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Collagen cross-linking agents + dimethyl sulfoxide improving the adhesive properties of erosive lesion dentin

Abstract

To investigate the effect of the dimethyl sulfoxide combined with cross-linking agents on microtensile bond strength, silver nitrate penetration and in situ degree of conversion analysis of adhesives to the erosive dentin treatment with Cola-based soft drink. One hundred and sixty-six molars were assigned to 20 groups: (1) Treatment: Sound dentin; Erosive dentin; Erosive dentin treated with primer of dimethyl sulfoxide; Erosive dentin treated with DMSO primer containing proanthocyanidin and rivoflavin; (2) Adhesive systems: iBond Universal and Scotchbond Universal; and (3) adhesive strategy: etch-and-rinse or self-etch strategy. After restoration, specimens were sectioned into sticks to be tested. The data from microtensile bond strength (MPa), silver nitrate penetration (%) and in situ degree of conversion (%) were analyzed by (three- and two-factor ANOVA; Tukey's test α=5%). The application of dimethyl sulfoxide combined of not with cross-linkers improved all properties evaluated when compared to only erosive dentin treatment with Cola-based soft drink. However, only when dimethyl sulfoxide was combined to cross-linkers, the values of the microtensile bond strength, silver nitrate penetration and in situ degree of conversion in erosive dentin treatment with Cola-based soft drink was similar to sound dentin, for both adhesives and adhesive strategies. The application of dimethyl sulfoxide combined with the collagen cross-linking agent contributed to increasing the bond strength and degree of conversion in erosive lesion dentin, at the same time that significantly reduction of nanoleakage in this substrate.

Key Words:
Erosive tooth wear; Microtensile bond strength, Nanoleakage; Cross-linking agents, DMSO

Resumo

Este estudo investigou o efeito do dimetil sulfóxido combinado a agentes de reticulação de colágeno na resistência de união à microtração, infiltração de nitrato de prata e análise do grau de conversão por Micro-Raman de sistemas adesivos universais para a dentina erosionada por refrigerante a base de Cola. Cento e sessenta molares foram divididos em 20 grupos: (1) Tratamento: Dentina sadia; Dentina erosionada; Dentina erosionada tratada com primer de dimetil sulfóxido; Dentina erosionada tratada com primer contendo 6,5% de proantocianidina e; Dentina erosionada tratada com primer contendo 0,1% de rivoflavina; (2) Sistemas adesivos: iBond Universal e Scotchbond Universal; e (3) estratégia adesiva: estratégia condicionamento e lavagem ou autocondicionate. Após a restauração, os espécimes foram seccionados em palitos e testados. Os dados dos três testes foram analisados estatisticamente (ANOVA de 2 e 3 fatores e teste de Tukey; α = 5%). A aplicação de dimetil sulfóxido combinado ou não agentes de reticulação de colágeno melhorou todas as propriedades avaliadas quando comparado a dentina erosionada. Entretanto, apenas quando o dimetil sulfóxido foi combinado com agentes de reticulação de colágeno, os valores de adesão a dentina, infiltração de nitrato de prata e grau de conversão em dentina erosionada foi semelhante a dentina sadia, para os dois adesivos e estratégias adesivas. A aplicação de dimetil sulfóxido combinado com agentes de reticulação de colágeno contribuiu para aumentar a resistência de união e o grau de conversão dentro da camada híbrida na dentina erodida, ao mesmo tempo que reduziu significativamente a nanoinfiltração neste substrato.

Introduction

Preventive therapies based on fluoride application and improvements in hygiene practices have allowed a significant reduction in the incidence of caries 11. Bedran-Russo A, Leme-Kraus AA, Vidal CMP, Teixeira EC. An overview of dental adhesive systems and the dynamic tooth-adhesive interface. Dent Clin North Am. 2017;61(4):713-31. around the world. However, the increase acidic/erosive food ingestion, as is seen in many contemporary life , has led to an increment in the prevalence of lesion erosive 22. Schlueter N, Luka B. Erosive tooth wear - a review on global prevalence and on its prevalence in risk groups. Br Dent J. 2018;224(5):364-70..

Erosive tooth wear is a condition involving erosion with causes lying in attrition and abrasion, both of which impact quality of life, especially due to visibly shorter teeth and exposure of dentin, causing hypersensitivity (3). All these features complicate restorative management and contribute to the lower durability of restorations 33. Bartlett D. A personal perspective and update on erosive tooth wear - 10 years on: Part 2 - Restorative management. Br Dent J. 2016;221(4):167-71..

Erosive lesion dentin is a biological, chemical and structurally modified substrate 44. Lussi A, Schlueter N, Rakhmatullina E, Ganss C. Dental erosion--an overview with emphasis on chemical and histopathological aspects. Caries Res. 2011;45Suppl 1:2-12. recognized by demineralization and successive alteration of the organic matrix 55. Tjaderhane L, Buzalaf MA, Carrilho M, Chaussain C. Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'. Caries Res. 2015;49(3):193-208.. Furthermore, there are an increased micro- and nanoporosity and exposed denatured collagen fibers 44. Lussi A, Schlueter N, Rakhmatullina E, Ganss C. Dental erosion--an overview with emphasis on chemical and histopathological aspects. Caries Res. 2011;45Suppl 1:2-12.,55. Tjaderhane L, Buzalaf MA, Carrilho M, Chaussain C. Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'. Caries Res. 2015;49(3):193-208.. This process leads to occur a frequent pH drops and, consequently, promote higher proteolytic activity in the dentin matrix 55. Tjaderhane L, Buzalaf MA, Carrilho M, Chaussain C. Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'. Caries Res. 2015;49(3):193-208.. All these changes compromise resinous monomers infiltration into to erosive lesion dentin 66. Zimmerli B, De Munck J, Lussi A, Lambrechts P, Van Meerbeek B. Long-term bonding to eroded dentin requires superficial bur preparation. Clin Oral Investig. 2012;16(5):1451-61..

To increase the mechanical properties of collagen fibrils and make them less susceptible to endogenous protease action, some researchers have focused their studies on the use of collagen crosslinking agents like as carbodiimide, glutaraldehyde, riboflavin and proanthocyanidins 77. Bedran-Russo AK, Pashley DH, Agee K, Drummond JL, Miescke KJ. Changes in stiffness of demineralized dentin following application of collagen crosslinkers. J Biomed Mater Res B Appl Biomater. 2008;86(2):330-4..

Recently published studies have shown promising adhesive results from the application of an additional water-based primer containing proanthocyanidin and riboflavin to eroded dentin (8, 9). However, according to Siqueira et al.,88. Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Reis A, et al. Effect of phosphoric acid containing MMP-inactivator on the properties of resin bonding to eroded dentin. J Adhes Dent. 2019;21(2):149-58.,99. de Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Gomes JC, et al. Improving bonding to eroded dentin by using collagen cross-linking agents: 2 years of water storage. Clin Oral Investig. 2020;24(2):809-22., it was not possible to recover all adhesive and mechanical properties in eroded dentin compared to sound dentin. This may be due to the organic nature and higher molecule size of the collagen crosslinking agents. Both factors pose difficulties in the dissolution of polar solvents, limiting proper dissolution and infiltration within the demineralized collagen matrix; especially in eroded dentin wear 1010. Bedran-Russo AK, Pauli GF, Chen SN, McAlpine J, Castellan CS, Phansalkar RS, et al. Dentin biomodification: strategies, renewable resources and clinical applications. Dent Mater. 2014;30(1):62-76..

An interesting alternative is the combination of collagen crosslinking agents with more potent solvents, such as dimethyl sulfoxide. It is a polyfunctional molecule, fully mixable in most solvents and in hydrophilic and hydrophobic monomers utilized in adhesive dentistry 1111. Marren K. Dimethyl sulfoxide: an effective penetration enhancer for topical administration of NSAIDs. Phys Sportsmed. 2011;39(3):75-82.. The polar characteristic of dimethyl sulfoxide combined with the small, compact structure is responsible for its capacity to penetrate biological surfaces 1111. Marren K. Dimethyl sulfoxide: an effective penetration enhancer for topical administration of NSAIDs. Phys Sportsmed. 2011;39(3):75-82. and results in its ability to associate with water, proteins, carbohydrates, ionic substances and other constituents.

Additionally, dimethyl sulfoxide has the ability to contend with water molecules in an interpeptide hydrogen bond to dissociate extracellular collagen into a more dispersed network of fibrils 1212. Stape TH, Tjaderhane L, Tezvergil-Mutluay A, Yanikian CR, Szesz AL, Loguercio AD, et al. Dentin bond optimization using the dimethyl sulfoxide-wet bonding strategy: A 2-year in vitro study. Dent Mater. 2016;32(12):1472-81., consequently improving the infiltration of demineralized dentin 1313. Mehtala P, Agee K, Breschi L, Pashley DH, Tjäderhane L. Proprietary solvent enhances dentin wettability. Dental Materials. 2010;26:e12-e3. and maintaining the bond to dentin 1212. Stape TH, Tjaderhane L, Tezvergil-Mutluay A, Yanikian CR, Szesz AL, Loguercio AD, et al. Dentin bond optimization using the dimethyl sulfoxide-wet bonding strategy: A 2-year in vitro study. Dent Mater. 2016;32(12):1472-81.. Due to the amphiphilic nature of dimethyl sulfoxide, it promotes better dissolution and infiltration of crosslinking agents into the demineralized matrix of eroded dentin 1414. Butler WT. Dentin matrix proteins and dentinogenesis. Connect Tissue Res. 1995;33(1-3):59-65.. However, there is still a lack of knowledge; no research has evaluated an additional primer containing dimethyl sulfoxide combined with collagen crosslinking agents.

Therefore, this study evaluated the effect of dimethyl sulfoxide combined with collagen crosslinking agents on microtensile bond strength, silver nitrate penetration and in situ degree of conversion analysis of universal adhesive systems to the erosive dentin treatment with Cola-based soft drink. The experimental hypotheses evaluated were that regardless of the adhesive system, the application of dimethyl sulfoxide combined with the collagen crosslinking agents would 11. Bedran-Russo A, Leme-Kraus AA, Vidal CMP, Teixeira EC. An overview of dental adhesive systems and the dynamic tooth-adhesive interface. Dent Clin North Am. 2017;61(4):713-31. increase the bond strength, 22. Schlueter N, Luka B. Erosive tooth wear - a review on global prevalence and on its prevalence in risk groups. Br Dent J. 2018;224(5):364-70. decrease the silver nitrate penetration values of the adhesive interface and 33. Bartlett D. A personal perspective and update on erosive tooth wear - 10 years on: Part 2 - Restorative management. Br Dent J. 2016;221(4):167-71. improve in situ degree of conversion in erosive dentin treatment with Cola-based soft drink.

Materials and methods

Selection and Preparation of Teeth

One hundred and sixty-six extracted caries-free third molars were collected. After approved by the Commission for Medical Ethics (2.851.586), the molars were disinfected in 0.1% thymol solution and stored in deionized water for no more that 6 months. The occlusal third was removed using a diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). Then, a standardized smear layer was created using 600-grit silicon carbide for 60s.

Experimental Groups

One hundred and sixty molars were randomly divided to 20 experimental conditions (n = 8 teeth for microtensile bond strength, silver nitrate penetration and in situ degree of conversion analysis inside the hybrid layer measurements, according to the combination of the independent variables: 11. Bedran-Russo A, Leme-Kraus AA, Vidal CMP, Teixeira EC. An overview of dental adhesive systems and the dynamic tooth-adhesive interface. Dent Clin North Am. 2017;61(4):713-31.Treatment: Sound dentin (control group); Erosive dentin treatment with Cola-based soft drink; Erosive dentin treated with DMSO primer of dimethyl sulfoxide (Sigma-Aldrich; St. Louis, MO, USA; pH 8.2); Erosive dentin treated with Primer containing 6.5% by weight proanthocyanidin (Mega Natural Gold, Madera, USA; Batch number 05592502-01), DMSO Primer containing 0.1% by weight riboflavin (Fisher Scientific GmbH, Schwerte, Germany) Batch number 070046); 22. Schlueter N, Luka B. Erosive tooth wear - a review on global prevalence and on its prevalence in risk groups. Br Dent J. 2018;224(5):364-70. Adhesive systems: iBond Universal (Heraeus Kulzer; Hanau, Germany); Scotchbond Universal (3M Oral Care; St Paul, MN, USA); and 33. Bartlett D. A personal perspective and update on erosive tooth wear - 10 years on: Part 2 - Restorative management. Br Dent J. 2016;221(4):167-71.adhesive strategy: etch-and-rinse or self-etch strategy. The materials used, batch numbers, composition, and application modes are detailed in box 1.

Box 1
Adhesive system (Batch number), groups, composition, and application modes

Box 1
Continuation

Sample size calculation

The sample size calculation was performed on a free website, www.sealedenvelope.com. The sample size was determined considering the microtensile bond strength values of the Scotchbond Universal. The mean and standard deviation of Scotchbond Universal reported in the literature are 49.8 ± 5.3 MPa. 1515. Siqueira FSF, Cardenas AM, Ocampo JB, Hass V, Bandeca MC, Gomes JC, et al. Bonding performance of universal adhesives to eroded dentin. J Adhes Dent. 2018;20(2):121-32.,1616. Munoz MA, Luque I, Hass V, Reis A, Loguercio AD, Bombarda NH. Immediate bonding properties of universal adhesives to dentine. J Dent. 2013;41(5):404-11.,1717. Chen C, Niu LN, Xie H, Zhang ZY, Zhou LQ, Jiao K, et al. Bonding of universal adhesives to dentine--Old wine in new bottles? J Dent. 2015;43(5):525-36.. To detect a difference of 8 MPa among the tested groups at a significance level of 5%, a power of 80%, and using a two-sided test, the minimum sample size was 8 teeth per group.

Erosion model

Before erosive model, the lateral and root areas were covered with nail varnish, allowing erosive demineralization to occur only on the occlusal surface. The specimens were immersed in a cola drink (Coca-Cola, pH 2.6) 4 times a day for 90 s each (10 ml/specimen) for 5 days 99. de Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Gomes JC, et al. Improving bonding to eroded dentin by using collagen cross-linking agents: 2 years of water storage. Clin Oral Investig. 2020;24(2):809-22.. The cola drink was renewed after each erosive demineralization. Then, the specimens were washed in deionized water (10 s) and immersed in a remineralizing solution (pH 6.7, 10 ml/specimen) for 1 h 99. de Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Gomes JC, et al. Improving bonding to eroded dentin by using collagen cross-linking agents: 2 years of water storage. Clin Oral Investig. 2020;24(2):809-22., between erosive demineralization. The remineralization solution was replaced daily. The pH levels of all solutions were checked periodically.

Restorative procedures

After erosive model, the teeth were distributed according to the combination of variables. For groups where the primer of the dimethyl sulfoxide alone or combined to the cross-linking agents were applied, the experimental primers containing dimethyl sulfoxide was done according to Stape et al., 1212. Stape TH, Tjaderhane L, Tezvergil-Mutluay A, Yanikian CR, Szesz AL, Loguercio AD, et al. Dentin bond optimization using the dimethyl sulfoxide-wet bonding strategy: A 2-year in vitro study. Dent Mater. 2016;32(12):1472-81.. For this, 50 μL of dimethyl sulfoxide (Sigma-Aldrich; St. Louis, MO, USA; pH 8.2) was mixed to a base of water 50% (v/v) added or not the 6.5 wt% proanthocyanidin or 0.1 wt% riboflavin. The primers were renewed daily.

For etch-and-rinse strategy, the dimethyl sulfoxide primer was applied after acid-etching. The teeth were conditioned with 37% phosphoric acid etching (Condac, FGM Dental Products; Joinville, SC, Brazil) for 15 s, rinsed and kept slightly moist. For both strategies, the experimental primers were then applied using a microbrush by 60 s (Brush, KG Sorensen, Cotia, SP, Brazil). An air stream was used for 5 to 10 s to remove the excess. For the dimethyl sulfoxide associated with riboflavin primer application, the dentin surfaces were photoactivated using a curing-light unit set at 1200 mW/cm2 (Radii, SDI; Bayswater, Victoria, Australia) 99. de Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Gomes JC, et al. Improving bonding to eroded dentin by using collagen cross-linking agents: 2 years of water storage. Clin Oral Investig. 2020;24(2):809-22. and air-dried. Independent of the group, the dentin surface was kept slightly moist before the application of the adhesive (Box 1).

The adhesive systems were applied following the manufacturer's recommendations; further, composite resin buildup (Opallis, FGM, Joinville, Brazil) were placed in layer of 2 mm each, and individually photoactivated for 40 s (Radii, SDI; Bayswater, Victoria, Australia). After 24 h, composite-dentin bonded sticks (cross-sectional area 0.8 mm2) were prepared using a slow-speed diamond saw (Isomet, Buehler) and measured by a digital caliper (Digimatic Caliper, Mitutoyo, Tokyo, Japan) to calculate the bond strength in MPa. The number of sticks showing premature failure (PF) during specimen preparation was recorded for each tooth.

Two composite-dentin bonded sticks per tooth from each experimental group were used to evaluate the in situ degree of conversion within the adhesive/hybrid layers. Three composite-dentin bonded sticks per tooth were used to evaluate silver nitrate penetration, and the remaining composite-dentin bonded sticks were tested for microtensile bond strength.

Microtensile bond strength

The composite-dentin bonded sticks were fixed to a Geraldeli’s jig using cyanoacrylate glue and stressed under tension (Instron, Instron Inc., Canton, USA) at 1.0 mm/min until fracture occurred. The microtensile bond strength values (MPa) derived by dividing the imposed force by bonding area.

The fracture mode of the composite-dentin bonded sticks were examined with a light microscope at 100X magnification (Olympus SZ40, Tokyo, Japan) and categorized as cohesive (failure exclusively within the dentin or the resin composite) or adhesive (failure at the resin-dentin interface or with partial cohesive failure of the neighboring substrates). Specimens with premature fracture were included in the tooth mean for statistical analysis.

Silver nitrate penetration analysis

Composite-dentin bonded sticks (n = 3 per each tooth) were immersed in an aqueous solution of ammoniacal silver nitrate solution for 24 h, followed by 8h in photo-developing solution under a fluorescent lamp. Specimens were wet-polished using SiC papers and polished used diamond paste (Buehler Ltd., Lake Bluff, IL, USA).

The composite-dentin interfaces were observed using a field-emission scanning electron microscope (VEGA 3 TESCAN, Shimadzu, Tokyo, Japan) at 15 kV with backscattering mode. The amount of SNP within the adhesive layer, hybrid layer of each stick was measured in three regions (5 μm × 5 μm) of the bonded stick. Acquisition mode of images and calculate percentage of SNP was according to Hass et al., 1818. Hass V, Dobrovolski M, Zander-Grande C, Martins GC, Gordillo LA, Rodrigues Accorinte Mde L, et al. Correlation between degree of conversion, resin-dentin bond strength and nanoleakage of simplified etch-and-rinse adhesives. Dent Mater. 2013;29(9):921-8.. ImageJ software were used to calculate the percentage of SNP within hybrid layers in each specimen.

In situ degree of conversion by Micro-Raman analysis

Composite-dentin bonded sticks (n = 2 per each tooth) were prepared as previously described by Hass et al., 1818. Hass V, Dobrovolski M, Zander-Grande C, Martins GC, Gordillo LA, Rodrigues Accorinte Mde L, et al. Correlation between degree of conversion, resin-dentin bond strength and nanoleakage of simplified etch-and-rinse adhesives. Dent Mater. 2013;29(9):921-8.. In situ degree of conversion analysis was measured inside the hybrid layer of the adhesive interfaces using a micro-Raman spectrometer (XploRA ONETM Raman microscope, HORIBA Scientific, New Jersey, NY, USA). Previously, the micro-Raman spectrometer was calibrated for zero. Them, the Raman spectrometer was configured to use a 638-nm diode laser, 100x objective, 600-lines/mm grafting centered between 500 and 1800 cm-1, using 100 mW power, spatial resolution of approximately 3 µm, spectral resolution of 5 cm-1, and accumulation time of 25 s with 3 co-additions. The spectra were acquired at 3 different sites for each specimen, in the middle of the hybrid layer, and the values averaged for statistical purpose. Post-processing of the spectra was performed using the Opus Spectroscopy Software version 6.5. The average of the values was used for statistical analysis and the spectra of uncured adhesives were considered as references.

The ratio of the double-bond content of monomer to polymer in the adhesive were quantified by calculating ratio derived from the aliphatic C=C (vinyl) absorption (1638 cm-1) to the aromatic C=C absorption (1608 cm-1) signals for both polymerized and unpolymerized samples. The DC was calculated, according to the following formula:

D C ( % ) = ( 1 - & # 091 ; R c u r e d / R u n c u r e d & # 093 ; ) × 100

Where “R” is the ratio of aliphatic and aromatic peak intensities at 1638 cm-1 and 1608 cm-1 in cured and uncured adhesives, in accordance with Hass et al. 1818. Hass V, Dobrovolski M, Zander-Grande C, Martins GC, Gordillo LA, Rodrigues Accorinte Mde L, et al. Correlation between degree of conversion, resin-dentin bond strength and nanoleakage of simplified etch-and-rinse adhesives. Dent Mater. 2013;29(9):921-8.. In addition, the more intense peaks observed for all materials and the corresponding chemical bonding were recorded.

Occlusal and lateral morphological modification by erosion model

Six teeth were used in this part of the study. The teeth were sectioned parallel to the occlusal surface using a low-speed diamond saw (IsoMet 1000; Buehler, Lake Bluff, IL, USA) under cooling water to expose the mid-coronal dentin. After that, each tooth was transversely sectioned in a buccal-to-lingual direction to obtain two halves per teeth (n=12 specimens). For lateral analysis of the morphological modification, in the half the teeth a precut groove was made on the pulpar side to allow segmentation.

Specimens from each tooth were divided according to sound dentin and erosive dentin treatment with Cola-based soft drink. To allowing erosive demineralization to occur only on the occlusal surface, lateral and pulpar areas were covered with two layers of nail varnish. After that, the specimens were submitted to erosive demineralization according to erosion model section.

After erosive demineralization, the surfaces were rinsed with tap water for 30 seconds and air-dried for 5 seconds, keeping the dentin moist. The specimens were treated according to Siqueira et al. 1919. Siqueira F, Cardenas A, Gomes GM, Chibinski AC, Gomes O, Bandeca MC, et al. Three-year effects of deproteinization on the in vitro durability of resin/dentin-eroded interfaces. Oper Dent. 2018;43(1):60-70. and Kenshima et al. 2020. Kenshima S, Reis A, Uceda-Gomez N, Tancredo Lde L, E Filho LE, Nogueira FN, et al. Effect of smear layer thickness and pH of self-etching adhesive systems on the bond strength and gap formation to dentin. J Adhes Dent. 2005;7(2):117-26. and the entire surface was examined under a scanning electron microscope (MIRA3 LM, Tescan Orsay Holding, Warrendale, PA, USA). Three photomicrographs of representative occlusal and lateral areas were taken at 5000X and 20.000X magnification. It was possible to see a totally removal of the smear layer with a higher opening of the dentin tubules in dentin submitted to erosion model in comparison to sound dentin (Figure 1).

Figure 1
Representative SEM images of the occlusal and cross-sectional view of sound (A to C) and eroded (D to F) dentin specimens. In sound dentin, it was possible to see an obliteration of the dentin tubules (see B and white hands in C). On the other hand, after the erosive model, it was occurring a totally removal of the smear layer with a higher opening of the dentin tubules (see E and white hands in F). This confirm that the present model produced the desired erosive effect on dentin.

Statistical analysis

The Shapiro-Will test was employed to assess whether the data from these tests followed a normal distribution. The Barlett’s test was performed to determine the validity of the assumption of equal variances. After confirmed the normal distribution and homoscedasticity, the mean of microtensile bond strength (MPa), silver nitrate penetration (%) and in situ degree of conversion (%) of all bonded sticks from the same tooth were averaged for statistical purposes. Therefore, the experimental unit in this study was the tooth. The value attributed to PFs specimens was according to described in the previous studies (18, 21). In this study, the value was 4.2 MPa. The microtensile bond strength (MPa), silver nitrate penetration (%) and in situ degree of conversion (%) means for every test group resulted from the average of the eight teeth used per group. The microtensile bond strength (MPa) and silver nitrate penetration (%) data were analyzed by three-factor ANOVA (treatment vs. adhesive systems vs. adhesive strategies). The in situ degree of conversion (%) data for each adhesive were analyzed by two-factor ANOVA (treatment vs. adhesive strategies). For all tests, a post-hoc Tukey's test with of significance (alpha=0.05) was applied.

Results

Microtensile bond strength

Approximately 20-25 composite-dentin bonded sticks were obtained per tooth, including the premature failures. Regarding microtensile bond strength values, the cross-product interaction was not significant, nor was the main factor adhesive strategy (Table 1; p = 0.42 and p = 0.72, respectively). However, the main factors treatment (p = 0.002) and adhesive (p = 0.0001) were statistically significant (Table 1). A significantly lower microtensile bond strength value was observed in erosive dentin treatment with Cola-based soft drink compared to dimethyl sulfoxide groups (Table 1; p = 0.002). However, a significant increase in microtensile bond strength values was observed when dimethyl sulfoxide was combined with proanthocyanidin and riboflavin-UVA in comparison to only dimethyl sulfoxide (Table 1; p = 0.002). Actually, when dimethyl sulfoxide was combined with proanthocyanidin and riboflavin-UVA similar microtensile bond strength results between erosive dentin treatment with Cola-based soft drink and sound dentin were observed (Table 1; p > 0.05). Usually, SBU showed higher microtensile bond strength values compared to IBU (Table 1; p = 0.001).

Table 1
Means and standard deviations of resin-dentin bond strength values, as well as statistical analysis (MPa) for all experimental groups (*).

The most common fracture mode observed was adhesive/mixed for all experimental groups (Table 2). For Sound dentin, 99% of failures were considered A/M and only 1% were PF (Table 2). For eroded dentin, higher number of premature failures were observed (26%) (Table 2). When eroded dentin was treated with dimethyl sulfoxide or combined with proanthocyanidin and riboflavin-UVA only 2% of premature failures were observed (Table 2).

Table 2
Number of specimens (%) according to fracture mode.

Silver nitrate penetration analysis

Neither the cross-product interaction nor the main factor adhesive strategy was significant (Table 3; p = 0.47 and p = 0.55, respectively). On the other side, treatment (p = 0.02) and adhesive (p = 0.01), as main factors, were considered statistically significant (Table 3). A significantly higher silver nitrate penetration value was observed in eroded dentin compared to dimethyl sulfoxide groups (Table 3; p = 0.02; Figure 2). Contrastingly, the application of dimethyl sulfoxide decreased silver nitrate penetration values in eroded dentin, combined or not with proanthocyanidins and riboflavin-UVA (Table 3; p = 0.02; Figure 2). However, the addition of proanthocyanidins and riboflavin-UVA showed silver nitrate penetration values compared to observed in sound dentin (Table 3; p > 0.05). For all comparisons, Scotchbond Universal showed lower silver nitrate penetration values compared to IBond Universal (Table 3; p = 0.02).

Figure 2
Representative SEM images of the resin-dentin interfaces of the experimental groups. Silver nitrate deposit was observed. However, this deposition was more pronounced in ED (white hands) when compared to sound dentin groups, as well as DMSO or DMSO combination to collagen cross-linking agent. (Co = composite resin; AL = adhesive layer, HL = hybrid layer, De = dentin).

Table 3
Means and standard deviations of silver nitrate penetration values (%), as well as statistical analysis for all experimental groups (*).

In situ degree of conversion by Micro-Raman analysis

Regarding in situ degree of conversion values, the cross-product interaction and main factor adhesive strategy were not significant for each adhesive (Table 4; p > 0.38 and p > 0.53, respectively). However, the main factor treatment was considered statistically significant (Table 4; p = 0.01 for both adhesives). A significantly lower in situ degree of conversion value was observed in eroded dentin compared to dimethyl sulfoxide groups (Table 4; p = 0.01), while the application of dimethyl sulfoxide increased in situ degree of conversion values in eroded dentin, independently combination with proanthocyanidin and riboflavin-UVA (Table 4; p = 0.01). Also, the addition of dimethyl sulfoxide in eroded dentin, independently combination with proanthocyanidin and riboflavin-UVA, showed similar in situ degree of conversion results when compared to sound dentin for both adhesives (Table 4; p > 0.05).

Table 4
Means and standard deviations of in situ degree of conversion by micro-raman values (%), as well as statistical analysis for all experimental groups (*)

Discussion

The results presented in this study showed that the application of dimethyl sulfoxide as an additional primer prior to an adhesive procedure on eroded dentin significantly increased the microtensile bond strength and in situ degree of conversion as well as decreasing the silver nitrate penetration values compared to the erosive dentin treatment with Cola-based soft drink group, leading to acceptance of all hypotheses of the present study.

As mentioned in the introduction, erosive tooth wear promotes demineralization and dissolution of the mineral component, and continued progression induces formation of a zone of dense, the fibrous collagen network 55. Tjaderhane L, Buzalaf MA, Carrilho M, Chaussain C. Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'. Caries Res. 2015;49(3):193-208.. An increased loss of collagen periodicity occurs in the collagen matrix 2121. Reis A, Grande RH, Oliveira GM, Lopes GC, Loguercio AD. A 2-year evaluation of moisture on microtensile bond strength and nanoleakage. Dent Mater. 2007;23(7):862-70., and these spaces between the collagen fibrils are occupied by water 55. Tjaderhane L, Buzalaf MA, Carrilho M, Chaussain C. Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'. Caries Res. 2015;49(3):193-208..

All these features hinder adequate infiltration by the adhesive monomers into the underlying eroded dentin 66. Zimmerli B, De Munck J, Lussi A, Lambrechts P, Van Meerbeek B. Long-term bonding to eroded dentin requires superficial bur preparation. Clin Oral Investig. 2012;16(5):1451-61.,88. Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Reis A, et al. Effect of phosphoric acid containing MMP-inactivator on the properties of resin bonding to eroded dentin. J Adhes Dent. 2019;21(2):149-58.,99. de Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Gomes JC, et al. Improving bonding to eroded dentin by using collagen cross-linking agents: 2 years of water storage. Clin Oral Investig. 2020;24(2):809-22.. Therefore, the hybrid layer formed in eroded dentin could result in areas of hydrophilic predominance and demineralized zones with collagen fibrils incompletely encapsulated by resin monomers 2222. Tjaderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. J Dent Res. 1998;77(8):1622-9., leading to the formation of a structurally imperfect and highly porous hybrid layer 2222. Tjaderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. J Dent Res. 1998;77(8):1622-9.. These characteristics significantly affect the bonding performance of adhesive systems in eroded dentin 66. Zimmerli B, De Munck J, Lussi A, Lambrechts P, Van Meerbeek B. Long-term bonding to eroded dentin requires superficial bur preparation. Clin Oral Investig. 2012;16(5):1451-61.,88. Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Reis A, et al. Effect of phosphoric acid containing MMP-inactivator on the properties of resin bonding to eroded dentin. J Adhes Dent. 2019;21(2):149-58.,99. de Siqueira FSF, Hilgemberg B, Araujo LCR, Hass V, Bandeca MC, Gomes JC, et al. Improving bonding to eroded dentin by using collagen cross-linking agents: 2 years of water storage. Clin Oral Investig. 2020;24(2):809-22.. These results can also confirm by major amount of premature failure for erosive dentin treatment with Cola-based soft drink group when compared with sound dentin.

Dimethyl sulfoxide is an ideal solvent for medical purposes with a special ability to penetrate the biological surface 1111. Marren K. Dimethyl sulfoxide: an effective penetration enhancer for topical administration of NSAIDs. Phys Sportsmed. 2011;39(3):75-82.. Due to this characteristic, dimethyl sulfoxide has been recommended to increase adhesive penetration into the exposed collagen matrix 1212. Stape TH, Tjaderhane L, Tezvergil-Mutluay A, Yanikian CR, Szesz AL, Loguercio AD, et al. Dentin bond optimization using the dimethyl sulfoxide-wet bonding strategy: A 2-year in vitro study. Dent Mater. 2016;32(12):1472-81. and, consequently, significantly increase the microtensile bond strength values compared to a erosive dentin treatment with Cola-based soft drink group when compared to sound dentin, as well as previously observed by several authors 1212. Stape TH, Tjaderhane L, Tezvergil-Mutluay A, Yanikian CR, Szesz AL, Loguercio AD, et al. Dentin bond optimization using the dimethyl sulfoxide-wet bonding strategy: A 2-year in vitro study. Dent Mater. 2016;32(12):1472-81.,2323. Stape TH, Tjaderhane L, Marques MR, Aguiar FH, Martins LR. Effect of dimethyl sulfoxide wet-bonding technique on hybrid layer quality and dentin bond strength. Dent Mater. 2015;31(6):676-83..

Furthermore, dimethyl sulfoxide is a special solvent that dissolves both non-polar and polar compounds. It is a polyfunctional molecule with a highly polar S=O group and two hydrophobic groups. The partial negative charge of the oxygen atom of the dimethyl sulfoxide molecule favors the formation of hydrogen bridges with water molecules 2323. Stape TH, Tjaderhane L, Marques MR, Aguiar FH, Martins LR. Effect of dimethyl sulfoxide wet-bonding technique on hybrid layer quality and dentin bond strength. Dent Mater. 2015;31(6):676-83., thereby reducing the self-associative tendency of water 2424. Vishnyakov A, Laaksonen A, Widmalm G. Molecular dynamics simulations of alpha-D-Manp-(1-->3)-beta-D-Glcp-OMe in methanol and in dimethyl sulfoxide solutions. J Mol Graph Model. 2001;19(3-4):338-42, 96-7.. Also, taking into account the silver nitrate penetration within the hybrid layer representing water-rich interfacial regions, the interaction between dimethyl sulfoxide and water could be responsible for the lower silver nitrate penetration values when compared to the erosive dentin treatment with Cola-based soft drink group. In addition, the application of dimethyl sulfoxide groups in erosive dentin treatment with Cola-based soft drink showed similar silver nitrate penetration as sound dentin, in accordance with a previously published study 2323. Stape TH, Tjaderhane L, Marques MR, Aguiar FH, Martins LR. Effect of dimethyl sulfoxide wet-bonding technique on hybrid layer quality and dentin bond strength. Dent Mater. 2015;31(6):676-83..

Once dimethyl sulfoxide reduces the number of water molecules entrapped between polymeric chains, an increase in the degree of conversion would be expected 2525. Stape THS, Tjaderhane L, Abuna G, Sinhoreti MAC, Martins LRM, Tezvergil-Mutluay A. Optimization of the etch-and-rinse technique: New perspectives to improve resin-dentin bonding and hybrid layer integrity by reducing residual water using dimethyl sulfoxide pretreatments. Dent Mater. 2018;34(7):967-77., as observed in the present study. However, despite improvement in bonding performance promoted by dimethyl sulfoxide in an eroded-dentin interface, there is no cross-linking effect reinforcing the mechanical properties of the dentinal matrix 1010. Bedran-Russo AK, Pauli GF, Chen SN, McAlpine J, Castellan CS, Phansalkar RS, et al. Dentin biomodification: strategies, renewable resources and clinical applications. Dent Mater. 2014;30(1):62-76.. Thus, this is the first study evaluating the effect of dimethyl sulfoxide combined with collagen crosslinking agents on the bonding performance of universal adhesives in eroded dentin.

According to the results of this study, the combination of dimethyl sulfoxide with riboflavin or proanthocyanidin agents promoted a significant increase of microtensile bond strength values compared erosive dentin treatment with Cola-based soft drink group or even when only dimethyl sulfoxide was applied. An increase in bond strength values occurred for all dimethyl sulfoxide groups. However, only in the dimethyl sulfoxide- combined cross-linking agents, the results in the erosive dentin treatment with Cola-based soft drink were similar to sound dentin.

This improvement in bond strength could be attributed to the cross-linking effect of both collagen crosslinking agents used in the present study. Riboflavin is a known collagen crosslinking agent when combined with UVA or LED lights 2424. Vishnyakov A, Laaksonen A, Widmalm G. Molecular dynamics simulations of alpha-D-Manp-(1-->3)-beta-D-Glcp-OMe in methanol and in dimethyl sulfoxide solutions. J Mol Graph Model. 2001;19(3-4):338-42, 96-7.. Due to the photo-oxidative cross-linking effect, riboflavin showed stability and resistance to collagen degradation via the increased mechanical properties of dentin collagen 2626. Cova A, Breschi L, Nato F, Ruggeri A, Jr., Carrilho M, Tjaderhane L, et al. Effect of UVA-activated riboflavin on dentin bonding. J Dent Res. 2011;90(12):1439-45.. On the other hand, proanthocyanidins improves the biomechanical properties of collagen fibrils through several chemical mechanisms 2727. Fawzy A, Nitisusanta L, Iqbal K, Daood U, Beng LT, Neo J. Characterization of riboflavin-modified dentin collagen matrix. J Dent Res. 2012;91(11):1049-54., which produce collagen cross-linking. This increase of the mechanical properties of dentin collagen was also reflected in the increase in adhesive/mixture failures in these experimental groups when compared to erosive dentin treatment with Cola-based soft drink.

However, the combination of dimethyl sulfoxide with riboflavin or proanthocyanidin agents did not significantly affect the silver nitrate penetration or in situ degree of conversion values. These results were expected, as the collagen crosslinking agents tested herein cannot alter the hydrophilic property of adhesive, improve adhesive evaporation or produce more permeable adhesive layers 1212. Stape TH, Tjaderhane L, Tezvergil-Mutluay A, Yanikian CR, Szesz AL, Loguercio AD, et al. Dentin bond optimization using the dimethyl sulfoxide-wet bonding strategy: A 2-year in vitro study. Dent Mater. 2016;32(12):1472-81.. Also, due to the molecular size of organic substances such as riboflavin and proanthocyanidin, dissolution in polar solvents is difficult, limiting proper infiltration within the demineralized collagen matrix 1010. Bedran-Russo AK, Pauli GF, Chen SN, McAlpine J, Castellan CS, Phansalkar RS, et al. Dentin biomodification: strategies, renewable resources and clinical applications. Dent Mater. 2014;30(1):62-76..

Actually, some collagen crosslinking agents have potential to reduce the polymerization of adhesive systems 2828. Liu Y, Wang Y. Effect of proanthocyanidins and photo-initiators on photo-polymerization of a dental adhesive. J Dent. 2013;41(1):71-9.. For instance, proanthocyanidins donate H+ ions to free radicals and inhibit the initiation and propagation of the polymerization process 2828. Liu Y, Wang Y. Effect of proanthocyanidins and photo-initiators on photo-polymerization of a dental adhesive. J Dent. 2013;41(1):71-9.. However, at concentrations as low as those used, proanthocyanidins did not interfere with the degree of conversion, as previously observed in vitro studies 92. Schlueter N, Luka B. Erosive tooth wear - a review on global prevalence and on its prevalence in risk groups. Br Dent J. 2018;224(5):364-70.,1212. Stape TH, Tjaderhane L, Tezvergil-Mutluay A, Yanikian CR, Szesz AL, Loguercio AD, et al. Dentin bond optimization using the dimethyl sulfoxide-wet bonding strategy: A 2-year in vitro study. Dent Mater. 2016;32(12):1472-81..

On the other hand, this study provided evidence for better performance of Scotchbond Universal than IBond Universal for all comparisons. This is in agreement with a Jacker-Guhr et al., 2929. Jacker-Guhr S, Sander J, Luehrs AK. How “universal" is adhesion? Shear bond strength of multi-mode adhesives to enamel and dentin. J Adhes Dent. 2019;21(1):87-95.. According to this study, due to presence of acetone and absence of 2-hydroxyethyl methacrylate (HEMA), the IBond Universal application needs a strict protocol regarding correct moisture management 2929. Jacker-Guhr S, Sander J, Luehrs AK. How “universal" is adhesion? Shear bond strength of multi-mode adhesives to enamel and dentin. J Adhes Dent. 2019;21(1):87-95., mainly because HEMA-free universal adhesives are more susceptible to phase separation at the adhesive interface 2929. Jacker-Guhr S, Sander J, Luehrs AK. How “universal" is adhesion? Shear bond strength of multi-mode adhesives to enamel and dentin. J Adhes Dent. 2019;21(1):87-95..

In terms of self-etch strategy, it is worth mentioning that IBond Universal is an intermediary strong acidic adhesive in comparison to Scotchbond Universal, a mildly acidic adhesive. According to Van Meerbeek et al., 3030. Van Meerbeek B, Yoshihara K, Van Landuyt K, Yoshida Y, Peumans M. From Buonocore's pioneering acid-etch technique to self-adhering restoratives. A status perspective of rapidly advancing dental adhesive technology. J Adhes Dent. 2020;22(1):7-34., the most favorable adhesive performance in dentin was obtained with mild adhesives; intermediary strong adhesives showed more zones of partially demineralized but uninfiltrated dentine beneath hybrid layers, as observed in the silver nitrate penetration results of the present study. These areas are considered locations of potential degradation mechanisms with these intermediary strong adhesives 3030. Van Meerbeek B, Yoshihara K, Van Landuyt K, Yoshida Y, Peumans M. From Buonocore's pioneering acid-etch technique to self-adhering restoratives. A status perspective of rapidly advancing dental adhesive technology. J Adhes Dent. 2020;22(1):7-34..

The results of the present study may promote the development of novel strategies to improve eroded-dentin bonding performance. However, microtensile bond strength and chemical evaluations of adhesive interfaces can only provide limited information related to the interactions of these crosslinking agents in eroded dentin. Thus, more investigations are necessary to present the real advantages of the combination of dimethyl sulfoxide with cross-linking agents, especially after long-term water storage.

In the conclusion, the application of dimethyl sulfoxide combined with collagen crosslinking agents contributed to increasing the microtensile bond strength and in situ degree of conversion with a significant reduction of silver nitrate penetration in erosive dentin treatment with Cola-based soft drink.

Acknowledgment

Authors are grateful to State Foundation of Support to Research, Scientific and Technological Development of Maranhão (FAPEMA) and Central de Laboratórios Multiusuários/Universidade Estadual de Ponta Grossa (CLABMU/UEPG) for the support provided. Also, this study was partially supported by the National Council for Scientific and Technological Development (CNPq) under grants 303332/2017-4 and 308286/2019-7, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001

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Publication Dates

  • Publication in this collection
    05 Jan 2022
  • Date of issue
    Nov-Dec 2021

History

  • Received
    06 July 2021
  • Accepted
    13 Sept 2021
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