). In contrast, bottom-up methods assemble materials at the nanoscale to form larger structures. The ideal dentin remineralization method is the non-classical "bottom-up" approach. The bottom-up is a biomimetic method that imitate natural biological conditions (4). In this regard, a promising and innovative approach is the use of remineralizing materials, in which nanometer-sized apatite crystals replace the water in the dentin. Therefore, the structural integrity of caries-affected dentin is improved and the resin-dentin bond is strengthened(4). Non-collagen biomimetic analogs and molecules are commonly used for dentin remineralization. Common biomimetic molecules include casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), bioglass, polyacrylic acid (PAA), synthetic polypeptide, and polyamide-amine dendrimer(5). Cements derived from Portland CS are able to release Ca2 + and OH-, thus providing favorable conditions for the remineralization of dental hard tissues (4). CPP-ACP is a bioactive agent derived from milk products that can stabilize supersaturated calcium ion and phosphorus ion in the oral environment. When the pH drops, the stabilized ions are released. Phosphorus ion buffers pH conditions and calcium ion increases remineralization (6). Therefore, the incorporation of theses bioactive materials into dental adhesives might be of great benefit to stimulate mineralization and improve the mechanical properties of dentin without any adverse effect on the bonding agent properties including DC. The present study aimed to evaluate the effect of adding ACP and CS NPs to etch-and-rinse bonding agent on dentine flexural strength and the DC of the bonding agent. Accordingly, the null hypothesis was that the addition of ACP and CS NPs to the etch-and-rinse bonding agent has no effect on the dentine flexural strength and the DC.

This study was approved by the medical ethics committee of Islamic Azad University of Isfahan, Iran( IR.IAU.KHUISF.1401.300)

Synthesis of Calcium silicate nanoparticles

The CS NPs were synthesized using sol-gel method. Nitric acid was used as a catalyst for acid hydrolysis. Tetraethyl orthosilicate (TEOS) was added to 2 M nitric acid solution. This material is a precursor for silica. This mixture was stirred for 30 minutes and then Ca (NO3)2.4H2O was added as a source of calcium to the cell solution and stirred for 1 hour on a magnetic stirrer at ambient temperature. Afterwards, the solution was centrifuged at 6000 rpm for 8 minutes. The resulting sediments were washed with water and ethanol and then dried for 24 hours at 60°C in a vacuum oven. Finally, the samples were placed in the oven at 650°C for 5 hours until they were calcified and the final product of CS NPs with an average size of 100 nm was obtained (7).

Synthesis of amorphous calcium phosphate nanoparticles

ACP NPs were synthesized by reprecipitation method from a homogeneous solution containing calcium and phosphate ions. Briefly, (Ca₁₀ (PO₄)₆ (OH)₂ was prepared from hydroxyapatite nanoparticles containing calcium and phosphorus as a primary suspension in deionized water (2%w/w). The hydroxyapatite was dissolved in a solution containing 3 M hydrochloric acid. Afterward, 2 M sodium hydroxide solution was quickly added dropwise to form ACP. The final pH was adjusted using the diluted ammonium hydroxide and reached 8, 9, 10, and 11 respectively. ACP precipitates were separated by centrifugation and washed in deionized water for 30–40 minutes. Then, it was placed in a freeze dryer for 72 hours and dried in a vacuum oven at 80°C for one hour. Immediately after washing and before placing in the freezer, samples were frozen in liquid nitrogen. The final product of nanoparticles with an average size of 100 nm ACP was obtained (8).

Preparation of simulated body fluid

In order to investigate the bioactivity of synthesized nanoparticles, simulated body fluid (SBF) was prepared. After immersing and keeping the materials in the SBF, ion exchange takes place between the surface and the solution, and precipitates are formed on the sample surface, which, if analyzed, the bioactive mechanism of the used material is determined. In this research, the method proposed by Bohner and Lemaitre was used to prepare SBF(9). The protocol presented by Bohner and Lemaitre was prepared by following the recipe proposed by T. Kokubo and H. Takadama(10), which has a simpler manufacturing and maintenance method and the possibility of early precipitation of dissolved salts has decreased over time. In this method, two solutions are prepared separately, and when the SBF is needed, the temperature of aforementioned solutions is brought to 37°C and mixed together in equal proportions.

Flexural Strength

The 60 carious-free human third molars, extracted for periodontal reason or orthodontic treatment were collected. The excess tissue and debris on the surface of the teeth were cleaned and stored in 5% thymol solution at 4°C for a maximum of two months until the start of the experiment. A fully automatic cutting-off machine was used to prepare dentin slices with dimensions of 6 x 2 x 2 mm. Dentin specimen were mounted on a resin block. The one third occlusal crown was cut using a new diamond-cutting disc with cooling by a fully automatic cutting-off machine and the dentin samples with a thickness of 2 mm were then prepared, and both sections were made at the cementum enamel junction (CEJ). All specimens were checked for remnant of pulp or enamel tissue and any defects such as cracks and cavity. Then, the specimens were rubbed for 60 seconds under running water using 180 and 600 grit silicon carbide discs to create a smear layer similar to clinical conditions and stored in deionized water at 4^ºC. CS and ACP NPs with an average size of 100 nm were added separately to commercial two-step adhesive etch-and-rinse Tetric® N-bond, Ivoclar Vivadent, Liechtenstein at concentrations of 0 wt.%, 10 wt.%. They were mixed by an ultrasonic device in which the water temperature was controlled (cold water was added if needed to control the water temperature) for 30 minutes to obtain a homogeneous substance (11). Homogeneous and uniform distribution of nanoparticles in adhesive was confirmed by using by using scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. Disc-shaped specimen of all groups of adhesive, were examined using an environmental scanning electron microscope (ESEM) equipped with a secondary electron detector for the analyzer with an accelerating voltage of 25 kV. The specimens needed to be coated with gold for detailed examination. In order to demineralize the dentin samples, 37% phosphoric acid was applied on the samples for 15 seconds to etch them, and then they were washed with water for 15 seconds and finally dried for 5 seconds with a gentle dental air spray. The bonding agent of each group were rubbed on the dentine samples by the applicator and according to the manufacturer's instructions for 20 seconds. Then, the samples were dried by a gentle air spray and polymerized for 20 seconds by the light curing unit (intensity, 3200 mw/cm2; Valo, Ultradent, USA). The output light intensity of the light cure unit was evaluated using a light meter and recalibrated after polymerizing 20 samples. According to their groups, the samples were stored in simulated body fluid (SBF) for 24 hours and 2 months in an incubator at 37°C.The samples (n = 60) were randomly divided into the following groups:

G1: Bonding agent containing 10 wt. % ACP immersed in SBF for 24 hours

G2: Bonding agent containing 10 wt. % ACP immersed in SBF for 2 months.

G3: Bonding agent containing 10 wt. % CS immersed in SBF for 24 hours.

G4: Bonding agent containing 10 wt. % CS immersed in SBF for 2 months.

G5: Pure bonding agent immersed in SBF for 24 hours

G6: Pure bonding agent immersed in SBF for 2 months

sixty samples were prepared in the form of specimens with dimensions of 6x2x2 mm (length, width, thickness) and bonding agent was applied to each group. Then, the samples were subjected to a three-point flexural test using a universal test device to test the dentine flexural strength. In this device, a sample holder with two cylindrical supports with a 1-mm opening was used. The machine speed was set to 0.5 mm/min and the samples were tested until fracture. The testing machine software automatically recorded the amount of force at upon failure. The flexural strength was calculated using the 3PL/2bd2 formula, where P is the maximum load (N), L is the support span (mm), b is the width of the specimen, and d is the thickness of the specimen (12).

Degree of Conversion

DC was estimated using a Fourier transform infrared spectrometer (FTIR). Adhesives were applied according to their groups (D1: bonding agent containing ACP 10 wt. %, D2: bonding agent containing CS 10 wt. %, D3: pure bonding agent), on the potassium bromide discs of the spectroscope. While the adhesive is in contact with the FTIR sensor, the peak absorbance for c-c double bonds was measured for the unpolymerized resin. When the adhesive was polymerized for 40 seconds by the light cure, the peak absorbance of FTIR was measured again. The peak absorbance of aromatic c-c bonds (1607 cm-¹) and the peak absorbance of aliphatic bonds (1638 cm-¹) were measured using the aforementioned method (13). To determine DC, the FTIR spectrum was set in the range of 400–4000 cm-¹ (Fig. 1,2,3). DC was calculated by calculating the absorbance intensity ratio (C-C and C = C) (percentage of unreacted double bonds) before and after polymerization using the following Eq. (14).

DC = [1 - (C aliphatic/C aromatic)/(U aliphatic/U aromatic)] × 100%

Aliphatic C is described as the peak absorbance at 1638 cm of the polymerized resin.

Aromatic C is described as the peak absorbance at 1607 cm of the polymerized resin.

Aliphatic U is described as the peak absorbance at 1638 cm of the unpolymerized resin.

Aromatic U is described as the peak absorbance at 1607 cm of the unpolymerized resin.

Statistical analysis

Data analysis was carried out at two descriptive and inferential levels. At the descriptive level, frequency distribution tables and the mean and standard deviation indices were used to describe the situation of the sample. Also, Student's t-test and analysis of variance were used at the inferential level in SPSS ver. 26 at P < 0.05.

The mean and standard deviation of flexural strength index separately in each of the 6 groups are shown in Table 1. The results showed a significant difference between three ACP, CS and pure bonding agent groups, in terms of the average flexural strength, regardless of the time of evaluation (P < 0.05). The result of the post hoc test (pairwise comparison) of the groups indicated a significant difference between the bonding agent containing ACP and pure bonding agent groups. The average flexural strength in the bonding agent containing ACP group was higher (P = 0.02) than the pure bonding agent group that this difference was statistically significant and the average flexural strength in the bonding agent containing CS group was higher than the pure bonding group, but this difference was not statistically significant (P = 0.74). Also, the results indicated that the average flexural strength was lower in the bonding agent containing ACP and CS two months after immersion as compared to the 24-hour period (P < 0.05 for both) (Table 1), but this difference was not significant in the pure bonding groups(Table 1).

Table 1

Comparison of average flextural strength between three types of bonding agent at two immersion times
Bonding agent type	Immersion time	Average	standard deviation	Significant level ¹	Significant level ²	Significant level ³
ACP	24 h	242/34	23/50	0/007	0/802 ^a	0.05^$
ACP	2 m	193/45	44/84	0/007	0/802 ^a	0.528^$$
Cs	24 h	218/91	31/54	0/013	0/661 ^c
Cs	2 m	179/60	32/10	0/013	0/661 ^c
Pure	24 h	215/41	21/32	0/747	0/412 ^e
Pure	2 m	207/26	75/76	0/747	0/412 ^e

Degree of Conversion Test

According to Table 2, DC calculated in the pure bonding agent, bonding agent containing ACP and CS is equal to 32.56%, 38.13% and 44.93%, respectively. The highest and lowest DC is related to the bonding agent containing ACP and pure bonding agent, respectively.

Table 2

Degree of conversion in three bonding agent groups
Bonding agent	Degree of conversion
Pure	32/56%
ACP	38/13%
Cs	44/93%

Flexural Strength

Properties such as flexural strength are very important due to the mechanical loading that occurs during chewing (15).In the present study, flexural strength was studied by adding CS and ACP NPs to commercial etch-and-rinse bonding agent. The results revealed that the average flexural strength is lower in the bonding agents containing ACP and CS two months after immersion compared to 24 hours after immersion (P < 0.05 for both), but this difference was not statistically significant in the pure bonding agent (Table 1 and Diagram 1). Pairwise comparison of the groups indicated a significant difference between the bonding agents containing ACP and the pure bonding agent group. The average flexural strength in the bonding agent containing ACP group was higher (P = 0.02) than the pure bonding agent group that this difference was statistically significant and the average flexural strength in the bonding agent containing CS group was higher than the pure bonding group, but this difference was not statistically significant (P = 0.74). Therefore, the null hypothesis suggesting no change in flexural strength with the addition of 10 wt. % ACP and 10 wt. % CS to commercial two-step etch and rinse bonding agent during the immersion period of 24 hours and 2 months is partially rejected.

Many studies have been carried out on the induction of dentine remineralization by bioactive nanoparticles (16). They showed that mechanical properties of the bonding system such as flexural strength will increase when ACP and CS are added to it. These studies are consistent with the present study, which showed a significant increase in flexural strength in the bonding agent group containing ACP, but there was no significant increase in the bonding group containing CS, which, according to the above studies, could be due to the bioactivity of ACP. In a study on the flexural properties of dentin by calcium silicate and MTA-based materials, Allen et al. concluded that the material did not affect the flexural strength of dentin, while it significantly decreased over time. Although the flexural modulus was not affected by the material and time, the modulus of toughness was affected by the material and time and significantly decreased (17). In the present study, the flexural strength did not change significantly when CS NPs were added. Since the flexural strength decreased insignificantly after the use of CS in the study by Allen et al., but in the present study, the flexural strength increased insignificantly with the addition of CS. This issue could probably be attributed to the size of the particles. It should be noted that nano-sized particles were used in the present study, which in turn led to greater access of the particles to the dentin, more likelihood for the formation of apatite, and an increase in the flexural strength, although it was not significant. On the other hand, there was a significant increase in flexural strength in the present study with the addition of ACP NPs, which can probably be due to the greater bioactivity and bioavailability of ACP to dentin compared to CS. Dentin strength forms in the mineral phase of the dentin (apatite), while the dentin toughness occurs in the organic phase (collagen) (17). Allen et al. attributed the significant reduction of MOT to the fact that MOT is affected by the collagen matrix and since CS affects the mineral part of the dentin and the collagen-apatite ratio changes. As a result, a significant decrease in MOT was observed in their study, while flexural strength did not decrease significantly. As a result, it is very important to pay attention to the organic part of the dentin, regardless of the formation of apatite by ACP and CS. It has been found that materials similar to MTA can dissolve bioactive components from mineralized dentin due to the increase in pH after setting and lead to the dissolution of collagen from the mineralized surface of dentin. Since the dentin toughness is attributed to its collagen matrix, the amount of collagen extracted from demineralized dentin and the changes in the collagen structure should be investigated after exposure to these materials (17). Since ACP NPs also rapidly neutralize an acidic challenge caries and increases the pH from 4 to above 6(18), it can cause the dissolution of the collagen matrix and decrease the flexural strength over time. Therefore, a significant decrease was found in flexural strength after two months of immersion in the present study.

On the other hand, the effect of adding nanoparticles on flexural strength was evaluated in the present study during two immersion periods, i.e. 24 hours and 2 months. The results revealed lower average flexural strength two months after immersion than 24 hours after Immersion in ACP and CS bonding agent groups, while this difference was not significant in the pure type.

In their study, Zhu Qing et al. concluded that the flexural strength decreased significantly in the dentin samples that were in contact with calcium hydroxide and tricalcium silicate cement for three months compared to the control group. They attributed it to the penetration of calcium hydroxide-derived hydroxyl ions into the interfibrillar space of mineral collagen and the denaturation of collagen fibrils without affecting apatite minerals (19). In the present study, the lack of significant increase in flexural strength in the bonding agent groups containing ACP and CS NPs after 2 months of immersion in SBF could be due to the change in collagen structure. In fact, the mere formation of apatite by bioactive materials such as calcium phosphate and CS cannot be a guarantee for improving the mechanical properties of dentin in the long term, because in fact intrafibrillar minerals play an important role in the mechanical properties of dentin (20). Also, dentin mechanical properties are affected when collagen fibers are possibly degraded due to the etching process within two months, which shows the importance of preserving and maintaining collagen fibers in obtaining a durable bonding. The flexural strength also decreased by adding CS and ACP for two months of immersion in SBF in the present study. This event can be attributed to the non-silanization of nanoparticles and its hydrolytic degradation, while there was no significant decrease in the flexural strength of the pure bonding agent after two months of immersion. Mansuang et al. conducted a study on the effects of silane content on the mechanical properties and hydrolytic durability of composite resin after storage in hot water. They concluded that the flexural strength and flexural modulus of non-silanized filler after 28 days of storage in hot water decreased dramatically while silenated filler showed little change (21).

Degree of conversion

From a clinical point of view, the DC is one of the most important physicochemical properties of dental restorative materials (15). A high DC of monomers is necessary to reduce the risk of releasing toxic monomers and to provide sufficient mechanical strength (22). The release of non-bonded and uncured monomers due to insufficient adhesive polymerization can increase the risk of caries in the dental biofilm (23). The decrease in the DC can be due to the scattering and reduction of light penetration and polymerization in the inner layer (24). Therefore, one of the goals of this study is to evaluate the DC of bonding systems after adding ACP and CS NPs. According to the results of this study, the DC increased in ACP and CS NPs compared to pure bonding agent, and the DC in the bonding agent containing CS increased more than ACP; therefore, the second hypothesis of this study was rejected. The presence of filler based on weight percentage and surface chemistry, shape, size, uniform distribution, presence of hydrophobic resins and filler-resin interaction affects the bonding longevity (25). One of the reasons for reducing the DC is the formation of structural defects in cross-linked polymers (15). As mentioned earlier, nanoparticles had a uniform distribution in bonding agent in the current study, therefore, due to the absence of structural defects, the DC has increased in bonding agent groups containing ACP and CS. Another factor that affects the DC is the weight percentage of the filler. Adding fillers can increase the bonding viscosity, which has an inverse effect on the DC. However, Abdul et al. found that the addition of nanoparticles increased the DC. The weight concentration of 0.15 to 0.5% did not change the viscosity and did not negatively affect the DC. While it was found during the optimization process that fillers ( 1% w/w) negatively affect the DC; therefore, 0.5% (w/w) was considered as the maximum filler content in the resin matrix (26). In the present study, there was no significant change in viscosity during the optimization process after adding 10% ACP and CS NPs, but there was a noticeable change in bonding agent viscosity after adding 20% nanoparticles. Therefore, the 10% concentration was used in this study, and increased the DC because it did not affect the bonding agent viscosity, which is consistent with the above study.

Another important aspect to consider is the difference in refractive index of filler and resin (27). The mismatch of the refractive index between the monomer and the particles causes an increase in dispersion and, as a result, a decrease in the monomer-to-polymer conversion (28). In a study on the physical, mechanical and antibacterial properties and DC of orthodontic adhesives containing strontium-doped bioactive glass nanoparticles and calcium phosphate monohydrate, Wirinrat et al. concluded that the addition of these nanoparticles reduced the DC of adhesive, which can due to the increase in the mismatch of the refractive index (29). Since the DC has increased in the present study, it can be concluded that the refractive index of CS and ACP NPs (especially CS) matched the filler and bonding resin matrix and did not cause light scattering. Another factor that contributes to the reduction of DC is that nanoparticles prevent the conversion of monomers into polymers and reduce cross-link bonding (15). In a study, Abdul et al. found that addition of Zn-On and Zn-Mt particles to etch-and-rinse bonding had a negative effect on the DC. Accordingly, Zn-On and Zn-Mt prevented the conversion of monomers to polymers and reduced cross-link bonding, and the penetration of light rays in bonding was thus reduced due to the high opacities of ZnOn and the yellowness of Zn-Mt and the change in the overall transparency of bonding (26). Inconsistent with the above study, in the present study, the increase in DC can be due to the nature of ACP and CS NPs and their uniform distribution in bonding. This factor did not prevent the cross-link of bonding agent and caused no structural defects in the polymer, and also these particles, like Zn-On and Zn-Mt, did not change the bonding transparency.

The results of the present study revealed that the addition of ACP NPs to the dentin bonding system increased its flexural strength of dentin, while the addition of CS NPs did not lead to a significant increase in its flexural strength. On the other hand, DC increased with the addition of nanoparticles.

CS calcium silicate

ACP amorphous calcium phosphate

NP nanoparticles

DC degree of conversion

FTIR Fourier Transform Infrared

SBF simulated body fluid

CEJ cementum enamel junction

SEM scanning electron microscopy

EDX energy dispersive X-ray

ESEM environmental scanning electron microscope

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests

Funding

Not applicable.

Author contributions

All authors whose names are listed above participated in the conceptualization, methods, data analysis and writing the manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

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Effect of Infiltrating a Commercial Etch-and-Rinse Bonding Agent with Bioactive Nanoparticles on the Degree of Conversion of Adhesive and Flexural Strength of Dentin

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Version 1

Abstract

Figures

Background

Methods

Synthesis of Calcium silicate nanoparticles

Synthesis of amorphous calcium phosphate nanoparticles

Preparation of simulated body fluid

Flexural Strength

Degree of Conversion

Statistical analysis

Results

Degree of Conversion Test

Discussion

Flexural Strength

Degree of conversion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1